A method for quantitatively detecting the content of PreScission protease using double antibody sandwich

Abstract

The present invention discloses a method for quantitatively detecting the content of PreScission protease using a double antibody sandwich, S1, a ELISA plate coated with a capture antibody; S2, the sample to be tested is added to the coated ELISA plate; S3, HRP-conjugated antibody is incubated as an enzyme-labeled antibody, TMB chromogenic solution is used for color development, and the OD value is detected with a microplate reader at a wavelength of 450nm; S4, a standard curve is drawn using a standard substance, and the content of PreScission protease in the sample to be tested is calculated according to the standard curve. The present invention forms an "antibody-antigen-antibody" sandwich structure by a double antibody sandwich, SM17 is coated as a capture antibody, and SM10 is used as a detection antibody. By obtaining a standard curve, the content of PreScission protease is quantitatively detected, and the sensitivity, precision and accuracy of the detection method provided by the present invention are maintained at a high level.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for quantitatively detecting the content of PreScission protease by utilizing a double antibody sandwich. Background Art

[0002] Prescision Protease (PreScission Protease) is a fusion protein composed of the 3C protease of human rhinovirus type 14 and GST. It is designed for cleavage of glutathione S-transferase (GST) tags. It can specifically cleave GST-tagged proteins containing a substrate recognition peptide sequence expressed in vectors such as the pGEX-6P series. PreScission Protease itself is a fusion protein of GST and human rhinovirus 3C protease. The N-terminal GST tag facilitates purification of the enzyme and enables immobilization and on-column cleavage of GST-tagged proteins. The C-terminal 3C protease specifically recognizes and cleaves the corresponding amino acid sequence: Leu-Glu-Val-Leu-Phe-Gln-Gly-Pro. The cleavage site is between Gln and Gly. After cleavage, the target protein retains a five-amino acid residue sequence: Gly-Pro-Leu-Gly-Ser (GPLGS) at the N-terminus.

[0003] Currently, many genetically engineered vaccines contain GST-tagged fusion proteins as their antigenic components. Prescission protease is required to remove the GST tag during purification. Therefore, it is necessary to measure the amount of Prescission protease remaining in the target protein after GST removal. However, there are few methods available for measuring Prescission protease levels. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for quantitatively detecting the content of PreScission protease using a double antibody sandwich, 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 objectives, the present invention provides the following technical solutions:

[0006] The present invention provides a method for quantitatively detecting the content of PreScission protease using a double antibody sandwich, comprising the following steps:

[0007] S1. Coat the ELISA plate with capture antibody.

[0008] S2, adding the test sample to the coated ELISA plate;

[0009] S3 and HRP-conjugated detection antibodies were incubated as enzyme-labeled antibodies, and TMB colorimetric solution was used for color development. The OD value was detected using a microplate reader at a wavelength of 450 nm.

[0010] S4. Use the standard to draw a standard curve, and calculate the content of PreScission protease in the tested sample based on the standard curve.

[0011] In one embodiment of the present invention, the capture antibody is SM17, which includes a heavy chain and a light chain. The amino acid sequence of the heavy chain of SM17 is shown in SEQ ID NO: 2, QIQLVQSGPELKKPGETVKISCKASGYTFTNFGMDWVKQAPGKGLKWMGWINTYTGEPTYADDFKGRFAFSLETSASTAFLQINNLKNEDTATYFCARGGSWGQGTLVTVSA, and the amino acid sequence of the light chain of SM17 is shown in SEQ ID NO: 3, DIVMTQSPSSLSVSAGEKVTMSCKSSQSLLDSGNQMNYLTWYQQKPGQPPKLLIYGASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAIYYCQNDHSYPLTFGAGTKLDLK. The heavy chain and light chain respectively include three CDRs and four FRs, specifically heavy chain CDR1 NFGMD, heavy chain CDR2 WINTYTGEPTYADDFKG, heavy chain CDR3 GGS, heavy chain FR1 QIQLVQSGPELKKPGETVKISCKASGYTFT, heavy chain FR2WVKQAPGKGLKWMG, heavy chain FR3 RFAFSLETSASTAFLQINNLKNEDTATYFCAR, heavy chain FR4 WGQGTLVTVSA; light chain CDR1 KSSQSLLDSGNQMNYLT, light chain CDR2 GASTRES, light chain CDR3 QNDHSYPLT, light chain FR1DIVMTQSPSSLSVSAGEKVTMSC, light chain FR2 WYQQKPGQPPKLLIY, light chain FR3 GVPDRFTGSGSGTDFTLTISSVQAEDLAIYYC, light chain FR4 FGAGTKLDLK.

[0012] In one embodiment of the present invention, the detection antibody in the S3 is SM10, and the SM10 includes a heavy chain and a light chain. The amino acid sequence of the heavy chain of the SM10 is shown in SEQ ID NO: 4, EVKLVESGGGLAQPGGSLRLSCATSGFTFTDYHMSWFRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCASLYYDYDYYFEDWGQGTTLTVSS, and the amino acid sequence of the light chain of the SM10 is shown in SEQ ID NO: 5, DIVMSQSPSSLAVSPGEKVTMSCKSSQSLLNSSTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTINSVQAEDLAVYYCKQSYNLWTFGGGTKLEIK. The heavy chain and light chain respectively include three CDRs and four FRs, specifically heavy chain CDR1 DYHMS, heavy chain CDR2 FIRNKANGYTTEYSASVKG, heavy chain CDR3 LYYDYDYYFED, heavy chain FR1EVKLVESGGGLAQPGGSLRLSCATSGFTFT, heavy chain FR2 WFRQPPGKALEWLG, heavy chain FR3 RFTISRDNSQSILYLQMNTLRAEDSATYYCAS, heavy chain FR4 WGQGTTLTVSS; light chain CDR1 KSSQSLLNSSTRKNYLA, light chain CDR2WASTRES, light chain CDR3 KQSYNLWT, light chain FR1 DIVMSQSPSSLAVSPGEKVTMSC, light chain FR2WYQQKPGQSPKLLIY, light chain FR3 GVPDRFTGSGSGTDFTLTINSVQAEDLAVYYC, light chain FR4 FGGGTKLEIK.

[0013] In one embodiment of the present invention, the determination of the capture antibody and the detection antibody further comprises the following steps:

[0014] A1. Inoculate, culture, collect and purify the engineered bacteria (pGEX-PP / BL21) of PreScission protease to obtain untagged PreScission protease;

[0015] A2. Preparation and screening of mouse monoclonal antibodies against untagged PreScission protease;

[0016] A3. Preparation of mouse monoclonal antibodies, resulting in 13 purified antibodies that bind to untagged PreScission protease;

[0017] A4. Antibody pairing screening was performed through ELISA evaluation method to determine the capture antibody and detection antibody.

[0018] In one embodiment of the present invention, after coating in S1, the plate was refrigerated overnight, blocked with 2% BSA, sealed, and incubated at room temperature for 1 hour.

[0019] In one embodiment of the present invention, before adding the sample to be tested in S2, washing liquid needs to be added to wash the plate twice, and after adding the sample to be tested, the reaction is allowed to proceed at room temperature for 2 hours.

[0020] In one embodiment of the present invention, the washing solution is PBST: Na2HPO4 8mM, NaCl 0.136M, KH2PO4 2mM, KCl 2.6mM, Tween-20 0.05% v / v.

[0021] In one embodiment of the present invention, before adding the HRP-conjugated detection antibody to S3, it is necessary to add a washing solution and wash the plate three times. After adding the HRP-conjugated detection antibody, mix evenly, incubate at room temperature for 1 hour, wash the plate three times with the washing solution, add the color developing solution A and solution B mixed evenly in a ratio of 1:1, incubate at room temperature in the dark for 20 minutes, and add the stop solution to terminate.

[0022] In one embodiment of the present invention, the color developing 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; solution B is 0.2 g of disodium ethylenediaminetetraacetic acid, 0.95 g of citric acid, 50 mL of glycerol, and 0.15 g of TMB (tetramethylbenzidine) dissolved in 3 mL of DMSO, and distilled water is added to 500 mL. The stop solution is 2 M sulfuric acid.

[0023] In one embodiment of the present invention, the standard curve in S4 is obtained by fitting a four-parameter logistic curve using the concentration of the standard at each point as the abscissa and the signal value corresponding to each concentration of the standard as the ordinate.

[0024] In one embodiment of the present invention, the average absorbance value of each concentration gradient minus the average absorbance value of the blank control is the signal value of the concentration.

[0025] Based on the above technical solution, the embodiments of the present invention can produce at least the following technical effects:

[0026] To quantitatively detect residual PreScission protease using a double antibody sandwich assay, and to eliminate interference from GST-tagged proteins, untagged PreScission protease was prepared. Using untagged PreScission protease as an immunogen, monoclonal antibodies targeting multiple active epitopes of untagged PreScission protease were obtained through immunization, screening, and identification. These monoclonal antibodies can be used in a double antibody sandwich enzyme-linked immunosorbent assay (ELISA) to quantitatively detect PreScission protease levels.

[0027] The present invention forms an "antibody-antigen-antibody" sandwich structure by sandwiching two antibodies, encapsulating SM17 as a capture antibody and SM10 as a detection antibody. The obtained standard curve is used to quantitatively detect PreScission protease. The detection method provided by the present invention maintains high levels of sensitivity, precision and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0029] Figure 1 This is the electrophoresis diagram of purity detection of the label-free PreScission protease in Example 1 of the present invention;

[0030] Figure 2 is a size exclusion high performance liquid chromatogram of Example 3 of the present invention;

[0031] Figure 3 This is the standard curve of Example 5 of the present invention. DETAILED DESCRIPTION

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0034] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0035] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0036] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0037] The present invention uses GST-free PreScission protease as an immunogen to immunize mice. Through cell fusion and screening, multiple mouse monoclonal antibodies that specifically recognize PreScission protease were successfully obtained. High-affinity mouse monoclonal antibodies were screened, prepared from ascites, purified using Protein A, and subsequently labeled with HRP.

[0038] After pairing experiments and condition optimization, the detection method's paired antibody combination (coating antibody SM17, detection antibody SM10) was finally determined, and corresponding detection conditions and standard curves were established. The method achieved a sensitivity of 15.14 pg / mL and a linear detection range of 78.125 to 5000 pg / mL, fully meeting the requirements of quantitative detection.

[0039] The reagents and instruments mainly used in the present invention are as follows:

[0040] Reagents Item No. 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 culture 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 (14.4-116 kDa) 26610 Thermo Protein standards (20-200kDa) 26614 Thermo

[0041] 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. microplate reader Multiskan Fc Thermo Inverted microscope TS-100 Nikon CO2 incubator 3111 Thermo Peristaltic pump BT100-2J Lange Constant Flow Pump Co., Ltd. Microspectrophotometer 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 Qilin Bell Micro high-speed refrigerated centrifuge Sorvall LEGEND MICRO 17R Thermo

[0042] Example 1 Expression and purification of untagged PreScission protease

[0043] The engineered bacteria of PreScission protease (pGEX-PP / BL21) were inoculated into LB medium containing ampicillin and cultured overnight (37°C, 100 rpm). The next day, the culture was expanded at a ratio of 1:100 (V / V) to OD600 The expression was induced overnight by adding IPTG (4°C, 200 rpm). The bacteria were harvested by centrifugation and lysed by ultrasonication. Bacteria were harvested by centrifugation and disrupted using a homogenizer (700-800 bar, 4°C, three times). The homogenate was centrifuged and the supernatant was collected (11600g, 4°C, 30 min). The supernatant was filtered through a 0.8 μm filter membrane, and the PreScission protease-GST fusion protein in the filtered supernatant was captured by GST-4FF affinity chromatography column. The PreScission protease-GST fusion protein was eluted with 20 mM glutathione and digested with thrombin at a ratio of 6 U / mg overnight at 4°C. The digestion buffer was exchanged with 10 mM PB (pH 8.5, 3.01 g / L Na2HPO4, 0.25 g / L NaH2PO4, 0.215 g / L NaH2PO4.H2O) by G25 chromatography. The G25 chromatography sample was purified using a Q HP column, and the Q HP flow-through sample was collected as the untagged PreScission protease. The purity of untagged PreScission protease was detected by SDS-PAGE gel electrophoresis.

[0044] The PreScission protease expression plasmid pGEX-PP was obtained as a gift from the Department of Microbiology, Chinese Academy of Sciences. The host bacterial strain was E. coli / BL21 (DE3), purchased from Novagen. The amino acid sequence of the untagged PreScission protease is shown in SEQ ID NO: 1: GPNTEFALSLLRKNIMTITTSKGEFTGLGIHDRVCVIPTHAQPGDDVLVNGQKIRVKDKYKLVDPENINLELTVLTLDRNEKFRDIRGFISEDLEGVDATLVVHSNNFTNTILEVGPVTMAGLINLSSTPTNRMIRYDYATKTGQCGGVLCATGKIFGIHVGGNGRQGFSAQLKKQYFVEKQ. The purity of the untagged PreScission protease is shown in Figure 1 As shown, the results showed that the purity was 97.1%

[0045] Example 2 Preparation and Screening of Anti-Tagged PreScission Protease Mouse Monoclonal Antibodies

[0046] (1) Animal immunization

[0047] Five female Balb / c mice aged 6-8 weeks (purchased from the China Food and Drug Administration) were immunized with untagged PreScission protease. Each mouse received a single immunization dose of 50 μg. For the first immunization, the immunogen was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously at multiple points in the abdomen. Two weeks later, the same dose of immunogen was emulsified with an equal volume of Freund's incomplete adjuvant and injected subcutaneously at multiple points in the abdomen. Blood was collected one week after immunization, and serum titers were determined using indirect ELISA. The OD value of mouse serum diluted 1:8000 was used. 450 A value greater than 1.0 (absorbance at 450 nm minus the absorbance of the blank control) was considered acceptable.

[0048] (2) Serum titer detection

[0049] Untagged PreScission protease was coated with coating solution (PBS: 136.9 mM NaCl, 10.1 mM Na2HPO4 4， 2.7 mM KCl, 1.8 mM KH2PO4 4， The plate was diluted to 5 μg / mL with PBST (pH 7.4) and 100 μL was added to each well of the ELISA plate and coated at 4°C overnight. The plate was washed once with 300 μL / well of washing solution (PBST), and the plate was patted dry. The plate was blocked with 300 μL / well of blocking solution (2% BSA in PBST) and blocked at room temperature for 1 hour. The plate was washed twice with 300 μL / well of washing solution and the plate was patted dry. The serially diluted serum samples and sample diluent (0.1% BSA in PBST) were added at 100 μL / well, and the detection antibody (horseradish peroxidase-labeled goat anti-mouse IgG) was added. Fc), add 100 μL / well to a 96-well plate and incubate at room temperature for 2 hours; wash the plate 5 times with 300 μL / well of washing solution, pat dry the plate; add 200 μL / well of color development solution and let it stand at room temperature for 12 minutes; add 50 μL / well of stop solution to terminate the reaction, and then use a microplate reader to detect at a wavelength of 450 nm. When the mouse serum stock solution is diluted 1:8000, the OD 450 A -Blank greater than 1.0 is considered a qualified titer, and mice with high titers are preferred for hybridoma fusion.

[0050] Table 1 Determination of serum antibody titers in mice after immunization

[0051] Mouse number Dilution <![CDATA[OD 450 ]]> <![CDATA[OD 450 -Blank]]> 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

[0052] From the results in Table 1 above, it can be seen that the serum titers of the five mice all reached the qualified standard, of which 5 # Mouse serum had the highest titer.

[0053] (3) Mouse monoclonal antibody fusion and screening

[0054] Myeloma cells (SP2 / 0) were purchased from China Center for Type Culture Collection (CCTCC).

[0055] Hybridoma cell screening: select 5 # Mouse spleen cells were mixed with 5 # Mouse myeloma cells (SP2 / 0) were mixed and fused using electrofusion to obtain hybridomas. Approximately 6 and 8 days after fusion, the cells were fed with HAT selection medium. Approximately 10 days after fusion, the supernatant from the master clone stage was screened using indirect ELISA. A total of 76 clones were obtained from the master clone stage that positively bound to the antigen protein. Fifteen of these master clones were selected for further screening. The ELISA results of the supernatants from these 15 master clones are shown in Table 2.

[0056] Table 2 ELISA test results of the supernatant of the main clone

[0057] 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

[0058] Subclone Screening: 15 positive hybridoma cells were seeded into half a 96-well cell culture plate using HT selection medium at a seeding density of 0.75 cells per well. The cells were incubated at 37°C with 5% CO2. Approximately seven days after incubation, the supernatant was collected for indirect ELISA analysis. After three consecutive rounds of limiting dilution and subsequent screening, 13 positive monoclonal cells that bound to untagged PreScission protease were obtained. The results are shown in Table 3.

[0059] Table 3 ELISA test results of subclone supernatants

[0060] serial number <![CDATA[OD 450 -Blank <!-- 6 -->]]> 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

[0061] In summary, 13 hybridoma cells that bind to the untagged PreScission protease were obtained after one round of fusion screening.

[0062] Example 3 Preparation of mouse monoclonal antibodies

[0063] This invention discloses the sequences of the antibodies produced by the two hybridoma cell lines used. If a person skilled in the art wishes to produce such antibodies, they can directly reconstitute the antibody sequences into an expression plasmid using genetic engineering techniques, then transfer the recombinant plasmid into a corresponding host cell for culture and expression. Host cells are primarily mammalian cells, including Sp2 / 0 myeloma cells, NS0 mouse myeloma cells, HEK293 human embryonic kidney cells, and Chinese hamster ovary (CHO) cells.

[0064] (1) Hybridoma cell culture

[0065] Transfer 1 mL of hybridoma cells into a 100 mL culture flask. When the cells have doubled to a certain density, discard the supernatant and switch to MHYBII medium for continued culture for 7 to 9 days before harvesting.

[0066] (2) Purification of monoclonal antibodies

[0067] The collected hybridoma cell supernatant was centrifuged in a micro-high-speed refrigerated centrifuge at 4000g for 30 minutes, and the supernatant was collected. The supernatant was filtered through a 0.45μM filter. A protein A column of appropriate specifications was selected based on the expression level. The column was rinsed with ultrapure water for 2CV, and the 25% ethanol storage solution was replaced. The equilibration buffer was added and equilibrated for 10CV, and the cell supernatant was loaded. The column was then rinsed with eluent for 5-10CV until the column was equilibrated. The column was then eluted with eluent, discarding the first 1CV and collecting the last 1.5CV. Neutralization buffer was added to neutralize the eluted antibody. The column was then washed with CIP for at least 5CV. The equilibration solution was added until the pH at the outlet was neutral. The column was then equilibrated with 25% ethanol for 2CV, and the column was stored.

[0068] (3) Mouse monoclonal antibody detection

[0069] Antibody concentration determination: Using a micro-spectrophotometer, adjust the concentration to zero with the sample buffer. Spot the purified antibody samples sequentially at 280 nm and record the absorbance. Divide the absorbance by the IgG extinction coefficient (1.414) to obtain the sample concentration (in mg / mL). See Table 4 for purified antibody concentrations, which ranged from 0.22 to 1.54 mg / mL.

[0070] Table 4 Purified antibody concentration test results

[0071] 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

[0072] ELISA binding detection: Use the indirect ELISA method to detect the binding of purified mouse monoclonal antibodies to the immunogen. Dilute the unlabeled PreScission protease to 1μg / mL, add 100μL / well to the ELISA plate, and coat overnight at 4℃; pour out the liquid in the plate and pat dry, block with 2% BSA, 300μL / well, seal and incubate at room temperature for 1h; wash the plate twice with 300μL / well washing solution, and pat dry for the last time; dilute the purified antibody to 1μg / mL, add 100μL each to the ELISA plate, and react at room temperature for 2h; wash the plate three times with 300μL / well washing solution, and pat dry for the last time; Rabbit Anti-Mouse IgG Dilute F(ab')2 / HRP secondary antibody to 0.09 μg / mL, add 100 μL / well, mix well, and incubate at room temperature for 1 hour; wash the plate three times with 300 μL / well washing solution, and pat dry the last time; mix color development solution A and solution B in a 1:1 ratio, add 200 μL to each well, and incubate at room temperature in the dark for 20 minutes; add 50 μL of stop solution to each well, and detect the OD value at a wavelength of 450 nm.

[0073] The results of ELISA binding assay of purified antibodies are shown in Table 5 , which show that all 13 purified antibodies can bind to the untagged PreScission protease.

[0074] Table 5 ELISA binding test results of purified antibodies

[0075] 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

[0076] Antibody purity test: Based on the antibody pairing screening results of Example 4, the successfully paired antibodies (SM10 and SM17) were selected for purity testing. The detection method was size exclusion high performance liquid chromatography, where the purity of SM10 was 96.77% and the purity of SM17 was 97.83%. The chromatograms are shown in Figure 2 .

[0077] Example 4 Antibody Pairing Screening

[0078] SM10 was selected and labeled with HRP using the sodium periodate method, and then the binding ability of the labeled antibody to the unlabeled PreScission protease was evaluated by ELISA. The coating antibody was diluted to 2 μg / mL and added to the ELISA plate at 100 μL / well and coated at 4°C overnight. The liquid in the plate was poured out and patted dry, and the plate was blocked with 2% BSA at 300 μL / well. After sealing, it was incubated at room temperature for 1 hour. The plate was washed twice with 300 μL / well washing solution and patted dry for the final time. The unlabeled PreScission protease was diluted to 10 ng / mL and 100 μL was added to each well. The sample diluent was used as a blank control and reacted at room temperature for 2 hours. The plate was washed three times with 300 μL / well washing solution and patted dry for the final time. The HRP-labeled antibody was diluted to 0.5 μg / mL and 100 μL / well was mixed. The plate was incubated at room temperature for 1 hour. The plate was washed three times with 300 μL / well washing solution and patted dry for the final time. The color development solution A and solution B were mixed in a 1:1 ratio and 200 μL was added to each well. The plate was incubated at room temperature in the dark for 20 minutes. 50 μL of stop solution was added to each well and the OD value was immediately measured at a wavelength of 450 nm.

[0079] The results of the antibody ELISA pairing test are shown in Table 6. The results show that 5 combinations were successfully paired, among which the combination of SM17 + enzyme-labeled secondary antibody SM10-HRP had the best reaction effect and the purification rate of SM17 antibody was the highest. Therefore, SM17 was selected as the coating antibody and SM10 as the detection antibody.

[0080] Table 6 Antibody ELISA paired test results

[0081] 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

[0082] Example 5 Establishment of standard curve

[0083] Antibody SM17 was diluted to 2 μg / mL, added to the ELISA plate at 100 μL / well, and coated overnight at 4°C; the plate was washed four times with 300 μL / well washing buffer, and patted dry for the final time; 2% BSA blocking solution was added to the ELISA plate at 300 μL / well, sealed, and incubated at room temperature for 1 hour; the plate was washed four times, and patted dry for the final time; unlabeled PreScission protease was diluted to 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 312.5 pg / mL, 156.25 pg / mL, and 78.125 pg / mL, and 1× dilution buffer was used as a blank control (0 pg / mL), and 100 μL / well was added respectively. The plate was sealed and incubated at room temperature for 2 hours; the plate was washed three times and patted dry. Dilute the enzyme-labeled secondary antibody SM10-HRP to 0.5 μg / mL and add 100 μL / well to the plate. Seal the plate and incubate at room temperature for 1 hour. Discard the solution, wash the plate three times, and pat dry. Add 100 μL of colorimetric solution to each well. Seal the plate and incubate at room temperature, protected from light, for 20 minutes. Add 50 μL of stop solution to each well. Measure the OD value of each well using a microplate reader at a wavelength of 450 nm. Calculate the average absorbance. Subtract the average absorbance of the blank control from the average absorbance of each concentration gradient to determine the signal value for that concentration. Perform a four-parameter logistic curve fit, using the standard concentration at each point as the horizontal axis and the signal value corresponding to each standard concentration as the vertical axis.

[0084] The results of the standard curve test are shown in Table 7, and the four-parameter Logistic curve fitting is shown in Figure 3 The linear range is 78.125~5000pg / mL, and the correlation coefficient R 2 The value is 0.9999, which meets the requirements of quantitative detection.

[0085] Table 7 Standard curve detection results

[0086] Standard concentration - pg / mL <![CDATA[OD 450 -1]]> <![CDATA[OD 450 -2]]> <![CDATA[OD 450 Average]]> <![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

[0087] Example 6 Sensitivity Measurement

[0088] Following the method described in Example 5, 20 sample dilutions (0.1% BSA in PBST, pH 7.4) were added to the ELISA plate for measurement. The mean OD value and SD of the 20 sample dilutions were calculated. The mean OD value plus twice the SD value was substituted into the linear equation of the calibration curve to calculate the corresponding concentration, which was the sensitivity. The test results are shown in Table 8. The sensitivity of this method was 15.14 pg / mL.

[0089] Table 8 Sensitivity test results

[0090] 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

[0091] Example 7 Precision Determination

[0092] Three samples with different concentrations within the standard curve range (high - 4000 pg / mL, mid - 670 pg / mL, and low - 220 pg / mL) were selected as quality control samples for precision determination. The three quality control samples were each measured 20 times within the same experiment on the same plate, and the RSD of the actual content of the calibration was defined as the intra-plate precision. The three quality control samples were measured three times between experiments on different plates, and the RSD of the actual content of the calibration between the three experiments was defined as the inter-plate precision.

[0093] From the results in Table 9, it can be seen that the RSDs of intra-plate and inter-plate precision are both lower than 10%, indicating that the method has good precision.

[0094] Table 9 Precision determination results

[0095] - 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

[0096] Example 8 Accuracy Determination

[0097] Standards with concentrations of high (4000 pg / mL), medium (670 pg / mL), and low (220 pg / mL) were added to the sample diluent, and detection was performed according to the method described in Example 5.

[0098] From the results in Table 10, it can be seen that the recoveries of high, medium and low concentration samples are all between 98% and 105%, indicating that the method has high accuracy.

[0099] Table 10 Accuracy test results

[0100] Theoretical concentration (pg / mL) <![CDATA[OD 450 -1]]> <![CDATA[OD 450 -2]]> OD average 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

[0101] Finally, it should be noted that:

[0102] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to 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 using a double antibody sandwich, characterized in that: The following steps are involved: S1. Coat the ELISA plate with a capture antibody; the capture antibody is SM17, and the SM17 includes a heavy chain and a light chain, and the heavy chain and light chain respectively contain three CDRs and four FRs, specifically heavy chain CDR1 NFGMD, heavy chain CDR2 WINTYTGEPTYADDFKG, heavy chain CDR3 GGS, heavy chain FR1 QIQLVQSGPELKKPGETVKISCKASGYTFT, heavy chain FR2 WVKQAPGKGLKWMG, heavy chain FR3 RFAFSLETSASTAFLQINNLKNEDTATYFCAR, heavy chain FR4 WGQGTLVTVSA; light chain CDR1 KSSQSLLDSGNQMNYLT, light chain CDR2 GASTRES, light chain CDR3 QNDHSYPLT, light chain FR1DIVMTQSPSSLSVSAGEKVTMSC, light chain FR2 WYQQKPGQPPKLLIY, light chain FR3 GVPDRFTGSGSGTDFTLTISSVQAEDLAIYYC, light chain FR4 FGAGTKLDLK; S2, adding the sample to be tested into the coated ELISA plate; S3 and HRP-conjugated detection antibody were incubated as enzyme-labeled antibodies, TMB color development solution was used for color development, and the OD value was detected with a microplate reader at a wavelength of 450 nm; the detection antibody was SM10, and the SM10 included a heavy chain and a light chain, and the heavy chain and light chain respectively contained three CDRs and four FRs, specifically heavy chain CDR1 DYHMS, heavy chain CDR2 FIRNKANGYTTEYSASVKG, heavy chain CDR3 LYYDYDYYFED, heavy chain FR1 EVKLVESGGGLAQPGGSLRLSCATSGFTFT, heavy chain FR2 WFRQPPGKALEWLG, heavy chain FR3 RFTISRDNSQSILYLQMNTLRAEDSATYYCAS, heavy chain FR4 WGQGTTLTVSS; light chain CDR1 KSSQSLLNSSTRKNYLA, light chain CDR2 WASTRES, light chain CDR3 KQSYNLWT, light chain FR1DIVMSQSPSSLAVSPGEKVTMSC, light chain FR2 WYQQKPGQSPKLLIY, light chain FR3 GVPDRFTGSGSGTDFTLTINSVQAEDLAVYYC, light chain FR4 FGGGTKLEIK; S4. Draw a standard curve using the standard product, and calculate the content of PreScission protease in the tested sample based on the standard curve.

2. The method for quantitatively detecting the content of PreScission protease using a double antibody sandwich according to claim 1, characterized in that: The determination of the capture antibody and the detection antibody further comprises the following steps: A1. Inoculate, culture, collect and purify the engineered bacteria of PreScission protease to obtain untagged PreScission protease; A2. Preparation and screening of mouse monoclonal antibodies against untagged PreScission protease; A3. Preparation of mouse monoclonal antibodies, resulting in 13 purified antibodies that bind to PreScission protease; A4. Antibody pairing screening was performed through ELISA evaluation method to determine the capture antibody and detection antibody.

3. The method for quantitatively detecting the content of PreScission protease using a double antibody sandwich according to claim 1, characterized in that: After coating in S1, the plate was refrigerated overnight, blocked with 2% BSA, sealed, and incubated at room temperature for 1 hour.

4. The method for quantitatively detecting the content of PreScission protease using a double antibody sandwich according to claim 1, characterized in that: Before adding the sample to be tested into S2, washing solution needs to be added to wash the plate twice, and after adding the sample to be tested, the plate is reacted at room temperature for 2 hours.

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

6. The method for quantitatively detecting the content of PreScission protease using a double antibody sandwich according to claim 1, characterized in that Before adding HRP-conjugated detection antibody to S3, it is necessary to add washing solution and wash the plate 3 times. After adding HRP-conjugated detection antibody, mix evenly, incubate at room temperature for 1 hour, wash the plate 3 times with washing solution, add color development solution A and solution B mixed evenly at a ratio of 1:1, incubate at room temperature in the dark for 20 minutes, and add stop solution to terminate.

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

8. The method for quantitatively detecting the content of PreScission protease using a double antibody sandwich according to claim 1, characterized in that: The standard curve in S4 is obtained by fitting a four-parameter logistic curve with the concentration of the standard at each point as the horizontal axis and the signal value corresponding to each concentration of the standard as the vertical axis.

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