Method for quantitatively detecting PreScision protease content by using double-antibody sandwich

Abstract

The invention discloses a method for quantitatively detecting the content of PreScision protease by using a double-antibody sandwich. The method comprises the following steps: S1, coating an elisa plate with a capture antibody; s2, adding a sample to be detected into the coated elisa plate; s3, incubating by taking the HRP binding antibody as an enzyme-labeled antibody, developing by using a TMB developing solution, and detecting an OD value by using a microplate reader with the wavelength of 450 nm; and S4, drawing a standard curve by using the standard substance, and calculating the PreScision protease content in the detected sample according to the standard curve. According to the present invention, the antibody-antigen-antibody sandwich structure is formed through the double antibody sandwich, the SM17 is adopted as the capture antibody for coating, the SM10 is adopted as the detection antibody, the PreScision protease content is quantitatively detected through the obtained standard curve, and the sensitivity, the precision and the accuracy of the detection method provided by the present invention are maintained at the high level.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically relates to a method for quantitatively detecting the content of PreScission protease by using a double-antibody sandwich method. Background Art

[0002] Prescision Protease is a fusion protein composed of the 3C protease of human rhinovirus type 14 and GST, which is designed specifically for the excision of the glutathione S-transferase (GST) tag. It can specifically separate the GST tag of the fusion protein with the enzyme substrate recognition polypeptide sequence expressed by vectors such as the pGEX-6P series. The PreScission protease itself is a fusion protein of GST and human rhinovirus 3C protease. The GST tag at the N-terminus is beneficial for the purification of the enzyme on the one hand, and can also achieve the immobilization of the enzyme and the on-column enzymatic cleavage of the protein with the GST tag on the other hand. The C-terminus is the 3C protease, which can specifically recognize the corresponding amino acid sequence and exert enzymatic cleavage activity. The specifically recognized amino acid sequence is Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro, and the cleavage site is between Gln and Gly. After cleavage, there is a residual sequence of 5 amino acids "Gly-Pro-Leu-Gly-Ser (GPLGS)" at the N-terminus of the target protein.

[0003] At present, the antigen components of many genetic engineering vaccines are GST-tagged fusion proteins, and PreScission protease is required to cleave and remove the GST-tagged protein during the purification process. Therefore, it is necessary to detect the residual amount of PreScission protease in the target protein after removing GST. However, there are few methods for detecting the content of PreScission protease in the prior art. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for quantitatively detecting the content of PreScission protease by using a double-antibody sandwich method, so as to solve the problem of the lack of methods for detecting the content of PreScission protease in the prior art.

[0005] To achieve the above purpose, the present invention provides the following technical solutions: A method for quantitatively detecting the content of PreScission protease by using a double-antibody sandwich method provided by the present invention includes the following steps: S1. Coat an enzyme-labeled plate with a capture antibody; S2. Add the sample to be detected to the coated enzyme-labeled plate; S3. Incubate the HRP-conjugated detection antibody as an enzyme-labeled antibody, develop the color using the TMB chromogenic solution, and detect the OD value with an ELISA reader at a wavelength of 450 nm; S4. Use the standard product to draw a standard curve, and calculate the content of PreScission protease in the sample to be measured according to the standard curve.

[0006] In one embodiment of the present invention, the capture antibody is SM17, the SM17 includes a heavy chain and a light chain, the amino acid sequence of the heavy chain of SM17 is as shown in SEQ ID NO: 2, QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMDWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAFLQINNLKNEDTATYFCARGGSWGQGTLVTVSA, the amino acid sequence of the light chain of SM17 is as shown in SEQ ID NO: 3, DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLDSGNQMNYLTWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNDHSYPLTFGAGTKLDLK, the heavy chain and the light chain respectively contain three CDRs and four FRs. Specifically, heavy chain CDR1 is NFGMD, heavy chain CDR2 is WINTYTGEPTYADDFKG, heavy chain CDR3 is GGS, heavy chain FR1 is QIQLVQSGPELKKPGETVKISCKASGYTFT, heavy chain FR2 is WVKQAPGKGLKWMG, heavy chain FR3 is RFAFSLETSASTAFLQINNLKNEDTATYFCAR, heavy chain FR4 is WGQGTLVTVSA; light chain CDR1 is KSSQSLLDSGNQMNYLT, light chain CDR2 is GASTRES, light chain CDR3 is QNDHSYPLT, light chain FR1 is DIVMTQSPSSLSVSAGEKVTMSC, light chain FR2 is WYQQKPGQPPKLLIY, light chain FR3 is GVPDRFTGSGSGTDFTLTISSVQAEDLAIYYC, light chain FR4 is FGAGTKLDLK.

[0007] In one embodiment of the present invention, the detection antibody in S3 is SM10, and the SM10 includes a heavy chain and a light chain. The amino acid sequence of the heavy chain of SM10 is as shown in SEQ ID NO: 4, EVKLVESGGGLAQPGGSLRLSCATSGFTFTDYHMSWFRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCASLYYDYDYYFEDWGQGTTLTVSS. The amino acid sequence of the light chain of SM10 is as shown in SEQ ID NO: 5, DIVMSQSPSSLAVSPGEKVTMSCKSSQSLLNSSTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK. The heavy chain and the light chain each contain three CDRs and four FRs. Specifically, heavy chain CDR1 is DYHMS, heavy chain CDR2 is FIRNKANGYTTEYSASVKG, heavy chain CDR3 is LYYDYDYYFED, heavy chain FR1 is EVKLVESGGGLAQPGGSLRLSCATSGFTFT, heavy chain FR2 is WFRQPPGKALEWLG, heavy chain FR3 is RFTISRDNSQSILYLQMNTLRAEDSATYYCAS, heavy chain FR4 is WGQGTTLTVSS; light chain CDR1 is KSSQSLLNSSTRKNYLA, light chain CDR2 is WASTRES, light chain CDR3 is KQSYNLWT, light chain FR1 is DIVMSQSPSSLAVSPGEKVTMSC, light chain FR2 is WYQQKPGQSPKLLIY, light chain FR3 is GVPDRFTGSGSGTDFTLTINSVQAEDLAVYYC, light chain FR4 is FGGGTKLEIK.

[0008] In one embodiment of the present invention, the determination of the capture antibody and the detection antibody further includes the following steps: A1. Inoculate, culture, collect and purify the engineering bacteria (pGEX-PP / BL21) of PreScission protease to obtain tag-free PreScission protease; A2. Preparation and screening of mouse monoclonal antibodies against tag-free PreScission protease; A3. Preparation of mouse monoclonal antibodies to obtain 13 purified antibodies that can bind to tag-free PreScission protease; A4. Through the ELISA evaluation method for antibody pairing screening, the capture antibody and the detection antibody are determined.

[0009] In one embodiment of the present invention, after coating in S1, it is refrigerated overnight, blocked with 2% BSA, and incubated at room temperature for 1 h after sealing.

[0010] In one embodiment of the present invention, before adding the sample to be measured in S2, the plate needs to be washed twice with the washing solution, and after adding the sample to be measured, it reacts at room temperature for 2 h.

[0011] In one embodiment of the present invention, the washing solution is PBST: 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, 0.05% v / v Tween-20.

[0012] In one embodiment of the present invention, before adding the HRP-conjugated detection antibody in S3, the plate needs to be washed three times with the washing solution. After adding the HRP-conjugated detection antibody, it is mixed evenly, incubated at room temperature for 1 h, washed three times with the washing solution, and after adding the chromogenic solution A and B mixed evenly at 1:1, it is incubated at room temperature in the dark for 20 min, and then the termination solution is added to terminate.

[0013] In one embodiment of the present invention, the chromogenic solution A is 13.6 g of sodium acetate, 1.6 g of citric acid, 0.3 mL of 30% hydrogen peroxide, and distilled water is added to 500 mL; the chromogenic solution B is 0.2 g of disodium ethylenediaminetetraacetate, 0.95 g of citric acid, 50 mL of glycerol, 0.15 g of TMB (tetramethylbenzidine) dissolved in 3 mL of DMSO, and distilled water is added to 500 mL. The termination solution is 2 M sulfuric acid.

[0014] In one embodiment of the present invention, the standard curve in S4 is obtained by performing four-parameter Logistic curve fitting with the concentrations of each point of the standard product as the abscissa and the signal values corresponding to the standard products of each concentration as the ordinate.

[0015] In one embodiment of the present invention, the average absorbance value of each concentration gradient minus the average value of the blank control is the signal value of this concentration.

[0016] Based on the above technical solutions, the embodiments of the present invention can at least produce the following technical effects: In order to quantitatively detect the residual amount of PreScission protease by double antibody sandwich and exclude the interference of GST-tagged protein, PreScission protease without tag was prepared. Using PreScission protease without GST tag as an immunogen, monoclonal antibodies against multiple active epitopes of tagless PreScission protease were obtained after immunization, screening, and identification. The obtained monoclonal antibodies can be used for quantitative detection of PreScission protease content by double antibody sandwich enzyme-linked immunosorbent assay.

[0017] The present invention forms a "antibody-antigen-antibody" sandwich structure through a double antibody sandwich. SM17 is coated as a capture antibody, and SM10 is used as a detection antibody. Through the obtained standard curve, PreScission protease is quantitatively detected. The detection method provided by the present invention maintains high levels of sensitivity, precision, and accuracy. Brief Description of the Drawings

[0018] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on the structures shown in these drawings.

[0019] Figure 1 It is the electrophoresis diagram of the purity detection of tagless PreScission protease in Example 1 of the present invention; Figure 2 It is the size exclusion high performance liquid chromatography diagram of Example 3 of the present invention; Figure 3 It is the standard curve of Example 5 of the present invention. Detailed Embodiments

[0020] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0021] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0022] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0023] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0024] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0025] The present invention uses PreScission protease without GST tag as an immunogen to immunize mice. Through cell fusion and screening, multiple strains of mouse monoclonal antibodies that can specifically recognize PreScission protease are successfully obtained. High-affinity mouse monoclonal antibodies are screened out, ascites is prepared, and purified by Protein A, and then HRP-labeled respectively.

[0026] After paired experiments and condition optimization, the paired antibody combination (coating antibody SM17, detection antibody SM10) of the detection method is finally determined, and the corresponding detection conditions and standard curve are established. The detection sensitivity of this method is 15.14 pg / mL, and the linear detection range is 78.125 - 5000 pg / mL, fully meeting the requirements of quantitative detection.

[0027] The main reagents and instruments used in the present invention are as follows: Reagents Part Number Manufacturer Freund's complete adjuvant F5881 Sigma Freund's incomplete adjuvant 263910 BD Horseradish peroxidase-conjugated goat anti-mouse IgG Fc GGFC-90P ICL 1640 Basic Medium 31800-105 GIBCO Fetal bovine serum SA201.02 CELLMAX HAT H0262-10VL Sigma HT H0137-10VL Sigma Horseradish peroxidase (HRP)-labeled rabbit anti-mouse IgG secondary antibody 50323-T16-H Sino Biological Horseradish peroxidase (HRP)-labeled rabbit anti-mouse Fc secondary antibody 10690-T00-H Sino Biological Fetal bovine serum SA201.02(20211110) CELLMAX MHYBII medium / Sino Biological Rabbit Anti-Mouse IgG F(ab')2 / HRP 315-035-047 Jackson Protein standards (14.4-116kDa) 26610 Thermo Protein Standards (20-200kDa) 26614 Thermo Instrument Name Specifications Manufacturer Clean bench DL-CJ-2NDI Beijing Donglian Har Instrument Manufacturing Co., Ltd. Injection emulsifier Dual channel CNC Lange Automation Technology Co., Ltd. ELISA reader Multiskan Fc Thermo Inverted microscope TS-100 Nikon Carbon dioxide incubator 3111 Thermo Peristaltic Pumps BT100-2J Lange Constant Flow Pump Co., Ltd. Micro-volume spectrophotometer NANoDROP8000 Thermo High-efficiency high-speed refrigerated centrifuge Avantij-26XP Beckman Nucleic acid protein detector HD-21-1 Shanghai Qingpu Huxi Instrument Factory Macromolecular Interaction Analyzer Biacore T200 Cytiva Electrophoresis Apparatus EPS600 Tianneng Decolorization shaker TS-8S Chilin Bell Micro high speed refrigerated centrifuge Sorvall LEGEND MICRO17R Thermo

[0028] Example 1 Expression and Purification of Tagless PreScission Protease Inoculate the engineering bacteria (pGEX-PP / BL21) of PreScission protease into LB medium containing ampicillin and culture overnight (37 °C, 100 rpm). The next day, expand the culture to OD at a ratio of 1:100 (V / V) 600Approximately 0.8, add IPTG to induce expression overnight (4°C, 200 rpm), centrifuge to collect bacteria and lyse the bacteria by sonication. Centrifuge to collect bacteria and use a homogenizer to break the bacteria (700 - 800 bar, 4°C, 3 times), centrifuge the homogenate to take the supernatant (11600 g, 4°C, 30 min), and filter using a 0.8 μm filter membrane. Capture the PreScission protease-GST fusion protein in the filtered supernatant through a GST-4FF affinity chromatography column, elute with 20 mM glutathione to obtain the PreScission protease-GST fusion protein, add Thrombin at a ratio of 6 U / mg and digest at 4°C overnight. Replace the buffer of the digested sample with 10 mM PB (pH 8.5, 3.01 g / L Na2HPO4, 0.25 g / L NaH2PO4, 0.215 g / L NaH2PO4.H20) through G25 chromatography, and purify the G25 chromatography sample using a Q HP chromatography column. Collect the Q HP flow-through sample, which is the tag-free PreScission protease. Detect the purity of the tag-free PreScission protease by SDS-PAGE gel electrophoresis.

[0029] The PreScission protease expression plasmid pGEX-PP was obtained as a gift from the Microbiology Teaching and Research Section of the Chinese Academy of Sciences. The host bacterial strain is E.coli / BL21(DE3), purchased from Novagen. The amino acid sequence of the tag-free PreScission protease is as shown in SEQ ID NO: 1, GPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEKQ. The purity of the tag-free PreScission protease is as Figure 1 shown, and the result shows that the purity is 97.1%

[0030] Example 2 Preparation and Screening of Mouse Monoclonal Antibodies Against Tag-Free PreScission Protease (1) Animal Immunization Five 6-8-week-old female Balb / c mice (purchased from the National Institutes for Food and Drug Control, China) were immunized with tagless PreScission protease. The single immunization dose for each mouse was 50 μg. For the first immunization, the immunogen was made into an emulsion with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points on the abdomen. After a 2-week interval, the same dose of immunogen was made into an emulsion with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points on the abdomen. Blood was collected one week after immunization, and the serum titer was measured using indirect ELISA. When the mouse serum was diluted 1:8000 times, the OD 450 -Blank (the value obtained by subtracting the absorbance of the blank control from the absorbance at 450 nm) greater than 1.0 was used as the qualified standard for the titer.

[0031] (2) Serum titer detection The tagless PreScission protease was diluted to 5 μg / mL with coating buffer (PBS: 136.9 mM NaCl, 10.1 mM Na2HPO 4， 2.7 mM KCl, 1.8 mM KH2PO 4， pH 7.4), and 100 μL per well was added to the enzyme-linked immunosorbent assay (ELISA) plate and coated overnight at 4 °C. The plate was washed once with washing buffer (PBST) at 300 μL / well, patted dry, and then blocked with blocking buffer (PBST solution containing 2% BSA) at 300 μL / well for 1 h at room temperature. The plate was washed twice with washing buffer at 300 μL / well and patted dry. The serially diluted serum samples and sample diluent (PBST solution containing 0.1% BSA) were added at 100 μL / well, and then the detection antibody (horseradish peroxidase-labeled goat anti-mouse IgG Fc) was added at 100 μL / well to the 96-well plate and incubated together for 2 h at room temperature. The plate was washed five times with washing buffer at 300 μL / well and patted dry. The chromogenic solution was added at 200 μL / well and left at room temperature for 12 min. The reaction was terminated by adding the stop solution at 50 μL / well, and then detected using an ELISA reader at a wavelength of 450 nm. When the mouse serum undiluted was diluted 1:8000 times, the OD 450 -Blank greater than 1.0 was used as the qualified standard for the titer. Mice with higher titers were preferably selected for hybridoma fusion.

[0032] Table 1 Determination of serum antibody titers of mice after immunization Mouse number Dilution <![CDATA[OD 450 > <![CDATA[OD 450 -Blank <!-- 5 -->]]> blank / 0.04 / <![CDATA[1 # > 8000 2.27 2.23 <![CDATA[1 # > 16000 1.15 1.11 <![CDATA[2 # > 8000 2.18 2.14 <![CDATA[2 # > 16000 1.95 1.91 <![CDATA[3 # > 8000 2.04 2.00 <![CDATA[3 # > 16000 1.66 1.62 <![CDATA[4 # > 8000 1.34 1.30 <![CDATA[4 # > 16000 0.99 0.95 <![CDATA[5 # > 8000 2.53 2.49 <![CDATA[5 # > 16000 2.12 2.08 Pre-immune serum 8000 0.04 0.00 Pre-immune serum 16000 0.04 0.00

[0033] As can be seen from the results in Table 1 above, the serum titers of all five mice reached the qualified standard, and among them, the serum titer of mouse No. 5 was the highest. # The serum titer of mouse No. 5 was the highest.

[0034] (3) Mouse monoclonal antibody fusion and screening Myeloma cells (SP2 / 0): Purchased from the China Center for Type Culture Collection (CCTCC).

[0035] Hybridoma cell screening: Select 5 # mouse spleen cells and mix them with 5 # mouse myeloma cells (SP2 / 0) in a 1:1 ratio, and use electrofusion to perform fusion to obtain hybridoma cells. Approximately on the 6th and 8th days after fusion, change the cell culture medium with HAT selection medium once each; approximately on the 10th day after cell fusion, use the indirect ELISA method to screen and detect the cell supernatant in the main clone stage. A total of 76 positive clones that bind to the antigen protein were obtained in the main clone stage of fusion. Select 15 of the main clone cells for the next screening. The ELISA detection results of the 15 main clone supernatants are shown in Table 2.

[0036] Table 2 ELISA detection results of main clone supernatants name <![CDATA[OD 450 -Blank]]> 1-H8 0.82 5-C5 0.55 6-C4 4.06 6-A1 3.47 14-C10 0.86 14-H3 3.88 15-C8 0.71 15-C7 3.95 16-H4 3.77 16-H1 3.32 17-D3 2.39 18-D1 3.68 18-G4 2.57 18-D2 0.98 18-B1 3.45 Subclone screening: Use HT selection medium to inoculate the 15 positive hybridoma cells obtained by screening at an inoculation density of 0.75 cells per well on half of a 96-well cell culture plate, and statically culture them under the conditions of 5% CO2 and 37°C. Approximately on the 7th day after culture, take the supernatant for indirect ELISA detection. After 3 consecutive rounds of limited dilution and corresponding screening, 13 positive monoclonal cells that bind to the tag-free PreScission protease were obtained. The detection results are shown in Table 3.

[0037] Table 3 ELISA detection results of subclone supernatants serial number <![CDATA[OD 450 -Blank]]> SM01 3.18 SM02 0.75 SM03 3.14 SM04 2.79 SM05 3.26 SM06 3.49 SM07 3.94 SM08 3.71 SM09 3.71 SM10 3.83 SM11 3.21 SM12 3.81 SM17 2.91 In summary, a total of 13 hybridoma cells that bind to the tag-free PreScission protease were obtained through 1 round of fusion screening.

[0038] Example 3 Preparation of murine monoclonal antibody The present invention discloses the sequences of the antibodies produced by the two hybridoma cells used. If those skilled in the art need to prepare the antibody, they can directly recombine the antibody sequence onto an expression plasmid through genetic engineering techniques, and then transfer the recombinant plasmid into the corresponding host cell for culture and expression. The host cells are mainly mammalian cells, including: Sp2 / 0 myeloma cells, NS0 mouse myeloma cells, HEK293 human embryonic kidney cells, and Chinese hamster ovary cells CHO, etc.

[0039] (1) Hybridoma cell culture Transfer 1 mL of hybridoma cells into a 100 mL culture flask. When the cells multiply to a certain density, discard the supernatant and replace it with MHYBII medium to continue culturing for 7 - 9 days and then harvest the cells.

[0040] (2)Purification of monoclonal antibody Centrifuge the supernatant of hybridoma cells collected using a micro high-speed refrigerated centrifuge under the centrifugation conditions of 4000g for 30 min, and collect the cell supernatant after centrifugation; filter the cell supernatant after centrifugation using a 0.45 μM filter membrane. Select a protein A column of appropriate specification according to the expression level, wash it with ultrapure water for 2 CV to replace the 25% ethanol preservation solution; add equilibration buffer and equilibrate for 10 CV, then pour in the cell supernatant for loading; wash with eluent for 5 - 10 CV until the protein column is equilibrated; elute with elution buffer, discard the first 1 CV, and collect the subsequent 1.5 CV; add neutralization buffer to neutralize the antibody after elution; wash with CIP for more than 5 CV; add equilibration solution until the pH at the outlet is neutral; equilibrate with 25% ethanol for 2 CV and store the column.

[0041] (3)Detection of murine monoclonal antibody Antibody concentration detection: Use a micro-spectrophotometer, zero it with the corresponding buffer of the test sample, and under the wavelength condition of 280 nm, spot the purified antibody sample in sequence, record the absorbance value, divide the detected data by the IgG extinction coefficient (1.414), and the obtained value is the concentration of the test sample (unit: mg / mL). The concentrations of the purified antibodies are shown in Table 4, and the concentrations are all between 0.22 and 1.54 mg / mL.

[0042] Table 4 Detection results of purified antibody concentrations name Concentration (mg / mL) SM01 0.32 SM05 0.37 SM06 0.83 SM07 1.03 SM08 0.34 SM09 0.22 SM10 0.81 SM11 1.26 SM02 0.28 SM03 0.43 SM04 0.69 SM12 1.54 SM17 0.89

[0043] ELISA binding detection: Use the indirect ELISA method to detect the binding of the purified murine monoclonal antibody to the immunogen. Dilute the tagless PreScission protease to 1 μg / mL and add 100 μL per well to the ELISA plate, and coat it overnight at 4°C; pour out the liquid in the plate and pat it dry, then block it with 2% BSA at 300 μL per well, incubate it at room temperature for 1 h after sealing; wash the plate 2 times with 300 μL per well of washing solution and pat it dry for the last time; dilute the purified prepared antibody to 1 μg / mL and add 100 μL each to the ELISA plate, and react at room temperature for 2 h; wash the plate 3 times with 300 μL per well of washing solution and pat it dry for the last time; dilute the secondary antibody Rabbit Anti-Mouse IgG F(ab')2 / HRP to 0.09 μg / mL, add 100 μL per well, mix well, and incubate at room temperature for 1 h; wash the plate 3 times with 300 μL per well of washing solution and pat it dry for the last time; mix the chromogenic solution A and B in a ratio of 1:1, add 200 μL per well, and incubate at room temperature in the dark for 20 min; add 50 μL of stop solution per well and detect the OD value at a wavelength of 450 nm.

[0044] The ELISA binding detection results of the purified antibodies are shown in Table 5, and the results show that all 13 purified antibodies can bind to the tagless PreScission protease.

[0045] Table 5 Purified Antibody ELISA Binding Test Results name <![CDATA[OD 450 -Blank]]> SM01 4.15 SM02 4.12 SM03 4.06 SM04 4.19 SM05 3.99 SM06 4.33 SM07 3.85 SM08 4.11 SM09 4.02 SM10 4.14 SM11 3.93 SM12 3.89 SM17 3.95 Antibody purity detection: According to the antibody pairing screening results in Example 4, the successfully paired antibodies (SM10 and SM17) were selected for purity detection. The detection method was size exclusion high performance liquid chromatography. The purity of SM10 was 96.77% and the purity of SM17 was 97.83%. See the chromatogram in Figure 2 .

[0046] Example 4 Antibody Pairing Screening Select SM10 and use sodium periodate method for HRP labeling, and then evaluate the binding ability of the labeled antibody to tag-free PreScission protease by ELISA. Dilute the coated antibody to 2 μg / mL, add 100 μL per well to the enzyme-linked immunosorbent assay (ELISA) plate, and coat overnight at 4 °C; pour out the liquid in the plate and pat it dry, block with 2% BSA, 300 μL per well, incubate at room temperature for 1 h after sealing; add 300 μL per well of washing solution, wash the plate 2 times, and pat it dry for the last time; dilute the tag-free PreScission protease to 10 ng / mL, add 100 μL to each well, and use the sample diluent as a blank control, react at room temperature for 2 h; add 300 μL per well of washing solution, wash the plate 3 times, and pat it dry for the last time; dilute the HRP-labeled antibody to 0.5 μg / mL respectively, add 100 μL per well, mix evenly, and incubate at room temperature for 1 h; add 300 μL per well of washing solution, wash the plate 3 times, and pat it dry for the last time; mix the chromogenic solution A and B in a ratio of 1:1, add 200 μL to each well, and incubate in the dark at room temperature for 20 min; add 50 μL of stop solution to each well and immediately measure the OD value at a wavelength of 450 nm.

[0047] The antibody ELISA pairing test results are shown in Table 6. The results show that 5 combinations were successfully paired. Among them, the combination of SM17 + enzyme-labeled secondary antibody SM10-HRP had the best reaction effect, and the purification yield of SM17 antibody was the highest. Therefore, SM17 was selected as the coated antibody and SM10 as the detection antibody.

[0048] Table 6 Antibody ELISA Pairing Test Results Coating antibody <![CDATA[Enzyme-labeled antibody: SM10-HRP (OD 450 -Blank)]]> SM11 3.34 SM10 0.00 SM09 0.02 SM08 0.01 SM07 0.00 SM06 0.84 SM05 0.03 SM01 1.15 SM17 3.57 SM12 0.03 SM04 3.41 SM03 0.01 SM02 2.82

[0049] Example 5 Establishment of Standard Curve Dilute the antibody SM17 to 2 μg / mL, add 100 μL per well to the ELISA plate, and coat overnight at 4°C; wash the plate 4 times with 300 μL per well of washing solution, and pat dry for the last time; add 300 μL per well of 2% BSA blocking solution to the ELISA plate, seal and incubate at room temperature for 1 h; wash the plate 4 times, and pat dry for the last time; dilute the tagless PreScission protease to 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156.25 pg / mL, 78.125 pg / mL, and use 1× dilution buffer as the blank control (0 pg / mL), add 100 μL per well respectively, seal the plate and incubate at room temperature for 2 h; wash the plate 3 times and pat dry the ELISA plate. Dilute the enzyme-labeled secondary antibody SM10-HRP to 0.5 μg / mL, add 100 μL per well to the wells of the plate, seal the plate and incubate at room temperature for 1 h. Discard the liquid and wash the plate 3 times, and pat dry the ELISA plate. Add 100 μL of chromogenic solution to each well, seal the plate and place at room temperature in the dark for color development for 20 min. Add 50 μL of stop solution to each well. Detect the OD value of each well with an ELISA reader at a wavelength of 450 nm, calculate the average value of the absorbance, subtract the average value of the blank control from the average absorbance of each concentration gradient, which is the signal value of this concentration. Use the concentration of each point of the standard product as the abscissa and the signal value corresponding to each concentration of the standard product as the ordinate for four-parameter Logistic curve fitting.

[0050] The detection results of the standard curve are shown in Table 7, and the four-parameter Logistic curve fitting is shown in Figure 3 . The linear range is 78.125 - 5000 pg / mL, and the correlation coefficient R 2 is 0.9999, meeting the requirements for quantitative detection.

[0051] Table 7 Detection Results of Standard Curve Standard concentration-pg / mL <![CDATA[OD 450 -1]]> <![CDATA[OD 450 -2]]> <![CDATA[OD 450 Average value]]> <![CDATA[OD 450 -Blank]]> Blank 0.0564 0.0667 0.062 / 78.125 0.0962 0.1100 0.103 0.041 156.25 0.1500 0.1677 0.159 0.097 312.5 0.2581 0.2471 0.253 0.191 625 0.4389 0.4555 0.447 0.385 1250 0.8063 0.7836 0.795 0.733 2500 1.5340 1.5222 1.528 1.466 5000 2.8451 2.9249 2.885 2.823

[0052] Example 6 Sensitivity Determination According to the method described in Example 5, add 20 sample dilutions (0.1% BSA + PBST pH 7.4) to the ELISA plate for determination, calculate the average OD value and SD of the 20 sample dilutions, and substitute the average OD value plus 2 times the SD value into the standard curve linear equation to calculate the corresponding concentration, which is the sensitivity. The detection results are shown in Table 8, and the sensitivity of this method is 15.14 pg / mL.

[0053] Table 8 Sensitivity Determination Results sample Sample diluent <![CDATA[OD 450 -1]]> 0.0688 <![CDATA[OD 450 -2]]> 0.0669 <![CDATA[OD 450 -3]]> 0.0675 <![CDATA[OD 450 -4]]> 0.062 <![CDATA[OD 450 -5]]> 0.0622 <![CDATA[OD 450 -6]]> 0.0741 <![CDATA[OD 450 -7]]> 0.0608 <![CDATA[OD 450 -8]]> 0.0685 <![CDATA[OD 450 -9]]> 0.0618 <![CDATA[OD 450 -10]]> 0.0698 <![CDATA[OD 450 -11]]> 0.0632 <![CDATA[OD 450 -12]]> 0.0627 <![CDATA[OD 450 -13]]> 0.0655 <![CDATA[OD 450 -14]]> 0.0659 <![CDATA[OD 450 -15]]> 0.0604 <![CDATA[OD 450 -16]]> 0.0645 <![CDATA[OD 450 -17]]> 0.0653 <![CDATA[OD 450 -18]]> 0.0712 <![CDATA[OD 450 -19]]> 0.0723 <![CDATA[OD 450 -20]]> 0.0738 Mean 0.066 SD 0.004 CV 6.5% Mean+2SD-Blank 0.019 Sensitivity (pg / mL) 15.14 Example 7 Precision Determination Samples with three different concentrations, high - 4000 pg / mL, medium - 670 pg / mL, and low - 220 pg / mL within the standard curve range were selected as quality control samples for the determination of precision. The three quality control samples were repeatedly measured 20 times respectively within the same experiment on the same plate, and the RSD of the calibrated actual content was the within - plate precision; the three quality control samples were repeatedly measured 3 times between different plate experiments, and the RSD of the calibrated actual content among the 3 experiments was the between - plate precision.

[0054] As can be seen from the results in Table 9, the RSDs of both within - plate and between - plate precisions are lower than 10%, indicating that the precision of this method is good.

[0055] Table 9 Results of Precision Determination - Intra-plate precision Intra-plate precision Intra-plate precision Inter-plate precision Inter-plate precision Inter-plate precision Sample (pg / mL) 220 670 4000 220 670 4000 Number of measurements 20 20 20 12 12 12 Mean 208 692 3990 209 702 4368 SD 19.34 34.85 157.60 14.48 18.05 184.33 RSD(%) 9.31 5.04 3.95 6.93 2.57 4.22

[0056] Example 8 Accuracy Determination Standard products with high - 4000 pg / mL, medium - 670 pg / mL, and low - 220 pg / mL concentrations were added to the sample diluent respectively, and detection was carried out according to the method described in Example 5.

[0057] As can be seen from the results in Table 10, the recoveries of high, medium, and low concentration samples are all between 98% and 105%, indicating that the accuracy of this method is relatively high.

[0058] Table 10 Results of Accuracy Determination Theoretical concentration (pg / mL) <![CDATA[OD 450 -1]]> <![CDATA[OD 450 -2]]> Average OD Concentration (pg / mL) Recovery rate (%) 220 0.2090 0.2040 0.2070 215.16 98 670 0.4350 0.4510 0.4430 686.85 103 4000 2.1100 2.2270 2.1690 4210.92 105 Finally, it should be noted that: The above - mentioned examples are only used to illustrate the technical solutions of the present invention, rather than to limit it; although the present invention has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that: they can still modify the technical solutions recorded in the foregoing examples, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for quantitatively detecting the content of PreScission protease by using a double antibody sandwich, characterized in that, It includes the following steps: S1. Coat an ELISA plate with a capture antibody; the capture antibody is SM17, and the SM17 includes a heavy chain and a light chain. The heavy chain and the light chain respectively contain three CDRs and four FRs. Specifically, the heavy chain CDR1 is NFGMD, the heavy chain CDR2 is WINTYTGEPTYADDFKG, the heavy chain CDR3 is GGS, the heavy chain FR1 is QIQLVQSGPELKKPGETVKISCKASGYTFT, the heavy chain FR2 is WVKQAPGKGLKWMG, the heavy chain FR3 is RFAFSLETSASTAFLQINNLKNEDTATYFCAR, the heavy chain FR4 is WGQGTLVTVSA; the light chain CDR1 is KSSQSLLDSGNQMNYLT, the light chain CDR2 is GASTRES, the light chain CDR3 is QNDHSYPLT, the light chain FR1 is DIVMTQSPSSLSVSAGEKVTMSC, the light chain FR2 is WYQQKPGQPPKLLIY, the light chain FR3 is GVPDRFTGSGSGTDFTLTISSVQAEDLAIYYC, the light chain FR4 is FGAGTKLDLK; S2. Add the sample to be measured into the coated ELISA plate; S3. Incubate with an HRP-conjugated detection antibody as an enzyme-labeled antibody, develop color using a TMB chromogenic solution, and detect the OD value at a wavelength of 450 nm with an ELISA reader; the detection antibody is SM10, and the SM10 includes a heavy chain and a light chain. The heavy chain and the light chain respectively contain three CDRs and four FRs. Specifically, the heavy chain CDR1 is DYHMS, the heavy chain CDR2 is FIRNKANGYTTEYSASVKG, the heavy chain CDR3 is LYYDYDYYFED, the heavy chain FR1 is EVKLVESGGGLAQPGGSLRLSCATSGFTFT, the heavy chain FR2 is WFRQPPGKALEWLG, the heavy chain FR3 is RFTISRDNSQSILYLQMNTLRAEDSATYYCAS, the heavy chain FR4 is WGQGTTLTVSS; the light chain CDR1 is KSSQSLLNSSTRKNYLA, the light chain CDR2 is WASTRES, the light chain CDR3 is KQSYNLWT, the light chain FR1 is DIVMSQSPSSLAVSPGEKVTMSC, the light chain FR2 is WYQQKPGQSPKLLIY, the light chain FR3 is GVPDRFTGSGSGTDFTLTINSVQAEDLAVYYC, the light chain FR4 is FGGGTKLEIK; S4. Draw a standard curve using the standard product, and calculate the content of PreScission protease in the sample to be measured according to the standard curve.

2. The method for quantitatively detecting the content of PreScission protease by using double antibody sandwich according to claim 1, wherein, The determination of the capture antibody and the detection antibody further includes the following steps: A1. Inoculate, culture, collect, and purify the engineering bacteria of PreScission protease to obtain tag-free PreScission protease; A2. Preparation and screening of anti-tagless PreScission protease murine monoclonal antibody; A3. Preparation of murine monoclonal antibody, obtaining 13 purified antibodies capable of binding to PreScission protease; A4. Antibody pairing screening was carried out by ELISA evaluation method to determine the capture antibody and detection antibody.

3. The method for quantitatively detecting the content of PreScission protease by using double antibody sandwich according to claim 1, characterized in that, After coating in S1, refrigerate overnight, block with 2% BSA, and incubate at room temperature for 1 h after sealing.

4. The method for quantitatively detecting the content of PreScission protease by using a double antibody sandwich according to claim 1, characterized in that, Before adding the sample to be measured in S2, the plate needs to be washed 2 times with the washing solution. After adding the sample to be measured, react at room temperature for 2 h.

5. The method for quantitatively detecting the content of PreScission protease by using double antibody sandwich according to claim 4, wherein, The washing solution is PBST: 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, 0.05% v / v Tween-20.

6. The method for quantitatively detecting the content of PreScission protease by using double antibody sandwich according to claim 1, wherein Before adding the HRP-conjugated detection antibody in S3, the plate needs to be washed 3 times with the washing solution. After adding the HRP-conjugated detection antibody, mix well, incubate at room temperature for 1 h, wash the plate 3 times with the washing solution, add the chromogenic solution A and B mixed evenly at 1:1, incubate at room temperature in the dark for 20 min, and add the stop solution to terminate.

7. The method for quantitatively detecting the content of PreScission protease by using a double antibody sandwich according to claim 6, wherein The washing solution is PBST: 8 mM Na2HPO4, 0.136 M NaCl, 2 mM KH2PO4, 2.6 mM KCl, 0.05% v / v Tween-20. The solution A is 13.6 g of sodium acetate, 1.6 g of citric acid, 0.3 mL of 30% hydrogen peroxide, and distilled water is added to 500 mL. The solution B is 0.2 g of disodium ethylenediaminetetraacetate, 0.95 g of citric acid, 50 mL of glycerol, 0.15 g of TMB dissolved in 3 mL of DMSO, and distilled water is added to 500 mL. The stop solution is 2 M sulfuric acid.

8. The method for quantitatively detecting the content of PreScission protease by using double antibody sandwich according to claim 1, characterized in that In S4, the standard curve is obtained by performing four-parameter Logistic curve fitting with the concentrations of each point of the standard product as the abscissa and the signal values corresponding to the standard products of each concentration as the ordinate.

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