Preparation of progastrin (PG80) antibodies and establishment of CLIA PG80 detection kit

Abstract

The present invention discloses a process for preparing progastrin (PG80) antibody and establishing a chemiluminescence immunoassay (CLIA) kit for detecting PG80. A PG80-N antigen epitope peptide is synthesized, and the amino acid sequence is SEQ ID NO.1. A PG80-C antigen epitope peptide is synthesized, and the amino acid sequence is SEQ ID NO.2. A carrier protein is coupled to prepare a PG80-N carrier protein antigen and a PG80-C carrier protein antigen. Animals are immunized to produce PG80-N or PG80-C monoclonal antibodies or polyclonal antibodies, which are used to establish a CLIA kit for detecting PG80, detect the PG80 level in blood or other body fluids, and can be used for early screening of various cancers, determining cancer progression or recurrence, and evaluating cancer treatment effects.

Description

Technical Field

[0001] The invention relates to the fields of immunology, peptide chemistry and photochemistry, and involves the process of chemically synthesizing PG80 antigen epitope peptides, preparing PG80-carrier protein antigens, immunizing animals to produce PG80 antibodies, and establishing a chemiluminescence immunoassay (CLIA) kit for detecting PG80. Technical Background

[0002] Gastrin is mainly produced in the duodenal G cells. A small amount of gastrin is produced by the upper small intestine, colon and pancreas, and multiple protease processing processes are required for its formation. First, the 80-amino acid precursor gastrin (Progastrin, PG80) passes through the Golgi network and undergoes alanine tyrosine sulfonation and serine phosphorylation. PG80 is cleaved at multiple sites such as Arg36 / Arg37, Lys54 / Lys55 or Arg73 / Arg74 to form multiple fragments of gastrin, which have the function of regulating gastric mucosal growth and gastric acid secretion. These gastrins are secreted into the blood, and the dominant fragments are gastrin-34 and gastrin-17, both of which exist in the form of tyrosine sulfate and non-sulfurized variants; in addition, there are also longer gastrin-52 or gastrin-71 and shorter gastrin-14 or gastrin-6 in the blood.

[0003] At present, due to the lack of effective screening indicators for most cancers, these cancers cannot be detected early and the survival rate is very low. Studies have found that in the early stages of almost all types of cancer, the WNT / β-catenin oncogenic pathway is activated, directly activating the GAST gene, and a variety of cancer cells secrete PG80. In the blood of early cancer patients, a significantly increased level of PG80 can be detected.

[0004] PG80 produced from primary tumor cells does not undergo protease processing, does not form gastrin, and has nothing to do with digestive function. PG80 directly enters the blood circulation. Therefore, PG80 in the blood becomes a biomarker for early cancer. Detecting the level of PG80 in the blood can be used for early screening of the occurrence of various cancers.

[0005] A study detected the PG80 level in the blood of patients with different types of cancer. The results showed that for breast cancer, AUC = 0.96, sensitivity was 70%; for colorectal cancer, AUC = 0.93, sensitivity was 73%; for melanoma, AUC = 0.96, sensitivity was 87%; for cervical cancer, AUC = 0.95, sensitivity was 84%. The specificity of detecting PG80 levels in the blood for diagnosing all stages of cancer was 97%, and the sensitivity for diagnosing early melanoma and breast cancer patients was 68% and 81%, respectively.

[0006] Other studies have tested the sensitivity of PG80 in the blood for early detection of colorectal cancer and breast cancer. The sensitivity for stage 0 and stage I breast cancer is 68.2%; the sensitivity for stage II colorectal cancer is increased to 82%, and the sensitivity for stage II breast cancer is increased to 78%. A study followed up patients with peritoneal cancer and found that after a series of treatments, the PG80 level in the blood of peritoneal cancer patients decreased from 4.4pM to 1.3pM, and the prognosis of patients with decreased PG80 levels in the blood after cancer surgery was better.

[0007] The above results indicate that PG80 in the blood can be used as a biomarker for the occurrence of various cancers, and can be used to screen early cancers, determine cancer progression and recurrence, and evaluate the effectiveness of cancer treatment. It is necessary to develop a simple, rapid and quantitative test kit for PG80 in blood or other body fluids. Summary of the invention

[0008] The present invention prepares an antibody that binds to the PG80 cleavage site, excludes Gastrin fragments of various lengths, and can specifically bind to full-length PG80. The antibody is used to establish a CLIA PG80 detection kit to quantitatively detect the PG80 level in a sample.

[0009] Specifically, the present invention discloses a PG80 antigen epitope peptide-carrier protein antigen preparation process, the steps are as follows:

[0010] The present invention synthesizes a PG80-N antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID NO.1, with GSGSGC amino acids added to the C-terminus and Cys amino acids at the outermost end, which is used for coupling with Maleimide activated carrier protein to prepare PG80-N-carrier protein antigen.

[0011] The PG80-C antigen epitope peptide was synthesized, and the amino acid sequence was shown in SEQ ID NO.2, with CGGSGG amino acid added to its N-terminus and Cys amino acid at the outermost end, which was used to couple with Maleimide activated carrier protein to prepare PG80-C-carrier protein antigen.

[0012] In the present invention, carrier proteins that can be used include bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), ovalbumin (OVA), blue carrier protein (BCP), etc., preferably Maleimide activated KLH.

[0013] The present invention prepares PG80-N-carrier protein antigen and PG80-C-carrier protein antigen, which are used for immunizing animals, including mice, rats, rabbits, goats, sheep, alpacas, donkeys, horses, etc., to produce PG80-N and PG80-C monoclonal antibodies or polyclonal antibodies.

[0014] The present invention synthesizes a PG80-N cross-linked peptide, the amino acid sequence of which is shown in SEQ ID NO.3, with GGSGGC amino acid added to the C-terminus and Cys amino acid at the outermost end, which is used to connect to a sulfonated coupling resin.

[0015] The present invention synthesizes a PG80-C cross-linked peptide, the amino acid sequence of which is shown in SEQ ID NO.4, with CGSGSG amino acid added to its N-terminus and Cys amino acid at the outermost end, which is used for connecting to a sulfonated coupling resin.

[0016] The present invention discloses a process for preparing PG80-N antibody, the steps of which are as follows:

[0017] (1) Prepare PG80-N-carrier protein antigen; (2) Immunize animals to produce PG80-N antiserum; (3) Determine the titer and specificity of PG80-N antiserum; (4) Connect PG80-N cross-linked peptide to sulfonated coupling resin; (5) Separate and purify PG80-N antibody with PG80-N resin; (6) Determine the titer and specificity of PG80-N antibody.

[0018] The present invention discloses a process for preparing PG80-C antibody, the steps of which are as follows:

[0019] (1) Prepare PG80-C-carrier protein antigen; (2) Immunize animals to produce PG80-C antiserum; (3) Determine the titer and specificity of PG80-C antiserum; (4) Connect PG80-C cross-linked peptide to sulfonated coupling resin; (5) Separate and purify PG80-C antibody with PG80-C resin; (6) Determine the titer and specificity of PG80-C antibody.

[0020] PG80-N antibody binds to the cleavage site of PG80 Arg36 / Arg37, and PG80-C antibody binds to the cleavage site of PG80 Arg73 / Arg74 and C-terminus. The kit established with PG80-N antibody and PG80-C antibody can only detect the full-length PG80 derived from tumors, but excludes the detection of gastrin of various lengths derived from the digestive system. Figure 1.

[0021] The present invention can use PG80-N or PG80-C monoclonal antibody or polyclonal antibody to establish a method for detecting PG80, including immunochromatography (Immunochromatography), electrochemiluminescence immunoassay (ECLIA), chemiluminescence immunoassay (CLIA), enzyme-linked immunosorbent assay (ELISA), microfluidic immunoassay, immunochip technology, immunobiosensor technology, etc.

[0022] The invention discloses a process for establishing a CLIA PG80 detection kit by using PG80-N or PG80-C antibodies.

[0023] The CLIA detection PG80 kit mainly contains PG80 calibrator, PG80 quality control, reagent R1, reagent R2 and reagent R3.

[0024] The present invention discloses a preparation process of Acridinium ester (AE)-PG80 antibody, and the steps are as follows:

[0025] (1) PBS is used to adjust the PG80 antibody to the required concentration; (2) Dimethylformamide (DMF) is used to dissolve the Acridinium NHS ester to the required concentration; (3) PG80 antibody is reacted with the Acridinium NHS ester; (4) After the reaction is terminated, the free Acridinium NHS ester is removed to obtain the purified AE-PG80 antibody; (5) The working solution is used to dilute the AE-PG80 antibody to a concentration of 10-100 ng / ml, which is reagent R1, and is stored at 2-8°C for future use.

[0026] Working solution: 10-100 mM NaHPO4, pH 6.5-8.5, 50-500 mM NaCl, 0.5-5.0% BSA, 0.2-2.0% Trehalose, 0.01-0.1% Tween-20, 0.01-0.1% ProClin300.

[0027] The present invention discloses a preparation process of Biotin-PG80 antibody, and the steps are as follows:

[0028] (1) Adjust the PG80 antibody to the required concentration with PBS; (2) Dissolve Sulfo-NHS-Biotin to the required concentration with H2O; (3) Mix the PG80 antibody and Sulfo-NHS-Biotin for reaction; (4) Remove free Sulfo-NHS-biotin to obtain purified Biotin-PG80 antibody; (5) Dilute the Biotin-PG80 antibody to a concentration of 10-100 ng / ml with the working solution, which is reagent R2, and store at 2-8°C for future use.

[0029] The present invention discloses a preparation process of a streptavidin-coated magnetic microparticle liquid, and the steps are as follows:

[0030] Streptavidin Coated Magnetic Particles (SMP), with a diameter range of 2.0-5.0 um, were washed three times with PBS, pH 7.4, 0.01% Tween 20, and then diluted with working solution to a concentration of 0.1-1.0%, which was reagent R3, and stored at 2-8°C for future use.

[0031] The present invention discloses a preparation process of PG80 calibrators and quality control products, and the steps are as follows:

[0032] The invention discloses a synthetic PG80 calibrator peptide, the amino acid sequence of which is shown in SEQ ID NO.5.

[0033] Dilute the PG80 calibrator peptide to 0-100pM / L with peptide preservation solution to become the PG80 calibrator; dilute the PG80 calibrator peptide to 1-4pM / L or 8-16pM / L with peptide preservation solution to become the PG80 quality control product.

[0034] Peptide storage solution: 10-100mM NaHPO4, pH6.0-8.0, 20-200mM NaCl, 0.2-2.0mM EDTA, 0.5-5.0mM DTT, 2.0-20% BSA, 0.2-2.0% Trehalose, 0.1-1.0% PVP, 0.01-0.1% Tween-20, 0.01-0.1% ProClin300.

[0035] The present invention discloses the application process of CLIA detection PG80 kit:

[0036] The CLIA analyzer automatically adds 5-50ul of the sample to be tested, 20-200ul of reagent R1, 20-200ul of reagent R2 and 10-100ul of reagent R3 to the reaction cup, reacts at 25-37°C for 5-30 minutes to form an AE-PG80 antibody-PG80-PG80 antibody-Biotin-SMP complex; the magnet adsorbs the SMP complex to the bottom surface of the reaction cup; the free AE-PG80 antibody, Biotin-PG80 antibody or other impurities are absorbed, and after washing the SMP complex, the luminescence reaction process of the Acridinium-H2O2-NaOH system is started, and the CLIA analyzer measures the light intensity value.

[0037] Establish an automatic procedure for CLIA analyzer to detect PG80 concentration:

[0038] The CLIA analyzer automatically adds 5-50ul PG80 calibrator, 20-200ul reagent R1, 20-200ul reagent R2 and 10-100ul reagent R3 to the reaction cup, reacts for 5-30 minutes, and after washing, the CLIA analyzer automatically detects the PG80 relative luminescence unit (RLU), and establishes a PG80 calibrator concentration standard curve with the PG80 calibrator concentration as the ordinate and the calibrator RLU as the abscissa. Based on this standard curve, an automatic conversion program between RLU and PG80 concentration is established, and the CLIA analyzer is set up to automatically detect the PG80 concentration system to further determine the PG80 concentration in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the PG80 antibody binding site

[0040] Figure 2 is the PG80 concentration standard curve, which is the PG80 concentration standard curve obtained by measuring the PG80 calibrator by the chemiluminescent immunoassay PG80 kit of the present invention, wherein the abscissa is the relative luminescence unit (RLU), the unit is RLU, and the ordinate is the PG80 concentration value in the PG80 calibrator of the present invention, the unit is pM / L. Specific implementation plan

[0041] The principles and results of the present invention are further described in conjunction with specific experiments. The multi-step experimental process of the present invention is listed below.

[0042] 1. Preparation of PG80-N Antibody (I) Preparation of PG80-N Antigen Epitope Peptide

[0043] The PG80-N antigen epitope peptide was synthesized, and the amino acid sequence was shown in SEQ ID NO.1, which contained 12 amino acids of human PG80 peptide and an additional GSGSGC amino acid sequence at the C-terminus.

[0044] (II) PG80-N antigen epitope peptide coupled to carrier protein

[0045] 1. KLH carrier protein solution: 4 mg Maleimide activated KLH carrier protein (ActiveHOOK KLH), purchased from G-Biosciences, dissolved in 0.5 ml ddH2O

[0046] 2.PG80-N epitope peptide solution: 2 mg synthetic PG80-N epitope peptide (>95%), dissolved in 0.5 ml PBS, pH 7.2

[0047] 3. Mix 0.5ml KLH carrier protein solution with 0.5ml PG80-N antigen epitope peptide solution and react for 2 hours at room temperature.

[0048] 4. Add the above reaction solution to the protein concentration and purification centrifugal column (Microcon-30KDa CentrifugalFilter)

[0049] 5. Centrifuge at 14000g for 2-8 minutes to reduce the reaction solution to 50-100ul

[0050] 6. Remove the liquid in the collection tube and add 400ul PBS (pH 7.4) to the protein concentration and purification centrifuge column

[0051] 7. Centrifuge at 14000g for 2-8 minutes to reduce the volume of the liquid in the centrifuge column to 50-100ul

[0052] 8. Repeat steps 6-7 5 times or more

[0053] 9. Aspirate about 50-100ul PG80-N-KLH solution from the protein concentration and purification centrifuge column

[0054] 10. Determine the concentration of PG80-N-KLH and store at -80℃.

[0055] (III) Production of PG80-N antiserum

[0056] Day 0: Select New Zealand white rabbits between 3 and 6 months old and collect 2.0 ml of blood as negative control

[0057] Day 1: 0.3 ml PG80-N-KLH, 1.5 mg / ml, mixed with 0.3 ml Freund's complete adjuvant (CFA), injected subcutaneously into New Zealand rabbits at 20 points, 30 ul / point

[0058] Day 14: 0.15 ml PG80-N-KLH was mixed with 0.15 ml Freund's incomplete adjuvant (IFA) and injected subcutaneously into New Zealand rabbits at 10 points, 30 ul / point

[0059] Day 28: 0.15ml PG80-N-KLH was mixed with 0.15ml IFA and injected subcutaneously into New Zealand rabbits at 10 points, 30ul / point. Day 35: 20ml New Zealand rabbit blood was collected to detect the titer and specificity of PG80-N antiserum.

[0060] Day 42: 0.15ml PG80-N-KLH was mixed with 0.15ml IFA and injected subcutaneously into New Zealand rabbits at 10 points, 30ul / point. Day 56: Blood was collected from New Zealand rabbits and the experiment was terminated.

[0061] (IV) Determination of PG80-N antiserum titer and specificity

[0062] 1. Prepare 10ug / ml PG80 calibrator peptide and 10μg / ml BSA coating solution with 50mM carbonate buffer, pH9.6, add 1OOul to the wells of the enzyme-linked reaction plate, 2-8℃, overnight, wash twice with PBS, pH7.4, 0.01% Tween20 (PBST)

[0063] 2. Add 200ul blocking solution to each well, PBS, pH 7.4, 1.0% BSA, room temperature, 2 hours, wash twice with PBST

[0064] 3. Dilute PG80-N antiserum at 1:10, 1:100, 1:1000, add 100ul to the reaction well, room temperature, 60 minutes

[0065] 4. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0066] 5. 1:4000 dilution of anti-rabbit IgG antibody-HRP, purchased from Promega, add 100ul to the reaction well, room temperature, 60 minutes

[0067] 6. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0068] 7. Add 100ul HRP substrate, TMB Substrate buffer, purchased from Santa Cruz Biotech Inc., and react for 10 minutes

[0069] 8. Add 50ul 2N H2SO4 to terminate the reaction

[0070] 9. Use an enzyme-linked reaction analyzer and select a wavelength of 450nm to measure the OD value.

[0071] PG80-N antiserum 1:10 1:100 1:1000 0 PG80 peptide 1.687 0.654 0.215 0.078 OD value 1.542 0.585 0.187 0.087 1.854 0.621 0.175 0.082 Average 1.694333 0.62 0.192333 0.082333 BSA 0.138 0.125 0.089 0.091 OD value 0.128 0.104 0.094 0.082 0.145 0.138 0.114 0.071 Average 0.137 0.122333 0.099 0.081333

[0072] (V) PG80-N cross-linked peptide linked to sulfonated coupling resin

[0073] The PG80-N cross-linked peptide was synthesized, and the amino acid sequence was shown in SEQ ID NO.3, which contained 12 amino acids of human PG80 peptide and an additional GGSGGC amino acid sequence at the C-terminus.

[0074] 1. 2 mg PG80-N cross-linked peptide dissolved in 2.0 ml binding buffer, 50 mM Tris, pH 8.5, 5 mM EDTA

[0075] 2. Sulfonated coupling resin (SulfoLink Coupling Resin) purchased from ThermoFisher Scientific, add 2 ml of sulfonated coupling resin to the chromatography column, and drain the liquid.

[0076] 3. Wash the sulfonated coupling resin column with 20 ml of binding solution

[0077] 4. Add 2 ml of PG80-N cross-linked peptide solution to the resin chromatography column, shake up and down for 30 minutes, and let it stand for 30 minutes.

[0078] 5.10ml binding solution to wash PG80-N-resin chromatography column

[0079] 6. Prepare 50mM L-Cysteine ​​solution with the binding solution, add 2.0ml L-Cysteine ​​solution to the PG80-N-resin chromatography column, shake up and down for 30 minutes, and let it stand for 30 minutes.

[0080] 7. Wash the PG80-N-resin column with 20ml 1.0M NaCl solution

[0081] 8. Wash the PG80-N-resin column with 10 ml of preservation solution, PBS, pH 7.4, 0.05% NaN3

[0082] 9. Add the preservation solution to the PG80-N-resin chromatography column and store at 2-8°C for future use.

[0083] (VI) Purification of PG80-N Antibody by PG80-N-Resin Chromatography Column

[0084] 1. Mix 5 ml of PG80-N antiserum with 5 ml of Tris diluent, 20 mM Tris, pH 7.5, 0.1 M NaCl, and filter through a 0.22 um filter membrane.

[0085] 2. 10 ml of PG80-N antiserum dilution was passed through the PG80-N resin column, and this step was repeated 8 times.

[0086] 3. Add 20 ml of washing solution A, 20 mM Tris, pH 7.5, 0.1 M NaCl to wash the PG80-N-resin column

[0087] 4. Add 20 ml of washing solution B, 20 mM Tris, pH 7.5, 0.5 M NaCl to wash the PG80-N-resin column

[0088] 5. Add 1.0 ml of eluent, 100 mM Glycine, pH 2.5, pass through the resin column, collect the eluent, put it into a microcentrifuge tube with 40 ul 2.0 M Tris, pH 9.0, mix well, and repeat this step 6 times

[0089] 6. After mixing the eluate, transfer it into a dialysis bag and dialyze overnight with dialysis solution, 20mM NaHPO4, pH7.5, 150mM NaCl, 1.0mM EDTA, 1.0mM DTT, 1.0mM PMSF, 5% Glycerol, 2-8℃

[0090] 7. Add the above antibody dialyzate to the protein microconcentrator centrifugal column (Microcon-10kDa CentrifugalFilter)

[0091] 8. Concentration and Purification of PG80-N Antibody

[0092] 9. Determine the concentration of PG80-N antibody and store at -20℃.

[0093] (VII) Determination of PG80-N antibody titer and specificity

[0094] 1. Add 100ul of 10ug / ml PG80 calibrator peptide and 10μg / ml BSA coating solution to the wells of the enzyme-linked reaction plate, incubate at 2-8°C overnight, and wash twice with PBST.

[0095] 2. Add 200ul blocking solution to each well, incubate at room temperature for 2 hours, and wash twice with PBST

[0096] 3. 0, 0.5, 5.0, 50ng / ml PG80-N antibody, add 100ul to the reaction well, react at room temperature for 60 minutes

[0097] 4. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0098] 5. Dilute anti-rabbit IgG antibody-HRP at 1:4000, add 100ul to the reaction well, room temperature, 60 minutes

[0099] 6. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0100] 7. Add 100ul HRP substrate, TMB Substrate buffer, react for 10 minutes

[0101] 8. Add 50ul 2N H2SO4 to terminate the reaction

[0102] 9. Use an enzyme-linked reaction analyzer and select a wavelength of 450nm to measure the OD value.

[0103]

[0104]

[0105] 2. Preparation of PG80-C Antibody (I) Preparation of PG80-C Antigen Epitope Peptide

[0106] The PG80-C antigen epitope peptide was synthesized, and the amino acid sequence was shown in SEQ ID NO.2, which contained 12 amino acids of human PG80 peptide and an additional CGGSGG amino acid sequence at the N-terminus.

[0107] (II) PG80-C antigen epitope peptide coupled to carrier protein

[0108] 1. KLH carrier protein solution: 4 mg Maleimide activated KLH carrier protein (ActiveHOOK KLH), dissolved in 0.5 ml ddH2O

[0109] 2.PG80-C epitope peptide solution: 2 mg synthetic PG80-C epitope peptide (>95%), dissolved in 0.5 ml PBS, pH 7.2

[0110] 3. Mix 0.5ml KLH carrier protein solution with 0.5ml PG80-C antigen epitope peptide solution and react for 2 hours at room temperature.

[0111] 4. Add the above reaction solution to Microcon-30KDa Centrifugal Filter

[0112] 5. Concentration and purification of PG80-C-KLH

[0113] 6. Determine the concentration of PG80-C-KLH and store at -80℃.

[0114] (III) Preparation of PG80-C antibody

[0115] Day 0: Select 8-10 month old Boer goats and collect 5 ml of goat blood as a negative control

[0116] Day 1: 0.6 ml PG80-C-KLH, 1.5 mg / ml, mixed with 0.6 ml Freund's complete adjuvant (CFA), injected into the goat's subcutaneous tissue at 30 points, 40 ul / point

[0117] Day 14: 0.3 ml PG80-C-KLH was mixed with 0.3 ml Freund's incomplete adjuvant (IFA) and injected into the goat's subcutaneous tissue at 15 points, 40 ul / point

[0118] Day 28: 0.3ml PG80-C-KLH mixed with 0.3ml IFA, injected into the goat subcutaneous tissue at 15 points, 40ul / point

[0119] Day 42: Collect 5 ml of goat blood and measure the titer and specificity of PG80-C antiserum

[0120] Day 45: 0.3ml PG80-C-KLH mixed with 0.3ml IFA, injected into the goat subcutaneous tissue at 15 points, 40ul / point

[0121] Day 59: Collect 200ml of goat blood

[0122] Day 62: 0.3ml PG80-C-KLH mixed with 0.3ml IFA, injected into the goat subcutaneous tissue at 15 points, 50ul / point

[0123] Day 76: Collect blood from goats and terminate the experiment.

[0124] (IV) Determination of PG80-C antiserum titer and specificity

[0125] 1. Add 100ul of 10ug / ml PG80 calibrator peptide and 10μg / ml BSA coating solution to the wells of the enzyme-linked reaction plate, incubate at 2-8°C overnight, and wash twice with PBST.

[0126] 2. Add 200ul blocking solution to each well, incubate at room temperature for 2 hours, and wash twice with PBST

[0127] 3. Dilute PG80-C antiserum at 1:10, 1:100, 1:1000, add 100ul to the reaction well, react at room temperature for 60 minutes

[0128] 4. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0129] 5. 1:3000 dilution of anti-goat IgG antibody-HRP, purchased from Promega, add 100ul to the reaction well, at room temperature for 60 minutes

[0130] 6. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0131] 7. Add 100ul HRP substrate, TMB Substrate buffer, react for 10 minutes

[0132] 8. Add 50ul 2N H2SO4 to terminate the reaction

[0133] 9. Use an enzyme-linked reaction analyzer and select a wavelength of 450nm to measure the OD value.

[0134] PG80-C antiserum 1:10 1:100 1:1000 0 PG80 peptide 1.758 0.776 0.178 0.086 OD value 1.662 0.825 0.194 0.094 1.876 0.818 0.225 0.078 Average 1.765333 0.806333 0.199 0.086 BSA 0.14 0.135 0.105 0.088 OD value 0.138 0.126 0.098 0.096 0.158 1.115 0.115 0.087 Average 0.147 0.25333 0.106 0.090333

[0135] (V) PG80-C cross-linked peptide linked to sulfonated coupling resin

[0136] The PG80-C cross-linked peptide was synthesized, and the amino acid sequence was shown in SEQ ID NO.4, which contained 12 amino acids of human PG80 peptide and an additional CGSGSG amino acid sequence at the N-terminus.

[0137] 1.2 mg PG80-C cross-linked peptide dissolved in 2.0 ml binding solution

[0138] 2. Add 2 ml of sulfonated coupling resin to the chromatography column and drain the liquid.

[0139] 3. Wash the sulfonated coupling resin column with 20 ml of binding solution

[0140] 4. Add 2 ml of PG80-C cross-linked peptide solution to the resin chromatography column, shake up and down for 30 minutes, and let it stand for 30 minutes.

[0141] 5.10ml binding solution to wash PG80-C-resin chromatography column

[0142] 6. Add 2.0 ml of L-Cysteine ​​solution to the PG80-C-resin column, shake up and down for 30 minutes, and let stand for 30 minutes.

[0143] 7. Wash the PG80-C-resin column with 20ml 1.0M NaCl solution

[0144] 8. Wash the PG80-C-resin column with 10 ml of preservation solution, PBS, pH 7.5, 0.05% NaN3

[0145] 9. Add the preservation solution to the PG80-C-resin chromatography column and store at 2-8℃ for future use.

[0146] (VI) Purification of PG80-C Antibody by PG80-C-Resin Chromatography Column

[0147] 1. Mix 5ml PG80-C antiserum with 5ml Tris diluent and filter through a 0.22um filter membrane.

[0148] 2. 10 ml of PG80-C antiserum dilution was passed through the PG80-C resin column, and this step was repeated 8 times.

[0149] 3. Add 20 ml of washing solution A to wash the PG80-C-resin chromatography column

[0150] 4. Add 20 ml of washing solution B to wash the PG80-C-resin chromatography column

[0151] 5. Add 1.0 ml of eluent through the resin column, collect the eluent, put it into a microcentrifuge tube with 40 ul 2.0 M Tris, pH 9.0, mix well, and repeat this step 6 times

[0152] 6. After mixing the eluate, transfer it into a dialysis bag and dialyze overnight at 2-8°C with dialysis solution.

[0153] 7. Add the above antibody dialyzate to the protein microconcentrator centrifugal column (Microcon-10kDa CentrifugalFilter)

[0154] 8. Concentration and Purification of PG80-N Antibody

[0155] 9. Determine the concentration of PG80-C antibody and store at -20℃.

[0156] (VII) Determination of PG80-C antibody titer and specificity

[0157] 1. Add 100ul of 10ug / ml PG80 calibrator peptide and 10μg / ml BSA coating solution to the wells of the enzyme-linked reaction plate, incubate at 2-8°C overnight, and wash twice with PBST.

[0158] 2. Add 200ul blocking solution to each well, incubate at room temperature for 2 hours, and wash twice with PBST

[0159] 3. 0, 0.5, 5.0, 50ng / ml PG80-C antibody, add 100ul to the reaction well, react at room temperature for 60 minutes

[0160] 4. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0161] 5. Dilute anti-goat IgG antibody-HRP at 1:3000, add 100ul to the reaction well, room temperature, 60 minutes

[0162] 6. Wash the reaction wells 4 times with PBST, 5 minutes each time

[0163] 7. Add 100ul HRP substrate, TMB Substrate buffer, react for 10 minutes

[0164] 8. Add 50ul 2N H2SO4 to terminate the reaction

[0165] 9. Use an enzyme-linked reaction analyzer and select a wavelength of 450nm to measure the OD value.

[0166] PG80-C Antibody 0 0.5 5 50ng / ml PG80 peptide 0.078 0.264 1.586 4.128 OD value 0.093 0.318 1.478 3.642 0.085 0.275 1.583 3.875 Average 0.085333 0.285667 1.549 3.881667 BSA 0.085 0.095 0.136 0.138 OD value 0.087 0.05 0.125 0.142 0.079 0.095 0.114 0.135 Average 0.083667 0.098333 0.125 0.138333

[0167] 3. Preparation of CLIA PG80 kit

[0168] The CLIA detection PG80 kit mainly contains PG80 calibrator, PG80 quality control, reagent R1, reagent R2 and reagent R3.

[0169] (A) Acridinium ester (AE) labeled PG80-N peptide antibody

[0170] 1. Dilute PG80-N antibody to 1.0 mg / ml in PBS, pH 7.4

[0171] 2. Take 1.0 mg of Acridinium NHS ester purchased from Cayman Chemical and dissolve it in 250 ul of DMF to obtain an Acridinium NHS ester concentration of 4 mg / ml

[0172] 3. Mix 2.0ml of the above PG80-N antibody solution and 80ul of the above Acridinium NHS ester solution, and react at room temperature for 30 minutes.

[0173] 4. Add 80ul 2.0M Glycine to the above reaction, let it act for 15 minutes at room temperature, and terminate the reaction.

[0174] 5. Use Zeba centrifugal desalting columns, purchased from ThermoScientific, to remove free Acridinium NHS ester as follows:

[0175] A. Take 5 ml of Zeba centrifugal desalting column, remove the bottom cover of the Zeba column, and place it in a 15 ml centrifuge tube

[0176] B. Centrifuge, 1000g, 2 minutes, remove the liquid in the 15ml centrifuge tube

[0177] C. Add 2.5 ml ddH2O to the Zeba column

[0178] D. Centrifuge, 1000g, 2 minutes, remove the liquid in the 15ml centrifuge tube

[0179] E. Repeat CD steps 2 or more times

[0180] F. Place the Zeba column into a new 15 ml centrifuge tube

[0181] G. Add the reaction liquid from step 4 above to the Zeba column and let it sit for 2 minutes

[0182] H. Centrifuge at 1000 g for 2 minutes and collect the liquid in the centrifuge tube for the purified AE-PG80-N antibody

[0183] I. Determine the AE-PG80-N antibody concentration.

[0184] 6. Dilute the AE-PG80-N antibody to a concentration of 50 ng / ml with the working solution, which is reagent R1. Store it at 2-8°C for future use.

[0185] Working solution: 20 mM NaHPO4, pH 7.0, 150 mM NaCl, 1.0% BSA, 1.0% Trehalose, 0.02% Tween-20, 0.03% ProClin300.

[0186] (II) Biotin labeled PG80-C antibody

[0187] 1. Take 1.0 mg of EZ-Link Sulfo-NHS-LC-Biotin, purchased from ThermoFisher Scientific, and dissolve it in 180 ul of ddH2O to obtain a Sulfo-NHS-LC-Biotin concentration of 10 mM

[0188] 2. Mix 2.0ml 1.0mg / ml PG80-C antibody and 30ul 10mM Sulfo-NHS-LC-Biotin, react at room temperature for 30 minutes

[0189] 3. Add 40ul 2.0M Glycine, and allow to react for 20 minutes at room temperature to terminate the reaction.

[0190] 4. Use Zeba centrifugal desalting column to remove free Sulfo-NHS-LC-Biotin

[0191] 5. Obtain purified Biotin-PG80-C antibody and determine the concentration of Biotin-PG80-C antibody

[0192] 6. Use working solution to dilute Biotin-PG80-C antibody to a concentration of 40 ng / ml, which becomes reagent R2, and store at 2-8°C for future use.

[0193] (III) Preparation of SMP solution

[0194] Streptavidin Coated Magnetic Particles (SMP), with a diameter of 3.0-3.9 μm, were purchased from Spherotech. Part of the SMP was taken out and placed in a centrifuge tube. The SMP was washed three times with PBS, pH 7.4, and the SMP concentration was diluted to 0.25% with working solution to become reagent R3. It was stored at 2-8°C for future use.

[0195] (IV) Preparation of PG80 Calibrators and Quality Controls

[0196] The PG80 calibrator peptide was synthesized, and the amino acid sequence was shown in SEQ ID NO.4, which contained 80 amino acids of human PG80 peptide.

[0197] Use peptide preservation solution to dilute the PG80 calibrator peptide concentration to 0, 1, 2, 4, 8, 16pM / L as PG80 calibrator; use peptide preservation solution to dilute the PG80 calibrator peptide concentration to 2.0pM / L and 8.0pM / L as PG80 quality control, store at 2-8℃ for future use.

[0198] Peptide storage solution: 20 mM NaHPO4, pH 7.0, 150 mM NaCl, 1.0 mM EDTA, 1.0 mM DTT, 10% BSA, 1.0% Trehalose, 0.2% PVP, 0.01% Tween-20, 0.025% ProClin300.

[0199] IV. Establishment of CLIA PG80 concentration testing procedure

[0200] (I) Application steps of CLIA PG80 test kit

[0201] 1. CLIA analyzer automatically adds 20ul sample, 50ul reagent R1, 50ul reagent R2, 25ul reagent R3 to the reaction cup, 37℃, react for 15 minutes

[0202] 2. Formation of AE-PG80-N antibody-PG80-PG80-C antibody-Biotin-SMP complex, the SMP complex is magnetically adsorbed on the bottom surface of the reaction cup

[0203] 3. Free AE-PG80-N antibody, Biotin-PG80-C antibody and other impurities are removed

[0204] 4. Add washing solution, 50mM NaHPO4, pH7.5, 500mM NaCl, 0.1% TritonX-100, 0.02% ProClin300, wash the SMP complex, and remove the washing solution

[0205] 5. Add H2O2 pre-stimulation solution, 1.25% H2O2, 0.1N Nitric acid to dissociate the SMP complex and release the AE-PG80-N antibody

[0206] 6. Add NaOH excitation solution, 0.25N NaOH, to start the luminescence reaction process of the Acridinium-H2O2-NaOH system and measure the relative luminescence unit (RLU) at a wavelength of 430nm.

[0207] (II) Establishment of an automatic procedure for CLIA analyzer to detect PG80 concentration

[0208] The CLIA analyzer automatically adds 20ul PG80 calibrator, 50ul reagent R1, 50ul reagent R2, and 25ul reagent R3 to the reaction cup, reacts at 37°C for 15 minutes, and after washing, the CLIA analyzer automatically measures PG80 RLU. The PG80 calibrator concentration is used as the ordinate and RLU is used as the abscissa to establish a PG80 calibrator concentration standard curve: y = 0.8712x + 0.0147, R 2 =0.9999, see Figure 2 Through this standard curve, a PG80 concentration standard card was obtained, and the value of this card was input into the CLIA analyzer, and an automatic conversion program between RLU and PG80 concentration was established. A CLIA analyzer automatic detection system for PG80 concentration was established to further determine the PG80 concentration in the sample.

[0209] PG80 0 1 2 4 8 16pM / L RLU 0.03 1.12 2.35 4.48 9.38 17.78 0.02 1.08 2.31 4.62 9.24 18.67 0.04 1.05 2.24 4.35 9.18 18.52 Average 0.03 1.08333 2.3 4.483333 9.266667 18.32333

[0210] (III) Determination of the repeatability and accuracy of the CLIA PG80 test kit

[0211] Using 2.0pM / L PG80 quality control products and 8.0pM / L PG80 quality control products as samples, the PG80 quality control product samples were repeatedly measured 20 times using the CLIA PG80 detection kit, and the mean (M), standard deviation (SD), coefficient of variation (CV) = SD / Mx100%, relative deviation (Bias) = ​​(1-M / sample concentration) x 100% were calculated respectively. The results are shown in the table below.

[0212] PG80 quality control product 2pM / L 8pM / L 2.08 7.87 2.12 8.18 1.95 8.15 1.82 7.74 2.08 7.98 1.97 8.12 1.93 7.84 1.88 7.95 1.85 8.11 1.84 8.05 1.92 7.71 2.08 7.85 2.03 7.72 1.94 7.88 1.85 7.82 2.12 8.18 2.18 7.85 1.95 7.85 1.82 7.93 2.07 7.84 M 1.974 7.931 SD 0.113 0.152 CV 5.72％ 1.90％ Bias 1.30％ 0.86％

[0213] The CV of the 2pM / L PG80 quality control product was 5.72%, and the Bias was 1.30%; the CV of the 8pM / L PG80 quality control product was 1.90%, and the Bias was 0.86%, indicating that this CLIA PG80 detection kit has good repeatability and accuracy.

[0214] (IV) Detection of PG80 concentration in plasma using CLIA kit

[0215] The CLIA analyzer automatically adds 20ul plasma, 50ul reagent R1, 50ul reagent R2, and 25ul reagent R3 to the reaction cup, reacts at 37°C for 15 minutes, and after washing, the CLIA analyzer automatically detects the PG80 concentration in the plasma. The results are as follows:

[0216] Plasma samples 1 2 3 4 5 6 7 8 9 10 PG80 concentration 1.6pM / L 0pM / L 0.87pM / L 2.48pM / L 0pM / L 6.78pM / L 3.68pM / L 1.85pM / L 8.54pM / L 5.42pM / L Plasma samples 11 12 13 14 15 16 17 18 19 20 PG80 concentration 9.76pM / L 1.68pM / L 0.50pM / L 3.84pM / L 2.65pM / L 0pM / L 6.28pM / L 2.84pM / L 9.84pM / L 2.36pM / L

Claims

1. A group of polypeptides for preparing PG80 antibodies, characterized in that: The group of polypeptides for preparing PG80 antibodies includes the following: PG80-N antigen epitope peptide, the amino acids of which are shown in SEQ ID NO.1; PG80-C antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID NO.2; PG80-N cross-linked peptide, the amino acid sequence of which is shown in SEQ ID NO.3; The amino acid sequence of the PG80-C cross-linked peptide is shown in SEQ ID NO.

4.

2. A method for preparing an antibody that specifically binds to PG80, characterized in that: The following steps are involved: (a) coupling the PG80-N antigen epitope peptide and the PG80-C antigen epitope peptide described in claim 1 to a carrier protein to form a PG80-N-carrier protein antigen and a PG80-C-carrier protein antigen; (b) immunizing an animal with the PG80-N-carrier protein antigen and the PG80-C-carrier protein antigen described in step (a) to produce PG80-N and PG80-C monoclonal antibodies or polyclonal antibodies; (c) connecting the PG80-N cross-linked peptide and the PG80-C cross-linked peptide described in claim 1 to a sulfonated coupling resin to prepare PG80-N-resin and PG80-C-resin, respectively; (d) using the PG80-N-resin and PG80-C-resin described in step (c) to separate and purify the PG80-N antibody and the PG80-C antibody, respectively.

3. The method according to claim 2, characterized in that The carrier protein in step (a) is selected from bovine serum albumin (BSA), kallikrein (KLH), ovalbumin (OVA) and blue carrier protein (BCP).

4. The method according to claim 2, characterized in that: The immunized animal in step (b) is selected from mice, rabbits, goats, sheep, alpacas, donkeys and horses.

5. A kit for detecting PG80 by chemiluminescent immunoassay (CLIA), characterized in that: Contains the following ingredients: A. PG80 calibrator, the PG80 calibrator peptide was diluted with peptide storage solution to a concentration of 0-16 pM / L as the PG80 calibrator; the amino acid sequence of the PG80 calibrator peptide is shown in SEQ ID NO.5; B.PG80 quality control: dilute the PG80 calibrator peptide to 1-4pM / L or 8-16pM / L with peptide storage solution as PG80 quality control; C. Reagent R1, i.e., chemiluminescent-PG80-N antibody reaction solution; D. Reagent R2, i.e. Biotin-PG80-C antibody reaction solution; E. Reagent R3, i.e., streptavidin-coated magnetic microparticle reaction solution; The PG80-N antibody and the PG80-C antibody are prepared by the preparation method described in claim 2.

6. Use of the PG80 antibody prepared by the method according to any one of claims 2 to 4 in preparing a kit for early screening of various cancers, determining cancer progression or recurrence, and evaluating cancer treatment effects.

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