Ultrap1 enzyme double-antibody sandwich ELISA kit and application thereof

Abstract

The invention belongs to the technical field of Ultrap1 enzyme quantitative detection, and particularly relates to an Ultrap1 enzyme double-antibody sandwich ELISA (enzyme-linked immuno sorbent assay) kit and application. Comprising an elisa plate coated with an anti-Ultrap1 enzyme mouse monoclonal antibody, an anti-Ultrap1 enzyme rabbit polyclonal antibody, an HRP-labeled goat anti-rabbit antibody, a diluent, a cleaning solution, a confining solution, a developing solution and a stop solution. According to the present invention, the complex of the Ulp1 enzyme and the SUMO tag protein is adopted as the immunogen to simultaneously immunize mice and New Zealand rabbits so as to prepare different species of the good affinity Ulp1 enzyme antibodies, and then the optimal reaction condition is further screened to develop the stable double-antibody sandwich ELISA detection kit with characteristics of high specificity and strong sensitivity, the defects in the prior art can be effectively overcome.

Description

TECHNICAL FIELD

[0001] 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. BACKGROUND

[0002] Escherichia coli is one of the important prokaryotic expression hosts in biological research and industrial application. Since the first biosimilar drug human insulin was launched in 1982, Escherichia coli has become the core tool for the production of recombinant protein drugs. Its advantages include: Fast growth: doubling time is only 20-30 minutes, suitable for industrial amplification; Clear genetic background: mature gene manipulation technology, rich expression vector types; Low cost: simple culture medium, mature fermentation process; High expression level: the proportion of recombinant protein can be more than 20% of total cell protein.

[0003] The main limitation of Escherichia coli as an expression host is the lack of eukaryotic post-translational modification function (such as glycosylation), and the folding auxiliary system is imperfect, which may lead to incorrect pairing of protein disulfide bonds or abnormal conformation, and under high expression conditions, recombinant proteins often accumulate in the form of inactive aggregates (inclusion bodies).

[0004] Strategies for Escherichia coli to reduce inclusion body formation include: 1. Reduce the culture temperature: reduce the culture temperature from the conventional 37℃ to 16-25℃, which can slow down the protein synthesis rate, prolong the correct folding time, and reduce the aggregation caused by hydrophobic interaction.

[0005] 2. Control the induction strength and timing: use low concentration of inducer (such as IPTG concentration) or mild induction strategy (such as auto-induction medium) to avoid aggregation caused by overexpression of exogenous proteins.

[0006] 3. Co-expression of chaperones and foldases: introduce GroEL / GroES, DnaK / DnaJ / GrpE, etc. chaperone system, or thioredoxin (Trx) foldase to assist the correct folding of recombinant proteins.

[0007] 4. Add folding auxiliary reagents: add sorbitol, sucrose, etc. osmotic pressure protectants, or appropriate L-arginine to improve the folding microenvironment.

[0008] 5. Optimize the design of signal peptide: direct the protein to the periplasmic space through secretion expression (such as using PelB signal peptide), and use the oxidative environment to promote correct pairing of disulfide bonds.

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

[0010] 7. Optimization of medium components: Provide a nutrient-rich medium (e.g., add glucose, trace elements), and strictly control the dissolved oxygen content (≥30%) and pH (6.8-7.4) to maintain cellular metabolic homeostasis.

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

[0012] Table 1 Properties of common solubility-enhancing tags Tag name Original source Characteristics Glutathione transferase GST Schistosoma japonicum Tag size is large, about 26 kDa. It can bind glutathione with high affinity, and the purification matrix has excellent selectivity. The substrate can be eluted by competition, and the conditions are mild. However, the fusion protein is prone to breakage, and the solubilization effect is general. Maltose binding protein MBP Escherichia coli Tag size is large, about 50 kDa. It can specifically bind maltose, and the purification matrix has excellent selectivity. The substrate can be eluted by competition, and the conditions are mild. The solubilization effect is good, but the fusion protein is prone to breakage. Thioredoxin Trx Escherichia coli Tag size is small, and the SDS-PAGE apparent molecular weight is close to 20 kDa. There is no corresponding affinity purification matrix, and it is usually used in combination with His-tag. It can promote protein expression, but the solubilization ability is general. Antibody binding domain Protein A Staphylococcus aureus Tag size is small, and the Z domain of 58 aa is usually selected. It can bind to the Fc region of IgG antibodies of various genera, and the purification matrix has good selectivity. However, the elution conditions are harsh, such as acid elution or denaturation elution, and the solubilization effect is general. It can be used in combination with proteases to achieve enzyme digestion elution. Ubiquitin-like protein SUMO Saccharomyces cerevisiae Tag size is small, and the SDS-PAGE apparent molecular weight is close to 20 kDa. There is no corresponding affinity purification matrix, and it is usually used in combination with His-tag. The solubilization ability is excellent, and it can be recognized by ULP protease conformation and efficiently removed. In the magnetic bead system, rapid enzyme digestion elution can be achieved. SUMO is a ubiquitin-like protein from Saccharomyces cerevisiae, and its ubiquitin-like domain can stabilize the folding intermediate state, promote correct disulfide bond pairing, and reduce hydrophobic region exposure, improving the conformational integrity of complex proteins and reducing the aggregation tendency of recombinant proteins, especially for the expression of difficult-to-solubilize eukaryotic proteins in prokaryotic systems.

[0013] Table 2 Advantages of the SUMO tag system Characteristics SUMO tag Traditional tag Solubilization effect High (broadly applicable) Medium (dependent on target protein characteristics) Risk of tag residue Can be completely removed May affect protein structure research Protease compatibility Strong specificity, mild conditions Optimized cleavage conditions are required Production cost Low (single column purification + one-step enzyme digestion) Higher (requires complex purification steps) SUMO tags can improve solubility and stability during recombinant protein expression, but may mask the natural active sites of target proteins or affect their higher-order structures, so precise removal is required to obtain target proteins with intact structures and undisturbed functions.

[0014] Specific proteases (Ulp1 protease) can be used to hydrolyze the peptide bond after the second glycine in the C-terminal Gly-Gly-x sequence, with higher efficiency than Factor Xa, Thrombin, TEV, 3C, and other tool proteases. After Ulp1 protease cleavage, there are no redundant tag amino acid residues at the N-terminus of the target protein, which is an advantage that other solubility-enhancing tags do not have.

[0015] Ulp1 enzyme needs to be completely removed after removing the SUMO tag, mainly based on the following four reasons: 1. Avoid non-specific protein degradation: Ulp1 is a cysteine protease, and even after completing SUMO tag cleavage, it still retains catalytic activity. If it remains in the sample, it may recognize and cleave other proteins containing SUMO modifications (such as endogenous SUMOylated proteins in cells), leading to non-target protein degradation or functional abnormalities. Although commercial Ulp1 enzymes claim to be "free of non-specific protease contamination", residual enzymes still pose a risk of accidental cleavage.

[0016] 2. Maintain the purity and stability of the target protein: Ulp1 enzyme molecular weight is about 45 kDa, containing His tag (common design of commercial enzyme). Residual enzyme molecules will contaminate the final product, especially when the target protein is used for structural analysis, drug development or functional experiments, impurity enzymes may interfere with experimental results or reduce protein uniformity. For example, His tag residues affect protein surface charge characteristics, and enzyme molecules may form dimers or multimers, increasing sample complexity.

[0017] 3. Prevent interference in downstream applications: In enzyme kinetics, cell experiments or animal models, residual Ulp1 enzyme may continue to cleave SUMO-modified proteins in the experimental system, changing signal pathway activity; in addition, in vivo applications may also induce immune responses.

[0018] 4. Comply with pharmaceutical protein production specifications: In the production of recombinant protein drugs, national pharmacopoeias have strict limits on impurity residues (such as host cell protein residue levels need to be less than ppm level). Even trace amounts of protease may affect product safety and batch consistency, so it must be removed.

[0019] Commercial Ulp1 enzyme is usually designed with His tag (6x or 8x His tag) at the N-terminus. After the enzyme digestion reaction, the mixture contains: target protein (without His tag), Ulp1 enzyme (His tag), free SUMO tag (His tag). At this time, nickel column can be used for secondary purification, allowing the target protein to flow through, and Ulp1 enzyme and free SUMO tag to be adsorbed and removed, reducing the risk of enzyme residues. Therefore, how to effectively detect Ulp1 enzyme from Saccharomyces cerevisiae of prokaryotic recombinant expression has become a technical problem to be solved in the field. SUMMARY

[0020] The purpose of the present application is to overcome the shortcomings in the prior art, provide a double antibody sandwich ELISA kit that can quickly, simply and high-throughput detect Ulp-1 enzyme residues in a sample, which can effectively detect Ulp1 enzyme from Saccharomyces cerevisiae of prokaryotic recombinant expression. The present application uses the complex of Ulp1 enzyme and SUMO tag protein as an immunogen, and simultaneously immunizes mice and New Zealand rabbits to prepare Ulp1 enzyme antibodies of different species and good affinity. Then, by further screening the optimal reaction conditions, a double antibody sandwich ELISA detection kit with high specificity, strong sensitivity and stability is developed, which can more effectively solve the shortcomings of the prior art.

[0021] The technical solution of the present application to solve the above technical problems is as follows: A Ulp1 enzyme double antibody sandwich ELISA kit, comprising: 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, washing solution, blocking solution, color developing solution and termination.

[0022] Preferably, the Ulp1 enzyme double-antibody sandwich ELISA kit provided by the present application 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 (PBST containing 0.1-0.5% BSA), cleaning solution (PBST), blocking solution (PBST containing 2-5% skimmed milk powder), color developing solution and termination solution.

[0023] The HRP-labeled goat anti-rabbit antibody is commercially available, for example, the following can be selected: (model / stock number D110058-0001, specification 1ml).

[0024] To achieve the above-mentioned purposes, the method provided by the present application comprises the following steps: The Ulp1 enzyme and SUMO-tagged protein complex is prepared by mixing 1mg Ulp1 enzyme with 200mg SUMO-tagged protein, and then incubating at 2-8°C for 16h. Since the N-terminal of the SUMO-tagged protein has a 6×His tag, affinity purification can be used. 10ml Ni NTA Beads 6FF is loaded into a gravity chromatography column, and 100ml of equilibrium solution (20mM Tris-HCI, 0.5M NaCl, pH8.0) is used to balance at a flow rate of 3ml / min. After reaction, the sample is loaded into the Ni NTA Beads 6FF gravity column at a flow rate of 2ml / min. After loading is completed, 50ml of impurity washing solution (20mM Tris-HCI, 0.5M NaCl, pH8.0) is used to wash the impurities. Elution is performed using elution solution (50mM Tris-HCl, 0.5M NaCl, 250mM imidazole pH8.0) to obtain the Ulp1 enzyme and SUMO-tagged protein complex (electrophoresis identification shows that Ulp1 enzyme and SUMO-tagged protein exist simultaneously in the elution).

[0025] (1) The Ulp1 enzyme and SUMO-tagged protein complex is used to immunize mice, and then a plurality of hybridoma cells are obtained by hybridoma. The desired clone is selected by affinity screening, and the antibody sequence is obtained by sequencing technology, and the sequence number is 7H2. The sequence is synthesized by gene synthesis and recombinant technology to construct a eukaryotic expression plasmid, which is expressed in Chinese hamster ovary cells; (2) A healthy adult New Zealand white rabbit is selected, and the Ulp1 enzyme and SUMO-tagged protein complex is used for immunization. After the first immunization and multiple booster immunizations, the antibody titer in the serum is determined, and the blood is collected for purification of the polyclonal antibody; the obtained antibody has a purity of more than 90% by SDS-PAGE; (3) After obtaining the two antibodies, a double-antibody sandwich ELISA kit is prepared by the following steps: 1. Anti-Ulp1 enzyme mouse monoclonal antibody is diluted to 6ug / ml with coating solution (CBS), 100ul per well is added to the enzyme-labeled plate for coating, the coating temperature is 4℃, and the coating time is 16-20h.

[0026] 2. After being washed with 100ul-300ul of cleaning solution per well for three times, blocking solution is added for blocking at 37℃ for 1-2h, and then dried.

[0027] 3. Ulp1 enzyme is diluted to 810ug / ml standard concentration with diluent, and then diluted with diluent according to a 3-fold gradient, and the diluted Ulp1 enzyme is added to the coated enzyme-labeled plate, and then incubated and combined at 37℃ for 1-2h.

[0028] 4. After being washed with 100ul-300ul of cleaning solution per well for three times, dried.

[0029] 5. Anti-Ulp1 enzyme rabbit polyclonal antibody diluent is added, and the final concentration is 6ug / ml, and then incubated and combined at 37℃ for 1h.

[0030] 6. After being washed with 100ul-300ul of cleaning solution per well for three times, dried.

[0031] 7. HRP-labeled goat anti-rabbit antibody diluent is added, and the dilution ratio is 1:5000, and then incubated and combined at 37℃ for 1-2h.

[0032] 8. After being washed with 100ul-300ul of cleaning solution per well for three times, dried.

[0033] 9. Color developing solution is added, and then developed at 25℃ in the dark for 5min.

[0034] 10. Stop solution is added for termination, and then read at OD450nm.

[0035] 11. According to the reading of the enzyme-labeled instrument, the standard curve is obtained by fitting the logarithmic value corresponding to the Ulp1 enzyme standard concentration.

[0036] Since Ulp1 enzyme specifically recognizes the tertiary structure of SUMO label for enzyme cutting, and a complex structure is formed after enzyme cutting, the application adopts Ulp1 enzyme and SUMO label complex protein as antigen for immunization, so as to ensure the structure of Ulp1 enzyme during immunization, and the obtained antibody has better specificity, which provides favorable conditions for the development of the kit.

[0037] Preferably, the anti-Ulp1 enzyme mouse monoclonal antibody coated enzyme-labeled plate, anti-Ulp1 enzyme rabbit polyclonal antibody, and HRP-labeled goat anti-rabbit antibody are applied in the detection of prokaryotic recombinant expressed Ulp1 enzyme from Saccharomyces cerevisiae.

[0038] The beneficial effects of the present application are: (1) Ulp1 enzyme and SUMO tag complex protein as immunogen, ensure that in mice and rabbits can produce specific antibodies, ensure the preparation of genetically engineered mouse monoclonal antibody, and rabbit serum purification of rabbit polyclonal antibody, which has specific recognition ability to Ulp1 enzyme, and lays a good foundation for subsequent development of high specificity, sensitivity and double antibody sandwich ELISA kit.

[0039] (2) The antibody obtained by recombinant expression or immunization is subjected to antigen-specific affinity purification, so as to ensure the specificity of the antibody and is not affected by host impurity proteins or other immunoglobulins.

[0040] (3) The kit prepared by the present application has the advantages of high sensitivity (the minimum concentration of the sample can be detected is 0.02 ng / ml), wide detection range, good repeatability, high specificity (low blank background of ELISA), good stability, etc., which can reduce the detection cost and make up for the defects of Ulp1 enzyme in the production process of drug proteins. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is the SDS-PAGE electrophoresis diagram of antigen affinity purification of rabbit polyclonal antibody provided in one specific embodiment of the present application; Figure 2 is the Ulp1 enzyme standard curve provided in one specific embodiment of the present application. DETAILED DESCRIPTION

[0042] The present application is further described below in combination with examples, but is not limited thereto.

[0043] Example 1: Preparation of mouse monoclonal antibody In this experiment, BALB / c mice immunization-hybridoma technology route was adopted, and it took about 5 months to complete the preparation of monoclonal antibody. After antibody sequencing, recombinant expression was realized. The key stages include: antigen emulsification, animal immunization, cell fusion, hybridoma screening, antibody sequencing and recombinant expression verification.

[0044] I. Mouse immunization stage Day 1-7 Antigen preparation: the recombinant protein antigen (150 μg) is mixed with the same volume of complete Freund's adjuvant, and emulsified by 20 times of push injection through a three-way valve under ice bath condition to form a water-in-oil structure. Emulsion verification: 5 μl of emulsion is dropped into water, and the complete emulsion droplet maintains for 30 seconds without diffusion.

[0045] Day 8 First immunization: intraperitoneal injection of 50 μl of emulsified antigen (containing 50 μg of protein), and observation of allergic reaction after injection.

[0046] Day 22 & Day 36 Boosting immunization: change to incomplete Freund's adjuvant, dose reduced to 30 μg / time, injection site rotated to back subcutaneously.

[0047] Day 50 Titer detection: orbital blood collection to separate serum, indirect ELISA detection of titer ≥ 1:6400 to enter the booster immunization.

[0048] Day 53 Booster immunization: intravenous injection of 100 μg of antigen solution without adjuvant.

[0049] II. Hybridoma preparation stage Day 56-58 Cell fusion: the mouse was executed by breaking the neck, and the spleen was aseptically prepared to prepare a single cell suspension (5 x 10 7 cells) mixed with SP2 / 0 myeloma cells at a ratio of 5:1, 50% PEG-1500 promoted fusion for 90 seconds, HAT medium screening, 37°C / 5% CO2 incubator culture Day 59-87 Hybridoma screening: on the 7th day of placing in a 37°C / 5% CO2 incubator, ELISA primary screening was performed on positive wells (OD450nm>1.0). On the 14th day, limited dilution method was used for cloning (average 0.8 cells / well). After three subcloning, a stable secretion cell strain was obtained.

[0050] III. Antibody sequencing stage Day 88-101 Gene cloning: TRIzol method was used to extract hybridoma RNA, and cDNA was obtained by reverse transcription; IgG specific primers were designed to amplify the light and heavy chain variable regions; after TA cloning, Sanger sequencing was performed, and sequence information was analyzed by software.

[0051] IV. Recombinant expression stage Day 102-122 Vector construction: the light and heavy chain genes were cloned into pcDNA3.4 vectors respectively; 293F cells were transfected (electroporation method), and the supernatant was collected 72 hours after transfection.

[0052] Day 123-129 Purification and identification: rProtein A column was used for purification, SDS-PAGE was used to verify the purity >95%, and indirect ELISA was used to verify the binding activity.

[0053] Example 2: Preparation of rabbit polyclonal antibody Ulp1 enzyme and SUMO tag complex protein as immunogen, using the method of subcutaneous injection of multiple points in the back of New Zealand white rabbits, the first immunization 400ug, 14 days interval booster immunization, booster immunization 200ug. The first immunization using equal volume of Freund's complete adjuvant and antigen, when the fourth immunization complete a week, the ear vein blood serum antibody titer, meet the immune serum titer requirements (using ELISA to detect the activity of antibody in serum, serum 1:64000 dilution, OD450 absorption value reached more than 0.5), ear artery blood, animal euthanasia, centrifugal separation of serum, and through the antigen specific purification, obtain anti-Ulp1 enzyme rabbit polyclonal antibody. Electrophoresis chart see Figure 1 Example 3: the establishment and optimization of double antibody sandwich ELISA method 1, the basic steps of test method 1) coated enzyme labeled plate: the anti-Ulp1 enzyme mouse monoclonal antibody was diluted to 6ug / ml with coating solution (CBS), 100ul per well was added to the 96 well enzyme labeled plate for coating, 4℃ coating 16h, then 300ul PBST per well was used to clean three times, 1-2min once, the water in the enzyme labeled plate was dried on the water absorption paper; 2) blocking enzyme labeled plate: 100ul blocking solution (blocking solution was 2-5% skim milk powder added in PBST, PBST composition: NaCl 137 mM, KCl 2.7 mM, Na2HPO410 mM, KH2PO41.8 mM, Tween-20 0.1% (w / v),) per well was used to block 37℃ for 1h, after the incubation was completed, the blocking solution was shaken off, then 300ul PBST per well was used to clean three times, 1-2min once, the water in the enzyme labeled plate was dried on the water absorption paper; 3) add Ulp1 enzyme standard curve: the Ulp1 enzyme solution was diluted to 810ug / ml standard concentration with diluent, then multiple gradients were diluted according to 3 times gradient ratio, the diluted Ulp1 enzyme of multiple gradients was added to the coated enzyme labeled plate, 37℃ incubation combined 1h, after the incubation was completed, the blocking solution was shaken off, then 300ul PBST per well was used to clean three times, 1-2min once, the water in the enzyme labeled plate was dried on the water absorption paper; 4) add detection antibody: the anti-Ulp1 enzyme rabbit polyclonal antibody was diluted with diluent, 100ul diluted antibody was added per well to ensure the final concentration of 6ug / ml, 37℃ incubation combined 1h, after the incubation was completed, the blocking solution was shaken off, then 300ul PBST per well was used to clean three times, 1-2min once, the water in the enzyme labeled plate was dried on the water absorption paper; 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, incubate at 37°C for 1-2 hours, remove the blocking solution after incubation, and then wash three times with 300 μL PBST per well for 1-2 minutes each time. Pat the microplate dry on absorbent paper. 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; 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; 8) Reading: Read and record the values ​​using a microplate reader at OD 450nm wavelength. Calculation and analysis results are shown below. Figure 2 .

[0054] 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.

[0055] Table 3 Selection of Plate Temperature and Time Blocking temperature and time P / N value N value 4℃ 16h 20.662 0.0668 37℃ 1h 14.152 0.0954 37℃ 2h 16.264 0.0750 37℃ 3h 12.275 0.0973 .

[0056] 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.

[0057] Table 4 Selection of Antibody Concentration for Plate Packaging Blocking antibody concentration ng / well P / N value N value 150 9.904 0.1130 300 8.517 0.1469 600 10.047 0.1362 .

[0058] 3) Screening of sealing fluid types Screening of 2.5% (W / V) skimmed milk powder, 5% (W / V) skimmed milk powder, 7.5% (W / V) skimmed 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 are aqueous solution), fetal bovine serum, etc. at 37°C for 1h, considering the N value, and selecting the condition with larger P / N value as the best blocking condition. The results are shown in Table 5, and the blocking solution is selected as 2.5% (W / V) skimmed milk powder.

[0059] Table 5 Screening of blocking liquid types Blocking solution P / N value N value 2.5% (W / V) skimmed milk powder 12.510 0.1163 5% (W / V) skimmed milk powder 12.755 0.1055 7.5% (W / V) skimmed milk powder 11.517 0.1209 2% (W / V) gelatin 6.225 0.1405 5% (W / V) gelatin 5.740 0.1412 2% (W / V) BSA 6.075 0.1447 5% (W / V) BSA 7.488 0.1177 7.5% (W / V) BSA 6.166 0.1360 2% (W / V) NH4Cl 7.765 0.1292 5% (W / V) NH4Cl 7.235 0.1198 Fetal bovine serum 6.142 0.1300 .

[0060] 4) Selection of blocking time Selecting the use of 5% (W / V) skimmed milk powder, blocking at 37°C for 1h, 1.5h and 2h, considering the N value, and selecting the condition with larger P / N value as the best blocking time. The results are shown in Table 6, and the blocking time is selected as 1h considering the saving of detection time.

[0061] Table 6 Screening of blocking time Time h P / N value N value 1 10.846 0.1177 2 10.385 0.1170 3 10.846 0.1199 .

[0062] 5) Selection of detection antibody concentration Selecting the use concentration of detection antibody anti-Ulp1 enzyme rabbit polyclonal antibody as 1.5ug / ml, 3.0ug / ml, 6.0ug / ml, Incubating at 37°C for 1h, considering the N value, and selecting the condition with largest P / N value as the best detection antibody use concentration. The results are shown in Table 7, which proves that the best concentration of detection antibody is 6.0 ug / ml.

[0063] Table 7 Screening of detection antibody concentration Antibody concentration ug / ml P / N value N value 1.5 10.371 0.1490 3.0 11.749 0.1304 6.0 12.177 0.1461 .

[0064] 6) Selection of enzyme-labeled antibody dilution ratio Selecting the dilution ratio of enzyme-labeled antibody as 1:5000, 1:10000, 1:20000, incubating at 37°C for 1h, considering the N value, and selecting the condition with largest P / N value as the best enzyme-labeled antibody dilution ratio. The results are shown in Table 8, which proves that the dilution ratio is 1:5000.

[0065] Table 8 Screening of enzyme-labeled antibody concentration .

[0066] 7) Selection of color development time Using TMB color developing solution, color development was performed for 5 min, 10 min, 15 min and 20 min at 25°C, respectively, and the condition with smaller N value and larger P / N value was selected as the optimal color development time. The results are shown in Table 9. According to the results, the color development time was selected as 5 min.

[0067] Table 9 Screening of color development time

[0068] Through condition optimization, the optimal preparation and use conditions of the kit were obtained as follows: the optimal time and temperature for plate coating were 16 h, the plate coating concentration was 600 ng / well at 4°C, the blocking solution was 2.5% (W / V) skimmed milk powder, and the blocking time was selected as 1 h. The optimal concentration of detection antibody was 6.0 ug / ml. The dilution ratio of enzyme-labeled antibody was 1:5000, and the color development time was 5 min. The above conditions can play the advantages of the kit.

[0069] 3. Experimental verification 1) Sensitivity verification Ulp1 enzyme was diluted by three gradients 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 sandwich ELISA method of the application was used to verify the sensitivity of the kit (the plate coating time and temperature were 16 h at 4°C, the plate coating concentration was 600 ng / well, the blocking solution was 2.5% (W / V) skimmed milk powder, the blocking time was 1 h, the concentration of detection antibody was 6.0 ug / ml, the dilution ratio of enzyme-labeled antibody was 1:5000, and the color development time was 5 min). The detection data are shown in Table 10. When the concentration was 0.02 ng / ml, the P / N value reached more than 2.1, and therefore the sensitivity of the kit was 0.02 ng / ml.

[0070] Table 10 Sensitivity verification

[0071] 2) Intra-batch stability verification The Ulp1 enzyme standard was diluted into multiple gradients, and each concentration was detected in triplicate to verify the stability of the same batch. The detection results were used to calculate the coefficient of variation of the intra-batch experiment. The formula for calculating the coefficient of variation is: CV (%) = SD / Mean x 100%. Wherein: SD is the standard deviation; Mean is the average value; the intra-batch repeatability verification results are shown in Table 11, and the intra-batch difference is less than 10%.

[0072] Table 11 Intra-batch stability verification Ulp1 enzyme concentration ng / ml Mean SD CV % 13.72 2.044 0.065 3.18% 4.57 1.617 0.055 3.41% 1.52 0.849 0.069 8.07% 0.51 0.562 0.044 7.76% 0.17 0.455 0.030 6.61% 0.06 0.477 0.013 2.73% 0.02 0.243 0.015 6.16% 3) Inter-batch stability verification The Ulp1 enzyme standard was diluted into multiple gradients, and each concentration was detected in 3 repeats, to verify the stability between different batches. The detection results were used to calculate the coefficient of variation of the inter-batch experiment. The formula for calculating the coefficient of variation is: CV (%) = SD / Mean x 100%. Wherein: SD is the standard deviation; Mean is the average value; the inter-batch repeatability verification results are shown in Table 12, and the inter-batch difference is less than 10%.

[0073] The intra-batch difference and the inter-batch difference are both less than 10%, indicating that the method has good repeatability.

[0074] Table 12 Inter-batch stability verification Ulp1 enzyme concentration ng / ml Mean SD CV % 13.72 2.070 0.022 1.07% 4.57 1.697 0.112 6.62% 1.52 0.911 0.052 5.68% 0.51 0.568 0.045 7.93% 0.17 0.464 0.043 9.17% 0.06 0.459 0.042 9.12% 0.02 0.254 0.015 5.99% The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the inventive concept, several modifications and improvements can be made, which are all within the protection scope of the present application.

Claims

1. A Ulp1 enzyme double antibody sandwich ELISA kit, characterized by, It comprises: Anti-Ulp1 enzyme mouse monoclonal antibody coated ELISA plate, anti-Ulp1 enzyme rabbit polyclonal antibody, HRP labeled goat anti-rabbit antibody, diluent, washing solution, blocking solution, color developing solution and termination solution.

2. The kit of claim 1, wherein The diluent is PBST containing 0.1-0.5% BSA.

3. The kit of claim 1, wherein The washing solution is PBST, the blocking solution is PBST containing 2-5% skimmed milk powder; the color developing solution is TMB color developing solution; and the termination solution is 0.5M-0.6M phosphoric acid termination solution.

4. The kit of claim 1, wherein The anti-Ulp1 enzyme mouse monoclonal antibody and the anti-Ulp1 enzyme rabbit polyclonal antibody are prepared by immunizing the complex protein of Ulp1 enzyme and SUMO tag protein, and the sequence number of the anti-Ulp1 enzyme mouse monoclonal antibody is 7H2; the mass ratio of the SUMO tag protein to the Ulp1 enzyme is 200:

1.

5. The kit according to any one of claims 1-4 is used for detecting prokaryotic recombinant expressed Ulp1 enzyme from Saccharomyces cerevisiae.

6. The anti-Ulp1 enzyme mouse monoclonal antibody, the anti-Ulp1 enzyme rabbit polyclonal antibody and the HRP labeled goat anti-rabbit antibody are used for detecting prokaryotic recombinant expressed Ulp1 enzyme from Saccharomyces cerevisiae.

7. Use of the kit according to claim 5 for the detection of prokaryotically recombinantly expressed Ulp1 enzymes of Saccharomyces cerevisiae origin, characterized in that, It comprises the following steps: (1) After the anti-Ulp1 enzyme mouse monoclonal antibody is diluted to 6-8ug / ml with the coating solution, 100-150ul per well is added to the ELISA plate for coating, the coating temperature is 4-37℃, and the coating time is 16-20h; (2) After each well is washed with 100ul-300ul of washing solution for three times, the blocking solution is added for blocking at 37℃ for 1-2h, and then it is patted dry; (3) The Ulp1 enzyme is diluted to 800-810ug / ml standard concentration with the diluent, and then it is further diluted with the diluent according to 3 times gradient, the diluted Ulp1 enzyme is added to the coated ELISA plate, and then it is incubated and combined at 37℃ for 1-2h; (4) After each well is washed with 100ul-300ul of washing solution for three times, it is patted dry; (5) The anti-Ulp1 enzyme rabbit polyclonal antibody diluent is added, and the final concentration is 1.5-6ug / ml, and then it is incubated and combined at 37℃ for 1h; (6) After each well is washed with 100ul-300ul of washing solution for three times, it is patted dry; (7) The HRP labeled goat anti-rabbit antibody diluent is added, and then it is incubated and combined at 37℃ for 1-2h; (8) After each well is washed with 100ul-300ul of washing solution for three times, it is patted dry; (9) The color developing solution is added, and then it is colored at 25℃ in the dark; (10) The termination solution is added for termination, and then it is read at OD450nm; (11) According to the reading of the enzyme label instrument, the standard curve is obtained by fitting the logarithmic value corresponding to the Ulp1 enzyme standard concentration.

8. Use according to claim 7, characterized in that, The blocking solution is selected from one or more of 5%(W / V) skimmed milk powder, 5%(W / V) skimmed milk powder, 7.5%(W / V) skimmed 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. Use according to claim 7, characterized in that, The coating antibody concentration is 600ng / well.

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

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