WO2019184249A1 - Direct chemiluminescence kit for detecting mechano-growth factor and e peptide thereof, and preparation method therefor - Google Patents

Direct chemiluminescence kit for detecting mechano-growth factor and e peptide thereof, and preparation method therefor Download PDF

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Publication number
WO2019184249A1
WO2019184249A1 PCT/CN2018/104107 CN2018104107W WO2019184249A1 WO 2019184249 A1 WO2019184249 A1 WO 2019184249A1 CN 2018104107 W CN2018104107 W CN 2018104107W WO 2019184249 A1 WO2019184249 A1 WO 2019184249A1
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Prior art keywords
mgf
antibody
solution
ct24e
labeled
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PCT/CN2018/104107
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French (fr)
Chinese (zh)
Inventor
李大军
蔡淑娟
徐霞飞
涂策
张金林
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苏州长光华医生物医学工程有限公司
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Publication of WO2019184249A1 publication Critical patent/WO2019184249A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • G01N33/54326—Magnetic particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
    • G01N21/76—Chemiluminescence; Bioluminescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G01N2333/65—Insulin-like growth factors (Somatomedins), e.g. IGF-1, IGF-2

Definitions

  • the present application relates to a direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof, and a preparation method thereof, and belongs to the technical field of immunoassay.
  • Force growth factor was first discovered in stretch-stimulated skeletal muscle and is an important allosteric variant produced by the selective splicing of insulin-like growth factor-I (IGF-I) gene under the induction of force stimulation. Splicing occurs at the site before exon 6 of the IGF-I gene, whereas in the region of exon 5, MGF inserts 49 bp and 52 bp, respectively, in humans and rodents, resulting in a frameshift mutation in translation of exon 6. A specific C-terminal 24-amino acid E peptide sequence (MGF-Ct24E) was generated.
  • MGF and MGF-Ct24E can activate muscle satellite cells, promote tissue regeneration, promote osteoblast proliferation, and play a role in stress stimulation and neuronal and muscle tissue responses to injury in osteoblasts or bone tissue, in response to mature motor neurons. Maintenance also plays an important role in promoting the proliferation and survival of a variety of cells. Other studies have found that MGF and MGF-Ct24 help to improve memory.
  • MGF can be modified by chemical modification of the C-terminus and/or N-terminus, such as PEGylation, LD-type amino acid conversion, glycosylation, sulfation, amidation, acetylation, etc., to increase the stability of MGF.
  • the modified MGF and MGF-Ct24E can be formulated into pharmaceutical preparations, such as aqueous and non-aqueous sterile injectable solutions, aqueous and non-aqueous sterile suspensions, which can be administered by skin, parenteral, intramuscular, subcutaneous or transdermal.
  • the drug is administered by direct injection into the blood or directly to the mucosal tissue for treating various diseases such as skeletal muscle disorders, myocardial disorders, and neurological disorders.
  • MGF and MGF-Ct24E can be applied to the field of tissue engineering, and bioactive biomimetic materials can be prepared by linking with modified polylactic acid materials in a suitable manner for bone regeneration and repair, vascular regeneration and the like.
  • MGF and MGF-Ct24E can be loaded onto the surface of the magnesium alloy in a suitable manner, and implanted in the body to release in time to stimulate the growth of osteoblasts to overcome the problems caused by insufficient stress.
  • MGF and MGF-Ct24E are so extensive. How to detect the presence of MGF and MGF-Ct24E when bone cells or bone tissue are damaged by stress or nerve or muscle tissue damage? How to monitor the metabolism of the preparation in the body to facilitate treatment/give
  • the drug program how to monitor the release rate of MGF and MGF-Ct24E in biomimetic materials, is an urgent problem to be solved.
  • only Western blotting is used to detect MGF and MGF-Ct24E, and the Western blotting detection is only qualitative analysis, which is not quantitative, and the speed is slow.
  • the technical problem to be solved by the present application is: to solve the technical problem of the method or the kit for the rapid and accurate quantitative detection of MGF and MGF-Ct24E in the prior art, and to provide a direct detection of the force growth factor and the E peptide thereof.
  • Chemiluminescence kit, method of preparation is: to solve the technical problem of the method or the kit for the rapid and accurate quantitative detection of MGF and MGF-Ct24E in the prior art, and to provide a direct detection of the force growth factor and the E peptide thereof.
  • a direct chemiluminescence kit for detecting a force growth factor and an E-peptide thereof comprising: streptavidin-coupled magnetic particle working solution, biotin-labeled MGF and/or MGF-Ct24E antibody working solution, chemiluminescent label Labeled MGF and / or MGF-Ct24E antibody working solution, chemiluminescent substrate solution, cleaning solution.
  • the MGF and/or MGF-Ct24E antibody is a monoclonal antibody, a polyclonal antibody, a monoclonal antibody Fab fragment or a polyclonal antibody Fab fragment, preferably derived from a mouse, rabbit, or sheep.
  • the chemiluminescent label is an acridinium ester, an acridinium ester derivative, an acridinesulfonamide, an isoluminol or an isoluminol derivative
  • the acridinium ester derivative is an acridinium ester- NHS ester, acridinium propane sulfonate or 9-acridine carboxylic acid
  • the isoluminol derivative is isoluminol (4-aminophthalic hydrazide), N-(4-amino Butyl)-N-ethylisoluminol, N-(6-aminohexyl)-N-ethylisoluminol, isoluminol isothiocyanate or N-(4-isothiocyanato Butyl)-N-ethylisoluminol.
  • the streptavidin-coupled magnetic microparticle working solution comprises a phosphate buffer solution having a pH of 6.8 to 7.6 and a concentration of 0.01 mol/L to 0.2 mol/L, a Tris buffer solution, a HEPES buffer solution, and a MOPSO.
  • buffers containing 0.05 to 2 wt% of bovine serum albumin and/or casein, 0.02 to 1 wt% of dodecyl polyethylene glycol ether (Brij35) and/or Polyethylene glycol p-isooctylphenyl ether (Triton X-100) and / or polyoxyethylene sorbitan monolaurate (Tween 20) and / or lauryl polyoxyethylene ether (flat plus O-20 ), 0.05 to 0.5 wt% of proclin 300 and/or sodium azide.
  • Brij35 dodecyl polyethylene glycol ether
  • Triton X-100 Polyethylene glycol p-isooctylphenyl ether
  • Tween 20 polyoxyethylene sorbitan monolaurate
  • lauryl polyoxyethylene ether flat plus O-20
  • the biotin-labeled MGF and/or MGF-Ct24E antibody and the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody working solution have a pH of 6.0-7.6 and a concentration of 0.01 mol/L to 0.2 mol.
  • Murine IgG 0.5 to 5 wt% sodium chloride, 1 to 5 wt% sucrose, 0.05 to 2 wt% calf serum, 0.02 to 1 wt% casein, 0.02 to 1 wt% Brij35 and/or Triton X-100 and / or Tween 20 and / or flat plus O-20, 0.05 ⁇ 0.5% by weight of proclin 300 and / or sodium azide.
  • the direct chemiluminescence kit for detecting a force growth factor and/or its E peptide further comprises an MGF and/or MGF-Ct24E calibrator working solution and/or a control working fluid, the MGF and/or MGF -Ct24E calibrator and / or control working fluid from a pH of 6.0 ⁇ 7.6, a concentration of 0.01mol / L ⁇ 0.2mol / L of phosphate buffer, Tris buffer, HEPES buffer, MES buffer
  • the buffer comprises 0.5-5 wt% of bovine serum albumin, 10-50 wt% of human serum, 0.5-3 wt% of sodium chloride, 2-20 wt% of ethylene glycol, 0.02-1 wt. % Brij35 and/or Triton X-100 and/or Tween 20 and/or plain plus O-20, 0.05-0.5 wt% proclin 300 and/or sodium azide.
  • the cleaning solution is a phosphate buffer solution or a Tris buffer solution having a pH of 6.8 to 7.6 and a concentration of 0.1 mol/L to 0.5 mol/L, and the buffer solution contains 0.2 to 10% by weight of Triton X-100 and / or Tween 20, 5-20% by weight of sodium chloride, 0.5 to 15% by weight of proclin 300 and / or sodium azide.
  • the chemiluminescent substrate liquid comprises a substrate liquid 1 and a substrate liquid 2, the substrate liquid 1 containing 0.1 to 1.5% by weight of dimethylformamide and/or ethanol, 0.1 to 1.5% by weight.
  • Triton X-100 and/or Tween 20 0.05 to 5 wt% of hydrogen peroxide and 1 mmol/L to 5 mmol/L of EDTA or a salt thereof;
  • the substrate 2 contains 0.1 to 1.5 wt% of dimethylformamide and/or Ethanol, 0.1-1.5 wt% Triton X-100 and/or Tween 20, 0.05-0.5 wt% proclin 300, 0.01 mol/L-1 mol/L sodium hydroxide, 1 mmol/L-5 mmol/L EDTA or Its salt.
  • the present application also provides a method for preparing a direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof, comprising: preparation of a streptavidin-coupled magnetic particle working solution, biotin-labeled MGF and/or Preparation of MGF-Ct24E antibody working solution, preparation of chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody working solution, preparation of cleaning solution, preparation of chemiluminescent substrate solution, dispensing of the above prepared reagent and Assembly.
  • the biotin-labeled MGF and/or MGF-Ct24E antibody is prepared by reacting N-hydroxysuccinimide-activated biotin with an antibody at a molar ratio of 1:10-100 at 2-8 °C. Or spin-mix the reaction for 0.5-2 hours at room temperature, and dialysis to remove excess biotin to obtain biotin-labeled MGF and/or MGF-Ct24E antibody.
  • the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody is prepared by: a chemiluminescent label The mixture is reacted with the antibody at a molar ratio of 1:10-80 at 2-8 ° C or room temperature for 0.5-2 hours, and the excess chemiluminescent label is removed by dialysis to obtain a chemiluminescent label labeled MGF and/or MGF-. Ct24E antibody.
  • the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody is prepared by adding solid EDC to the antibody in fractions.
  • solid EDC solid EDC
  • N-(4-aminobutyl)-N-ethylisoluminol (ABEI) solution was added dropwise, so that the molar ratio of antibody:ABEI:EDC was 1:30-70:250-350.
  • the present application provides a direct chemiluminescence kit for detecting a force growth factor and an E-peptide thereof, and a preparation method.
  • the luminescence system of the prepared kit is direct chemiluminescence, and the streptavidin-biotin signal amplification system is used for detection.
  • High sensitivity, wide linear range, and good repeatability of test results can accurately quantify force growth factors and / or its E peptide; especially the kit for fully automated chemiluminescence systems, loading, incubation, cleaning and detection
  • the steps can be automated, avoiding the deviation of results caused by human operation, improving the work efficiency.
  • the test sample can be used to quantitatively detect the force growth factor and/or the force growth factor E in the sample. Peptides make detection faster, more reliable, and more stable.
  • the streptavidin-coupled magnetic particle suspension was magnetically separated to remove the supernatant, and resuspended to a concentration of 0.5 mg/mL with a pH of 6.8 and a concentration of 0.1 mol/L in Tris buffer, the buffer containing 0.5 wt% bovine serum albumin, 0.05 wt% Triton X-100, 0.05% proclin 300 and 0.05% sodium azide.
  • NHS-Biotin N-hydroxysuccinimide-activated biotin
  • the NHS-Biotin aqueous solution was added to the antibody, and the NHS-Biotin and the antibody were spin-mixed at a molar ratio of 1:50 at 2-8 ° C for 1 hour to obtain a biotin-labeled MGF antibody solution;
  • the biotin-labeled MGF antibody solution is transferred to a dialysis bag for dialysis, and the dialysate is changed every 3-4 hours, and replaced 3-4 times.
  • the biotin-labeled MGF antibody is collected into a centrifuge tube or a cryotube, and added. 1/10 volume of PBS-BSA buffer (0.01M, pH 7.0-7.6) containing 5wt% BSA, and then add glycerin after mixing.
  • the volume of glycerol added is the volume of the collected labeled antibody plus the volume of PBS-BSA buffer. After mixing, put it at ⁇ -15 °C for storage.
  • the acridinium ester solution is added to the antibody, and the acridinium ester and the antibody are spin-mixed at a molar ratio of 1:50 at 2-8 ° C for 1 hour to obtain an acridinium ester-labeled MGF antibody solution;
  • acridinium ester-labeled MGF antibody solution to a dialysis bag for dialysis, change the dialysate every 3-4 hours, replace 3-4 times, and collect the acridinium ester-labeled MGF antibody into the centrifuge tube or cryotube after dialysis.
  • the volume of glycerol added is the volume of the collected labeled antibody plus PBS-BSA buffer. Volume, mix and put at ⁇ -15 ° C for storage.
  • a phosphate buffer solution having a pH of 6.0 and a concentration of 0.05 mol/L was prepared, and the buffer contained 3 wt% of bovine serum albumin, 0.5 wt% of casein, 50 mg/L of mouse IgG, and 0.5 wt% of chlorination.
  • the solution, and the antibody concentration was 0.25 mg/L acridinium ester labeled MGF antibody working solution.
  • a buffer of N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) having a pH of 7.4 and a concentration of 0.1 mol/L was prepared, and the buffer contained 3 wt% of bovine serum.
  • MGF calibrator and substance Control product working fluid were used to prepare MGF calibrator and substance Control product working fluid, calibrator a total of 6 points, MGF concentration of 0pmol / L, 0.8pmol / L, 4pmol / L, 16pmol / L, 80pmol / L, 200pmol / L, quality control products are divided into high, medium and low three
  • concentration of MGF was 4 pmol/L, 16 pmol/L, and 80 pmol/L, respectively.
  • a phosphate buffer solution having a pH of 7.4 and a concentration of 0.1 mol/L was prepared, and the buffer contained 10% by weight of Tween 20, 10% by weight of sodium chloride, and 10% by weight of proclin 300.
  • the substrate solution 1 was prepared to contain 1 wt% of dimethylformamide, 1 wt% of Tween 20, and 3 mmol/L.
  • the substrate solution 2 was prepared to contain 1 wt% of dimethylformamide, 0.5 mol/L of sodium hydroxide, 0.2 wt% of proclin 300, 1 wt% of Tween 20, and 2 mmol/L of EDTA disodium salt.
  • the streptavidin-coupled magnetic particle suspension was magnetically separated to remove the supernatant, and resuspended to a concentration of 1 mg/mL with a pH of 6.8, a concentration of 0.2 mol/L in HEPES buffer, and the buffer contained 2 wt. % bovine serum albumin, 0.02 wt% Triton X-100, 0.05% sodium azide.
  • NHS-Biotin N-hydroxysuccinimide-activated biotin
  • the NHS-Biotin aqueous solution was added to the antibody, and the NHS-Biotin and the antibody were spin-mixed at a molar ratio of 1:100 at 2-8 ° C for 2 hours to obtain a biotin-labeled MGF antibody solution;
  • the biotin-labeled MGF antibody solution is transferred to a dialysis bag for dialysis, and the dialysate is changed every 3-4 hours, and replaced 3-4 times.
  • the biotin-labeled MGF antibody is collected into a centrifuge tube or a cryotube, and added. 1/10 volume of PBS-BSA buffer (0.01M, pH 7.0-7.6) containing 5wt% BSA, and then add glycerin after mixing.
  • the volume of glycerol added is the volume of the collected labeled antibody plus the volume of PBS-BSA buffer. After mixing, put it at ⁇ -15 °C for storage.
  • the acridinium ester solution is added to the antibody, and the acridinium ester and the antibody are spin-mixed at a molar ratio of 1:80 at 2-8 ° C for 1 hour to obtain an acridinium ester-labeled MGF antibody solution;
  • acridinium ester-labeled MGF antibody solution to a dialysis bag for dialysis, change the dialysate every 3-4 hours, replace 3-4 times, and collect the acridinium ester-labeled MGF antibody into the centrifuge tube or cryotube after dialysis.
  • the volume of glycerol added is the volume of the collected labeled antibody plus PBS-BSA buffer. Volume, mix and put at ⁇ -15 ° C for storage.
  • a phosphate buffer solution having a pH of 6.0 and a concentration of 0.2 mol/L was prepared, and the buffer contained 5 wt% of bovine serum albumin, 0.0.02 wt% of casein, 1 mg/L of mouse IgG, and 5 wt% of chlorination.
  • a buffer of N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) having a pH of 7.6 and a concentration of 0.2 mol/L was prepared, and the buffer contained 5 wt% of bovine serum.
  • MGF-Ct24E 10% by weight of human serum, 0.5% by weight of sodium chloride, 20% by weight of ethylene glycol, 0.02% by weight of Tween-20, 0.5% by weight of proclin 300; then the buffer was used to prepare MGF-Ct24E calibrator And the quality control working fluid, calibrator working fluid a total of 6 points, MGF-Ct24E concentration of 0pmol / L, 0.8pmol / L, 4pmol / L, 16pmol / L, 80pmol / L, 200pmol / L, quality control The concentration of MGF-Ct24E was 4 pmol/L, 16 pmol/L and 80 pmol/L, respectively.
  • a phosphate buffer solution having a pH of 7.6 and a concentration of 0.2 mol/L was prepared, and the buffer contained 10% by weight of Tween 20, 5% by weight of sodium chloride, and 15% by weight of proclin 300.
  • the substrate solution 1 was prepared to contain 1.5 wt% of dimethylformamide, 1.5 wt% of Tween 20, 1 mmol/L of EDTA disodium salt, and 5 wt% of hydrogen peroxide, and stored in the dark;
  • the substrate solution 2 was prepared to contain 1.5% by weight of dimethylformamide, 1 mol/L of sodium hydroxide, 0.5% by weight of proclin 300, 1.5% by weight of Tween 20, and 5 mmol/L of EDTA disodium salt.
  • the streptavidin-coupled magnetic particle suspension was magnetically separated to remove the supernatant, and resuspended to a concentration of 0.1 mg/mL with a phosphate buffer having a pH of 7.6 and a concentration of 0.01 mol/L. Containing 0.05% by weight of casein protein, 1% by weight of Brij35, 0.5% proclin 300.
  • NHS-Biotin N-hydroxysuccinimide-activated biotin
  • the NHS-Biotin aqueous solution was added to the MGF-Ct24E goat monoclonal antibody, and the NHS-Biotin and MGF-Ct24E goat monoclonal antibody were vortexed and mixed at a molar ratio of 1:10 at room temperature for 0.5 hour to obtain biotin-labeled MGF-Ct24E antibody solution;
  • the solid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to the antibody solution in portions, and N-dissolved with 0.1 mol/L HCL was added dropwise.
  • EDC solid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
  • ABEI (4-aminobutyl)-N-ethylisoluminol
  • the antibody: ABEI: EDC molar ratio is 1:50:300
  • the pH is controlled between 3.5-4.0
  • the reaction is carried out at room temperature for 4.5 hours.
  • the reaction was stopped with a 1.0 mol/L acetate solution, and the reaction solution was dialyzed against a 1 mmol/L HCl solution for 48 hours, and then the free ABEI was removed by a dextran G-25 column equilibrated with 1 mmol/L HCl solution, and collected stepwise. Identification quality, after the required part of the package, ⁇ -15 ° C for storage.
  • a Tris buffer having a pH of 7.6 and a concentration of 0.01 mol/L was prepared, and the buffer contained 0.5 wt% of bovine serum albumin, 100 mg/L of mouse IgG, 0.5 wt% of sodium chloride, and 5% by weight of sucrose. 0.05 wt% of calf serum, 0.02 wt% of flattened plus O-20, 0.5 wt% of sodium azide; then, the buffer was used to prepare a biotin-labeled MGF-Ct24E antibody working solution at a concentration of 0.5 mg/L, and ABEI-labeled MGF-Ct24E antibody working solution at a concentration of 0.25 mg/L.
  • a MES buffer having a pH of 6.8 and a concentration of 0.01 mol/L was prepared.
  • the buffer contained 0.5 wt% of bovine serum albumin, 1 wt% of casein, 50 wt% of human serum, 3 wt% of sodium chloride, 2 wt.
  • MGF calibrator and quality control working fluid % ethylene glycol, 1wt% Triton X-100, 0.05wt% proclin 300; then use this buffer to prepare MGF calibrator and quality control working fluid, calibrator working solution a total of 6 points, MGF concentration
  • concentration of MGF was 4pmol/L, 16pmol/L and 80pmol, respectively, at 0pmol/L, 0.8pmol/L, 4pmol/L, 16pmol/L, 80pmol/L, and 200pmol/L. /L.
  • a phosphate buffer solution having a pH of 6.8 and a concentration of 0.5 mol/L was prepared, and the buffer contained 0.2 wt% of Triton X-100, 20 wt% of sodium chloride, and 0.5 wt% of sodium azide.
  • the substrate solution 1 was prepared to contain 0.1 wt% ethanol, 0.1 wt% Triton X-100, 5 mmol/L EDTA, 0.05 wt% hydrogen peroxide, and stored in the dark;
  • the substrate liquid 2 was prepared to contain 0.1 wt% of ethanol, 0.01 mol/L of sodium hydroxide, 0.05 wt% of proclin 300, 0.1 wt% of Triton X-100, and 1 mmol/L of EDTA.
  • Example 4 Method for Detecting MGF and/or MGF-Ct24E Using the Kit of the Present Application
  • AE-180 fully automated chemiluminescence immunoassay analyzer
  • the magnetic composite suspension is placed in a magnetic field, and the cleaning solution is diluted 10-30 times, the magnetic composite is washed;
  • the substrate solution 1 and the substrate solution 2 were successively injected into the washed magnetic composite to measure the photon intensity.
  • the S0 calibrator (0 ng/ml) was repeatedly measured 20 times by the detection method of Example 4, and the RLU values were: 3151, 3125, 3287, 3116, 3064, 3147, 2456, 2410, 2585. , 2552, 2683, 2964, 3333, 3197, 2913, 3291, 3187, 3235, 2533, 3024, RLU average (x) is 2963, standard deviation (s) is 308; repeat the same method to determine S1 calibrator (0.8pmol/L) 2 times, RLU value: 21028, 20015, RLU average value is 20522; According to the calibrator concentration-RLU average between S0 calibrator and S1 calibrator, two-point regression fitting is used to obtain one time. For the equation, the x+2s value (3579) is substituted into the first equation, and the corresponding concentration value, that is, the lowest detection limit, is obtained, and the result is 0.028 pmol/L.
  • the minimum detection limit obtained using the kit of Example 2 was 0.021 pmol/L
  • the minimum detection limit obtained using the kit of Example 3 was 0.035 pmol/L.
  • a high value calibrator of 80 pmol/L was added to a 0.8 pmol/L low value calibrator to recover the sample, the high value calibrator added and the volume of the low value calibrator added.
  • the ratio is 1:9.
  • the high-value calibrator, the low-value calibrator, and the recovered sample are repeatedly detected twice, and the corresponding RLU value is obtained, and the corresponding concentration value and concentration average are calculated.
  • the value, the test results are shown in Table 1, and the recovery rate R was calculated according to the following formula, and the result was 103.2%.
  • V 0 the volume of the low value calibrator
  • V the volume of the high value calibrator
  • the recovery obtained by using the kit of Example 2 was 98.2%, and the recovery obtained by using the kit of Example 3 was 99.5%.
  • the linear correlation coefficient obtained using the kit of Example 2 was 0.9996, and the linear correlation coefficient obtained using the kit of Example 3 was 0.9995.
  • the test solution having the concentrations of 4 pmol/L, 16 pmol/L, and 80 pmol/L was repeatedly measured 10 times each time, and the corresponding RLU value was obtained and calculated.
  • the coefficient of variation corresponding to the working fluid of the three control levels was 4.6%, 2.5%, and 5.8%, respectively, and three concentrations were obtained using the kit of Example 3.
  • the corresponding coefficients of variation for the horizontal control solution were 3.1%, 5.5%, and 2.8%, respectively.

Abstract

Disclosed are a direct chemiluminescence kit for detecting a mechano-growth factor and/or the E peptide thereof, and a preparation method therefor. The preparation method comprises firstly formulating the following components of the kit: a streptavidin-coupled magnetic microparticle working solution, a biotin-labeled MGF and/or MGF-Ct24E antibody working solution, a chemiluminescent marker-labeled MGF and/or MGF-Ct24E antibody working solution, an MGF and MGF-Ct24E calibration product and/or quality control product working solution, a chemiluminescent substrate solution and a cleaning fluid; and then dispensing and assembling each component formulated as described above to obtain the kit.

Description

检测力生长因子、其E肽的直接化学发光试剂盒、制法Direct chemiluminescence kit for detecting force growth factor and its E peptide, and preparation method 技术领域Technical field
本申请涉及一种检测力生长因子、其E肽的直接化学发光试剂盒、制法，属于免疫分析技术领域。The present application relates to a direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof, and a preparation method thereof, and belongs to the technical field of immunoassay.
背景技术Background technique
力生长因子(MGF)最初在拉伸刺激的骨骼肌中被发现，是肌肉在力刺激诱导下，由***-Ⅰ(IGF-Ⅰ)基因选择性剪接产生的重要变构体，选择剪接发生在IGF-Ⅰ基因外显子6之前的位点，而MGF在外显子5这一区域，人类和啮齿动物类分别***了49bp和52bp，导致外显子6的翻译发生移码突变，产生特异的C端24个氨基酸的E肽序列(MGF-Ct24E)。Force growth factor (MGF) was first discovered in stretch-stimulated skeletal muscle and is an important allosteric variant produced by the selective splicing of insulin-like growth factor-I (IGF-I) gene under the induction of force stimulation. Splicing occurs at the site before exon 6 of the IGF-I gene, whereas in the region of exon 5, MGF inserts 49 bp and 52 bp, respectively, in humans and rodents, resulting in a frameshift mutation in translation of exon 6. A specific C-terminal 24-amino acid E peptide sequence (MGF-Ct24E) was generated.
MGF及MGF-Ct24E能够激活肌肉卫星细胞，促进组织再生，促进成骨细胞增殖，在成骨细胞或骨组织对应力刺激以及神经、肌肉组织对损伤的响应中发挥作用，对成熟运动神经元的维持也有着重要作用，具有促进多种细胞增殖和存活的作用。还有研究发现，MGF及MGF-Ct24有助于改善记忆。MGF and MGF-Ct24E can activate muscle satellite cells, promote tissue regeneration, promote osteoblast proliferation, and play a role in stress stimulation and neuronal and muscle tissue responses to injury in osteoblasts or bone tissue, in response to mature motor neurons. Maintenance also plays an important role in promoting the proliferation and survival of a variety of cells. Other studies have found that MGF and MGF-Ct24 help to improve memory.
通过对C-末端和/或N-末端进行化学修饰，如聚乙二醇化、L-D型氨基酸转化、糖基化、硫酸化、酰胺化、乙酰化等方法修饰MGF，可增加MGF的稳定性，修饰后的MGF及MGF-Ct24E可制成药物制剂，如水性和非水性无菌注射液、水性和非水性无菌悬液，制剂可通过皮肤、肠胃外、肌肉、皮下或经皮肤等方式给药，或者通过直接注射到血液中或直接施用至粘膜组织给药，用来治疗骨骼肌障碍、心肌障碍、神经障碍等多种疾病。MGF can be modified by chemical modification of the C-terminus and/or N-terminus, such as PEGylation, LD-type amino acid conversion, glycosylation, sulfation, amidation, acetylation, etc., to increase the stability of MGF. The modified MGF and MGF-Ct24E can be formulated into pharmaceutical preparations, such as aqueous and non-aqueous sterile injectable solutions, aqueous and non-aqueous sterile suspensions, which can be administered by skin, parenteral, intramuscular, subcutaneous or transdermal. The drug is administered by direct injection into the blood or directly to the mucosal tissue for treating various diseases such as skeletal muscle disorders, myocardial disorders, and neurological disorders.
MGF及MGF-Ct24E可以应用到组织工程领域，通过合适的方式与改性的聚乳酸材料连接制备生物活性仿生材料，用于骨再生修复、血管再生修复等。另外，MGF及MGF-Ct24E可以通过合适的方式负载到镁合金表面，植入体内及时释放从而刺激成骨细胞的增长，来克服应力不足引起的问题。MGF and MGF-Ct24E can be applied to the field of tissue engineering, and bioactive biomimetic materials can be prepared by linking with modified polylactic acid materials in a suitable manner for bone regeneration and repair, vascular regeneration and the like. In addition, MGF and MGF-Ct24E can be loaded onto the surface of the magnesium alloy in a suitable manner, and implanted in the body to release in time to stimulate the growth of osteoblasts to overcome the problems caused by insufficient stress.
MGF及MGF-Ct24E的应用如此广泛，如何检测骨细胞或骨组织受到应力刺激或神经、肌肉组织受到损伤时是否有MGF及MGF-Ct24E产生，如何监控制剂在体内代谢程度以便于制定治疗/给药方案，如何监测仿生材料中MGF及MGF-Ct24E的释放速度，这些都是需要迫切解决的问题。然而，现有技术中仅有蛋白质免疫印迹法检测MGF及MGF-Ct24E，而该蛋白质免疫印迹检测只是定 性的分析，不能定量，且速度较慢，目前尚未有快速、准确地定量检测MGF及MGF-Ct24E的方法或试剂盒。The application of MGF and MGF-Ct24E is so extensive. How to detect the presence of MGF and MGF-Ct24E when bone cells or bone tissue are damaged by stress or nerve or muscle tissue damage? How to monitor the metabolism of the preparation in the body to facilitate treatment/give The drug program, how to monitor the release rate of MGF and MGF-Ct24E in biomimetic materials, is an urgent problem to be solved. However, in the prior art, only Western blotting is used to detect MGF and MGF-Ct24E, and the Western blotting detection is only qualitative analysis, which is not quantitative, and the speed is slow. Currently, there is no rapid and accurate quantitative detection of MGF and MGF. -Ct24E method or kit.
申请内容Application content
本申请要解决的技术问题是：为解决现有技术中尚未有快速、准确地定量检测MGF及MGF-Ct24E的方法或试剂盒的技术问题，提供一种检测力生长因子、其E肽的直接化学发光试剂盒、制法。The technical problem to be solved by the present application is: to solve the technical problem of the method or the kit for the rapid and accurate quantitative detection of MGF and MGF-Ct24E in the prior art, and to provide a direct detection of the force growth factor and the E peptide thereof. Chemiluminescence kit, method of preparation.
本申请解决其技术问题所采用的技术方案是：The technical solution adopted by the present application to solve its technical problems is:
一种检测力生长因子、其E肽的直接化学发光试剂盒，包括：链霉亲和素偶联的磁性微粒工作液，生物素标记的MGF和/或MGF-Ct24E抗体工作液，化学发光标记物标记的MGF和/或MGF-Ct24E抗体工作液，化学发光底物液，清洗液。A direct chemiluminescence kit for detecting a force growth factor and an E-peptide thereof, comprising: streptavidin-coupled magnetic particle working solution, biotin-labeled MGF and/or MGF-Ct24E antibody working solution, chemiluminescent label Labeled MGF and / or MGF-Ct24E antibody working solution, chemiluminescent substrate solution, cleaning solution.
优选地，所述MGF和/或MGF-Ct24E抗体为单克隆抗体、多克隆抗体、单克隆抗体Fab片段或多克隆抗体Fab片段，优选来源于鼠、兔、羊。Preferably, the MGF and/or MGF-Ct24E antibody is a monoclonal antibody, a polyclonal antibody, a monoclonal antibody Fab fragment or a polyclonal antibody Fab fragment, preferably derived from a mouse, rabbit, or sheep.
优选地，所述化学发光标记物为吖啶酯、吖啶酯衍生物、吖啶磺酰胺、异鲁米诺或异鲁米诺衍生物，所述吖啶酯衍生物为吖啶酯酸-NHS酯、吖啶酸丙磺酸钠盐或9-吖啶羧酸，所述异鲁米诺衍生物为异鲁米诺(4-氨基邻苯二甲酰肼)、N-(4-氨基丁基)-N-乙基异鲁米诺、N-(6-氨基已基)-N-乙基异鲁米诺、异硫氰酸异鲁米诺或N-(4-异硫氰基丁基)-N-乙基异鲁米诺。Preferably, the chemiluminescent label is an acridinium ester, an acridinium ester derivative, an acridinesulfonamide, an isoluminol or an isoluminol derivative, and the acridinium ester derivative is an acridinium ester- NHS ester, acridinium propane sulfonate or 9-acridine carboxylic acid, the isoluminol derivative is isoluminol (4-aminophthalic hydrazide), N-(4-amino Butyl)-N-ethylisoluminol, N-(6-aminohexyl)-N-ethylisoluminol, isoluminol isothiocyanate or N-(4-isothiocyanato Butyl)-N-ethylisoluminol.
优选地，所述链霉亲和素偶联的磁性微粒工作液由pH为6.8～7.6，浓度为0.01mol/L～0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPSO缓冲液中的一种配制而成，所述缓冲液含有0.05～2wt％的牛血清白蛋白和/或酪蛋白、0.02～1wt％的十二烷基聚乙二醇醚(Brij35)和/或聚乙二醇对异辛基苯基醚(Triton X-100)和/或聚氧乙烯失水山梨醇单月桂酸酯(Tween 20)和/或月桂醇聚氧乙烯醚(平平加O-20)、0.05～0.5wt％的proclin 300和/或叠氮钠。Preferably, the streptavidin-coupled magnetic microparticle working solution comprises a phosphate buffer solution having a pH of 6.8 to 7.6 and a concentration of 0.01 mol/L to 0.2 mol/L, a Tris buffer solution, a HEPES buffer solution, and a MOPSO. One of the buffers containing 0.05 to 2 wt% of bovine serum albumin and/or casein, 0.02 to 1 wt% of dodecyl polyethylene glycol ether (Brij35) and/or Polyethylene glycol p-isooctylphenyl ether (Triton X-100) and / or polyoxyethylene sorbitan monolaurate (Tween 20) and / or lauryl polyoxyethylene ether (flat plus O-20 ), 0.05 to 0.5 wt% of proclin 300 and/or sodium azide.
优选地，所述生物素标记的MGF和/或MGF-Ct24E抗体以及化学发光标记物标记的MGF和/或MGF-Ct24E抗体工作液由pH为6.0～7.6，浓度为0.01mol/L～0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPSO缓冲液中的一种配制而成，所述缓冲液含有0.5～5wt％的牛血清白蛋白、1mg/L～100mg/L的鼠IgG、0.5～5wt％的氯化钠、1～5wt％的蔗糖、0.05～2wt％的小牛血清、0.02～1wt％的酪蛋白、0.02～1wt％的Brij35和/或Triton X-100和/或Tween 20和/或平平加O-20、0.05～0.5wt％的proclin 300和/或叠氮钠。Preferably, the biotin-labeled MGF and/or MGF-Ct24E antibody and the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody working solution have a pH of 6.0-7.6 and a concentration of 0.01 mol/L to 0.2 mol. Prepared by one of /L phosphate buffer, Tris buffer, HEPES buffer, and MOPSO buffer containing 0.5 to 5 wt% of bovine serum albumin and 1 mg/L to 100 mg/L. Murine IgG, 0.5 to 5 wt% sodium chloride, 1 to 5 wt% sucrose, 0.05 to 2 wt% calf serum, 0.02 to 1 wt% casein, 0.02 to 1 wt% Brij35 and/or Triton X-100 and / or Tween 20 and / or flat plus O-20, 0.05 ~ 0.5% by weight of proclin 300 and / or sodium azide.
优选地，所述检测力生长因子和/或其E肽的直接化学发光试剂盒还包括MGF和/或MGF-Ct24E校准品工作液和/或质控品工作液，所述MGF和/或MGF-Ct24E校准品和/或质控品工作液由pH为6.0～7.6，浓度为0.01mol/L～0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MES缓冲液中的一种配制而成，所述缓冲液中含有0.5～5wt％的牛血清白蛋白、10～50wt％的人血清、0.5～3wt％的氯化钠、2～20wt％的乙二醇、0.02～1wt％的Brij35和/或Triton X-100和/或Tween 20和/或平平加O-20、0.05～0.5wt％的proclin 300和/或叠氮钠。Preferably, the direct chemiluminescence kit for detecting a force growth factor and/or its E peptide further comprises an MGF and/or MGF-Ct24E calibrator working solution and/or a control working fluid, the MGF and/or MGF -Ct24E calibrator and / or control working fluid from a pH of 6.0 ~ 7.6, a concentration of 0.01mol / L ~ 0.2mol / L of phosphate buffer, Tris buffer, HEPES buffer, MES buffer The buffer comprises 0.5-5 wt% of bovine serum albumin, 10-50 wt% of human serum, 0.5-3 wt% of sodium chloride, 2-20 wt% of ethylene glycol, 0.02-1 wt. % Brij35 and/or Triton X-100 and/or Tween 20 and/or plain plus O-20, 0.05-0.5 wt% proclin 300 and/or sodium azide.
优选地，所述清洗液为pH为6.8～7.6，浓度为0.1mol/L～0.5mol/L的磷酸盐缓冲液或Tris缓冲液，所述缓冲液含有0.2～10wt％的Triton X-100和/或Tween20、5～20wt％的氯化钠、0.5～15wt％的proclin 300和/或叠氮钠。Preferably, the cleaning solution is a phosphate buffer solution or a Tris buffer solution having a pH of 6.8 to 7.6 and a concentration of 0.1 mol/L to 0.5 mol/L, and the buffer solution contains 0.2 to 10% by weight of Triton X-100 and / or Tween 20, 5-20% by weight of sodium chloride, 0.5 to 15% by weight of proclin 300 and / or sodium azide.
优选地，所述化学发光底物液包括底物液1和底物液2，所述底物液1含有0.1～1.5wt％的二甲基甲酰胺和/或乙醇、0.1～1.5wt％的Triton X-100和/或Tween20、0.05～5wt％的双氧水以及1mmol/L-5mmol/L的EDTA或其盐；所述底物液2含有0.1～1.5wt％的二甲基甲酰胺和/或乙醇、0.1-1.5wt％的Triton X-100和/或Tween 20、0.05～0.5wt％的proclin 300、0.01mol/L-1mol/L的氢氧化钠、1mmol/L-5mmol/L的EDTA或其盐。Preferably, the chemiluminescent substrate liquid comprises a substrate liquid 1 and a substrate liquid 2, the substrate liquid 1 containing 0.1 to 1.5% by weight of dimethylformamide and/or ethanol, 0.1 to 1.5% by weight. Triton X-100 and/or Tween 20, 0.05 to 5 wt% of hydrogen peroxide and 1 mmol/L to 5 mmol/L of EDTA or a salt thereof; the substrate 2 contains 0.1 to 1.5 wt% of dimethylformamide and/or Ethanol, 0.1-1.5 wt% Triton X-100 and/or Tween 20, 0.05-0.5 wt% proclin 300, 0.01 mol/L-1 mol/L sodium hydroxide, 1 mmol/L-5 mmol/L EDTA or Its salt.
本申请还提供一种上述检测力生长因子、其E肽的直接化学发光试剂盒的制法，包括：链霉亲和素偶联的磁性微粒工作液的制备，生物素标记的MGF和/或MGF-Ct24E抗体工作液的制备，化学发光标记物标记的MGF和/或MGF-Ct24E抗体工作液的制备，清洗液的配制，化学发光底物液的配制，将上述制备的试剂进行分装及组装。The present application also provides a method for preparing a direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof, comprising: preparation of a streptavidin-coupled magnetic particle working solution, biotin-labeled MGF and/or Preparation of MGF-Ct24E antibody working solution, preparation of chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody working solution, preparation of cleaning solution, preparation of chemiluminescent substrate solution, dispensing of the above prepared reagent and Assembly.
优选地，所述生物素标记的MGF和/或MGF-Ct24E抗体的制备方法为：将N-羟基琥珀酰亚胺活化的生物素与抗体按照1:10-100的摩尔比在2-8℃或室温条件下旋转混匀反应0.5-2小时，透析除去多余的生物素，得生物素标记的MGF和/或MGF-Ct24E抗体。Preferably, the biotin-labeled MGF and/or MGF-Ct24E antibody is prepared by reacting N-hydroxysuccinimide-activated biotin with an antibody at a molar ratio of 1:10-100 at 2-8 °C. Or spin-mix the reaction for 0.5-2 hours at room temperature, and dialysis to remove excess biotin to obtain biotin-labeled MGF and/or MGF-Ct24E antibody.
优选地，所述化学发光标记物为吖啶酯、吖啶酯衍生物、吖啶磺酰胺时，化学发光标记物标记的MGF和/或MGF-Ct24E抗体的制备方法为：将化学发光标记物与抗体按照1:10-80的摩尔比在2-8℃或室温条件下旋转混匀反应0.5-2小时，透 析除去多余的化学发光标记物，得化学发光标记物标记MGF和/或MGF-Ct24E抗体。Preferably, when the chemiluminescent label is an acridinium ester, an acridinium ester derivative or an acridinesulfonamide, the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody is prepared by: a chemiluminescent label The mixture is reacted with the antibody at a molar ratio of 1:10-80 at 2-8 ° C or room temperature for 0.5-2 hours, and the excess chemiluminescent label is removed by dialysis to obtain a chemiluminescent label labeled MGF and/or MGF-. Ct24E antibody.
优选地，所述化学发光标记物为异鲁米诺、异鲁米诺衍生物时，化学发光标记物标记的MGF和/或MGF-Ct24E抗体的制备方法为：将固体EDC分次加入到抗体溶液中，再逐滴加入N-(4-氨丁基)-N-乙基异鲁米诺(ABEI)溶液，使抗体:ABEI:EDC的摩尔比为1:30-70:250-350，控制反应体系的pH在3.5-4.0之间，室温反应2-6小时，用醋酸盐溶液终止反应，除去多余的化学发光标记物和游离的ABEI，得化学发光标记物标记MGF和/或MGF-Ct24E抗体。Preferably, when the chemiluminescent label is an isoluminol or an isoluminol derivative, the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody is prepared by adding solid EDC to the antibody in fractions. In the solution, N-(4-aminobutyl)-N-ethylisoluminol (ABEI) solution was added dropwise, so that the molar ratio of antibody:ABEI:EDC was 1:30-70:250-350. Control the pH of the reaction system between 3.5 and 4.0, react at room temperature for 2-6 hours, stop the reaction with the acetate solution, remove the excess chemiluminescent label and free ABEI, and obtain the chemiluminescent label labeled MGF and/or MGF. -Ct24E antibody.
本申请的有益效果是：The beneficial effects of the application are:
本申请提供了一种检测力生长因子、其E肽的直接化学发光试剂盒、制法，制备的试剂盒的发光体系为直接化学发光，利用链霉亲和素-生物素信号放大***，检测灵敏度高、线性范围宽，检测结果重复性好，可以实现力生长因子和/或其E肽的准确定量；尤其是将该试剂盒用于全自动化学发光***，加样、孵育、清洗和检测等步骤均可实现自动化，避免了人为操作带来的结果偏差，提高了工作效率，通过内置标准曲线到测试软件，只需测试样本即可定量检测样本中力生长因子和/或力生长因子E肽，使检测更快速、更可靠、更稳定。The present application provides a direct chemiluminescence kit for detecting a force growth factor and an E-peptide thereof, and a preparation method. The luminescence system of the prepared kit is direct chemiluminescence, and the streptavidin-biotin signal amplification system is used for detection. High sensitivity, wide linear range, and good repeatability of test results, can accurately quantify force growth factors and / or its E peptide; especially the kit for fully automated chemiluminescence systems, loading, incubation, cleaning and detection The steps can be automated, avoiding the deviation of results caused by human operation, improving the work efficiency. By using the built-in standard curve to the test software, the test sample can be used to quantitatively detect the force growth factor and/or the force growth factor E in the sample. Peptides make detection faster, more reliable, and more stable.
具体实施方式detailed description
现在对本申请作进一步详细的说明。This application will now be described in further detail.
实施例1检测力生长因子和/或其E肽的化学发光试剂盒的制法Example 1 Method for Producing Chemiluminescence Kit for Detecting Power Growth Factor and/or Its E-peptide
1)链霉亲和素偶联的磁性微粒工作液的制备1) Preparation of streptavidin-coupled magnetic particle working solution
将链霉亲和素偶联的磁微粒悬浮液，磁分离去除上清，用pH为6.8，浓度为0.1mol/L的Tris缓冲液重悬至浓度为0.5mg/mL，所述缓冲液含有0.5wt％的牛血清白蛋白、0.05wt％的Triton X-100、0.05％proclin 300和0.05％叠氮钠。The streptavidin-coupled magnetic particle suspension was magnetically separated to remove the supernatant, and resuspended to a concentration of 0.5 mg/mL with a pH of 6.8 and a concentration of 0.1 mol/L in Tris buffer, the buffer containing 0.5 wt% bovine serum albumin, 0.05 wt% Triton X-100, 0.05% proclin 300 and 0.05% sodium azide.
2)生物素标记的MGF抗体的制备2) Preparation of biotinylated MGF antibody
取1mg MGF鼠单克隆抗体，加入适量PBS缓冲液(0.05M，pH7.0-7.6)调整抗体总浓度至1mg/ml，并加入透析袋中进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后将抗体转移至离心管或冻存管内；Take 1mg MGF mouse monoclonal antibody, add appropriate amount of PBS buffer (0.05M, pH7.0-7.6) to adjust the total antibody concentration to 1mg/ml, and add dialysis bag to dialysis, change dialysate every 3-4 hours , replace 3-4 times, transfer the antibody to the centrifuge tube or cryotube after dialysis;
准确称取1mg N-羟基琥珀酰亚胺活化的生物素(NHS-Biotin)，加入适量水调整其浓度为2mg/ml，得NHS-Biotin水溶液；Accurately weigh 1 mg of N-hydroxysuccinimide-activated biotin (NHS-Biotin), add appropriate amount of water to adjust the concentration to 2 mg / ml, to obtain NHS-Biotin aqueous solution;
将NHS-Biotin水溶液加入抗体中，使NHS-Biotin与抗体按照1:50的摩尔比在 2-8℃旋转混匀反应1小时，得生物素标记的MGF抗体溶液；The NHS-Biotin aqueous solution was added to the antibody, and the NHS-Biotin and the antibody were spin-mixed at a molar ratio of 1:50 at 2-8 ° C for 1 hour to obtain a biotin-labeled MGF antibody solution;
将生物素标记的MGF抗体溶液转移至透析袋进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后收集生物素标记的MGF抗体至离心管或冻存管内，加入1/10体积含5wt％BSA的PBS-BSA缓冲液(0.01M，pH7.0-7.6)，混匀后再加入甘油，所加甘油体积为所收集标记抗体体积加PBS-BSA缓冲液的体积，混匀后置于≤-15℃保存备用。The biotin-labeled MGF antibody solution is transferred to a dialysis bag for dialysis, and the dialysate is changed every 3-4 hours, and replaced 3-4 times. After dialysis, the biotin-labeled MGF antibody is collected into a centrifuge tube or a cryotube, and added. 1/10 volume of PBS-BSA buffer (0.01M, pH 7.0-7.6) containing 5wt% BSA, and then add glycerin after mixing. The volume of glycerol added is the volume of the collected labeled antibody plus the volume of PBS-BSA buffer. After mixing, put it at ≤ -15 °C for storage.
3)吖啶酯标记的MGF抗体的制备3) Preparation of acridinium ester labeled MGF antibody
取1mg MGF鼠单克隆抗体，加入适量CBS缓冲液(0.05M，pH8.0-9.6)调整抗体总浓度至1mg/ml，并加入透析袋中，进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后将抗体溶液转移至离心管或冻存管内；Take 1mg MGF mouse monoclonal antibody, add appropriate CBS buffer (0.05M, pH 8.0-9.6) to adjust the total antibody concentration to 1mg/ml, and add to the dialysis bag for dialysis, change dialysis every 3-4 hours Liquid, replace 3-4 times, transfer the antibody solution to the centrifuge tube or cryotube after dialysis;
准确称取1mg吖啶酯，加入二甲基甲酰胺调整其浓度为2mg/ml，得吖啶酯溶液；Accurately weigh 1 mg of acridinium ester, add dimethylformamide to adjust the concentration to 2 mg / ml, to obtain acridinium ester solution;
将吖啶酯溶液加入抗体中，使吖啶酯与抗体按照1:50的摩尔比在2-8℃下旋转混匀反应1小时，得吖啶酯标记的MGF抗体溶液；The acridinium ester solution is added to the antibody, and the acridinium ester and the antibody are spin-mixed at a molar ratio of 1:50 at 2-8 ° C for 1 hour to obtain an acridinium ester-labeled MGF antibody solution;
将吖啶酯标记的MGF抗体溶液转移至透析袋进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后收集吖啶酯标记MGF抗体至离心管或冻存管内，加入1/10体积含5wt％BSA的PBS-BSA缓冲液(0.01M，pH7.0-7.6)，混匀后再加入甘油，所加甘油体积为所收集标记抗体体积加PBS-BSA缓冲液的体积，混匀后置于≤-15℃保存备用。Transfer the acridinium ester-labeled MGF antibody solution to a dialysis bag for dialysis, change the dialysate every 3-4 hours, replace 3-4 times, and collect the acridinium ester-labeled MGF antibody into the centrifuge tube or cryotube after dialysis. Add 1/10 volume of PBS-BSA buffer (0.01M, pH 7.0-7.6) containing 5wt% BSA, mix and add glycerol. The volume of glycerol added is the volume of the collected labeled antibody plus PBS-BSA buffer. Volume, mix and put at ≤ -15 ° C for storage.
4)生物素标记的MGF抗体工作液以及吖啶酯标记的MGF抗体工作液的制备4) Preparation of biotin-labeled MGF antibody working solution and acridinium ester-labeled MGF antibody working solution
配制pH为6.0，浓度为0.05mol/L的磷酸盐缓冲液，所述缓冲液含有3wt％的牛血清白蛋白、0.5wt％的酪蛋白、50mg/L的鼠IgG、0.5wt％的氯化钠、1wt％的蔗糖、1wt％的小牛血清、0.5wt％的Tween-20、0.05wt％的proclin 300；然后用该缓冲液配制抗体浓度为0.5mg/L的生物素标记的MGF抗体工作液，以及抗体浓度为0.25mg/L吖啶酯标记的MGF抗体工作液。A phosphate buffer solution having a pH of 6.0 and a concentration of 0.05 mol/L was prepared, and the buffer contained 3 wt% of bovine serum albumin, 0.5 wt% of casein, 50 mg/L of mouse IgG, and 0.5 wt% of chlorination. Sodium, 1 wt% sucrose, 1 wt% calf serum, 0.5 wt% Tween-20, 0.05 wt% proclin 300; then the buffer was used to prepare a biotin-labeled MGF antibody with an antibody concentration of 0.5 mg/L. The solution, and the antibody concentration was 0.25 mg/L acridinium ester labeled MGF antibody working solution.
5)校准品和质控品工作液的配制5) Preparation of calibrator and quality control working fluid
配制pH为7.4，浓度为0.1mol/L的N-(2-羟乙基)哌嗪-N′-(2-乙磺酸)(HEPES)缓冲液，所述缓冲液含有3wt％的牛血清白蛋白、10wt％的人血清、0.5wt％的氯化钠、10wt％的乙二醇、0.5wt％的Tween-20、0.2wt％的proclin 300；然后用该缓 冲液配制MGF校准品和质控品工作液，校准品共6个点，MGF的浓度分别为0pmol/L、0.8pmol/L、4pmol/L、16pmol/L、80pmol/L、200pmol/L，质控品分高中低三个浓度，MGF的浓度分别为4pmol/L、16pmol/L、80pmol/L。A buffer of N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) having a pH of 7.4 and a concentration of 0.1 mol/L was prepared, and the buffer contained 3 wt% of bovine serum. Albumin, 10% by weight of human serum, 0.5% by weight of sodium chloride, 10% by weight of ethylene glycol, 0.5% by weight of Tween-20, 0.2% by weight of proclin 300; then the buffer was used to prepare MGF calibrator and substance Control product working fluid, calibrator a total of 6 points, MGF concentration of 0pmol / L, 0.8pmol / L, 4pmol / L, 16pmol / L, 80pmol / L, 200pmol / L, quality control products are divided into high, medium and low three The concentration of MGF was 4 pmol/L, 16 pmol/L, and 80 pmol/L, respectively.
6)清洗液的配制6) Preparation of cleaning solution
配制pH为7.4，浓度为0.1mol/L的磷酸盐缓冲液，所述缓冲液含有10wt％的Tween 20、10wt％的氯化钠、10wt％的proclin 300。A phosphate buffer solution having a pH of 7.4 and a concentration of 0.1 mol/L was prepared, and the buffer contained 10% by weight of Tween 20, 10% by weight of sodium chloride, and 10% by weight of proclin 300.
7)化学发光底物液的配制7) Preparation of chemiluminescent substrate solution
配制底物液1，使其含有1wt％的二甲基甲酰胺、1wt％的Tween 20、3mmol/LThe substrate solution 1 was prepared to contain 1 wt% of dimethylformamide, 1 wt% of Tween 20, and 3 mmol/L.
的EDTA二钠盐、1.5wt％双氧水，避光保存；EDTA disodium salt, 1.5wt% hydrogen peroxide, stored in the dark;
配制底物液2，使其含有1wt％的二甲基甲酰胺、0.5mol/L的氢氧化钠、0.2wt％的proclin 300、1wt％的Tween 20、2mmol/L的EDTA二钠盐。The substrate solution 2 was prepared to contain 1 wt% of dimethylformamide, 0.5 mol/L of sodium hydroxide, 0.2 wt% of proclin 300, 1 wt% of Tween 20, and 2 mmol/L of EDTA disodium salt.
8)试剂分装及组装，将链霉亲和素偶联的磁性微粒工作液3.0ml/瓶、生物素标记的MGF抗体工作液12ml/瓶、吖啶酯标记的MGF抗体工作液12ml/瓶、校准品/质控品工作液1.0ml/瓶分装后，组装在一起，保存于2-8℃，将清洗液500ml/瓶、化学发光底物液500ml/瓶单独包装，保存于20℃-25℃。8) Reagent packaging and assembly, streptavidin-coupled magnetic particle working solution 3.0ml/bottle, biotin-labeled MGF antibody working solution 12ml/bottle, acridinium-labeled MGF antibody working solution 12ml/bottle After calibrator/control solution working fluid 1.0ml/bottle is assembled, put it together and store it at 2-8°C. Pack 500ml/bottle of cleaning solution and 500ml/bottle of chemiluminescent substrate separately and store at 20°C. -25 ° C.
实施例2检测力生长因子和/或其E肽的化学发光试剂盒的制法Example 2 Method for Producing Chemiluminescence Kit for Detecting Power Growth Factor and/or Its E-peptide
1)链霉亲和素偶联的磁性微粒工作液的制备1) Preparation of streptavidin-coupled magnetic particle working solution
将链霉亲和素偶联的磁微粒悬浮液，磁分离去除上清，用pH为6.8，浓度为0.2mol/L的HEPES缓冲液重悬至浓度为1mg/mL，所述缓冲液含有2wt％的牛血清白蛋白、0.02wt％的Triton X-100、0.05％叠氮钠。The streptavidin-coupled magnetic particle suspension was magnetically separated to remove the supernatant, and resuspended to a concentration of 1 mg/mL with a pH of 6.8, a concentration of 0.2 mol/L in HEPES buffer, and the buffer contained 2 wt. % bovine serum albumin, 0.02 wt% Triton X-100, 0.05% sodium azide.
2)生物素标记的MGF抗体的制备2) Preparation of biotinylated MGF antibody
取1mg MGF羊单克隆抗体，加入适量PBS缓冲液(0.05M，pH7.0-7.6)调整抗体浓度至1mg/ml，并加入透析袋中进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后将抗体转移至离心管或冻存管内；Take 1mg MGF sheep monoclonal antibody, add appropriate amount of PBS buffer (0.05M, pH7.0-7.6) to adjust the antibody concentration to 1mg/ml, and add dialysis bag to dialysis, change dialysate every 3-4 hours, Replace 3-4 times, transfer the antibody to the centrifuge tube or cryotube after dialysis;
准确称取1mg N-羟基琥珀酰亚胺活化的生物素(NHS-Biotin)，加入适量水调整其浓度为2mg/ml，得NHS-Biotin水溶液；Accurately weigh 1 mg of N-hydroxysuccinimide-activated biotin (NHS-Biotin), add appropriate amount of water to adjust the concentration to 2 mg / ml, to obtain NHS-Biotin aqueous solution;
将NHS-Biotin水溶液加入抗体中，使NHS-Biotin与抗体按照1:100的摩尔比在2-8℃旋转混匀反应2小时，得生物素标记的MGF抗体溶液；The NHS-Biotin aqueous solution was added to the antibody, and the NHS-Biotin and the antibody were spin-mixed at a molar ratio of 1:100 at 2-8 ° C for 2 hours to obtain a biotin-labeled MGF antibody solution;
将生物素标记的MGF抗体溶液转移至透析袋进行透析，每隔3-4小时换一次 透析液，更换3-4次，透析后收集生物素标记的MGF抗体至离心管或冻存管内，加入1/10体积含5wt％BSA的PBS-BSA缓冲液(0.01M，pH7.0-7.6)，混匀后再加入甘油，所加甘油体积为所收集标记抗体体积加PBS-BSA缓冲液的体积，混匀后置于≤-15℃保存备用。The biotin-labeled MGF antibody solution is transferred to a dialysis bag for dialysis, and the dialysate is changed every 3-4 hours, and replaced 3-4 times. After dialysis, the biotin-labeled MGF antibody is collected into a centrifuge tube or a cryotube, and added. 1/10 volume of PBS-BSA buffer (0.01M, pH 7.0-7.6) containing 5wt% BSA, and then add glycerin after mixing. The volume of glycerol added is the volume of the collected labeled antibody plus the volume of PBS-BSA buffer. After mixing, put it at ≤ -15 °C for storage.
3)吖啶酯标记的MGF抗体的制备3) Preparation of acridinium ester labeled MGF antibody
取1mg MGF兔多克隆抗体，加入适量CBS缓冲液(0.05M，pH8.0-9.6)调整抗体浓度至1mg/ml，并加入透析袋中，进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后将抗体溶液转移至离心管或冻存管内；Take 1mg MGF rabbit polyclonal antibody, add appropriate CBS buffer (0.05M, pH 8.0-9.6) to adjust the antibody concentration to 1mg/ml, and add to the dialysis bag for dialysis, change the dialysate every 3-4 hours , replace 3-4 times, transfer the antibody solution to the centrifuge tube or cryotube after dialysis;
准确称取1mg吖啶酯，加入二甲基甲酰胺调整其浓度为2mg/ml，得吖啶酯溶液；Accurately weigh 1 mg of acridinium ester, add dimethylformamide to adjust the concentration to 2 mg / ml, to obtain acridinium ester solution;
将吖啶酯溶液加入抗体中，使吖啶酯与抗体按照1:80的摩尔比在2-8℃下旋转混匀反应1小时，得吖啶酯标记的MGF抗体溶液；The acridinium ester solution is added to the antibody, and the acridinium ester and the antibody are spin-mixed at a molar ratio of 1:80 at 2-8 ° C for 1 hour to obtain an acridinium ester-labeled MGF antibody solution;
将吖啶酯标记的MGF抗体溶液转移至透析袋进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后收集吖啶酯标记MGF抗体至离心管或冻存管内，加入1/10体积含5wt％BSA的PBS-BSA缓冲液(0.01M，pH7.0-7.6)，混匀后再加入甘油，所加甘油体积为所收集标记抗体体积加PBS-BSA缓冲液的体积，混匀后置于≤-15℃保存备用。Transfer the acridinium ester-labeled MGF antibody solution to a dialysis bag for dialysis, change the dialysate every 3-4 hours, replace 3-4 times, and collect the acridinium ester-labeled MGF antibody into the centrifuge tube or cryotube after dialysis. Add 1/10 volume of PBS-BSA buffer (0.01M, pH 7.0-7.6) containing 5wt% BSA, mix and add glycerol. The volume of glycerol added is the volume of the collected labeled antibody plus PBS-BSA buffer. Volume, mix and put at ≤ -15 ° C for storage.
4)生物素标记的MGF抗体工作液以及吖啶酯标记的MGF抗体工作液的制备4) Preparation of biotin-labeled MGF antibody working solution and acridinium ester-labeled MGF antibody working solution
配制pH为6.0，浓度为0.2mol/L的磷酸盐缓冲液，所述缓冲液含有5wt％的牛血清白蛋白、0.0.02wt％的酪蛋白、1mg/L的鼠IgG、5wt％的氯化钠、1wt％的蔗糖、2wt％的小牛血清、1wt％的Tween-20、0.05wt％的proclin 300；然后用该缓冲液配制浓度为0.5mg/L的生物素标记MGF抗体工作液，以及浓度为0.25mg/L吖啶酯标记MGF抗体工作液。A phosphate buffer solution having a pH of 6.0 and a concentration of 0.2 mol/L was prepared, and the buffer contained 5 wt% of bovine serum albumin, 0.0.02 wt% of casein, 1 mg/L of mouse IgG, and 5 wt% of chlorination. Sodium, 1% by weight of sucrose, 2% by weight of calf serum, 1% by weight of Tween-20, 0.05% by weight of proclin 300; then the buffer is used to prepare a concentration of 0.5 mg/L of biotin-labeled MGF antibody working solution, and The concentration of 0.25 mg/L acridinium ester labeled MGF antibody working solution.
6)校准品和质控品工作液的配制6) Preparation of calibrator and quality control working fluid
配制pH为7.6，浓度为0.2mol/L的N-(2-羟乙基)哌嗪-N′-(2-乙磺酸)(HEPES)缓冲液，所述缓冲液含有5wt％的牛血清白蛋白、10wt％的人血清、0.5wt％的氯化钠、20wt％的乙二醇、0.02wt％的Tween-20、0.5wt％的proclin 300；然后用该缓冲液配制MGF-Ct24E校准品和质控品工作液，校准品工作液共6个点，MGF-Ct24E的浓度分别为0pmol/L、0.8pmol/L、4pmol/L、16pmol/L、80pmol/L、 200pmol/L，质控品分高中低三个浓度，MGF-Ct24E的浓度分别为4pmol/L、16pmol/L、80pmol/L。A buffer of N-(2-hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) (HEPES) having a pH of 7.6 and a concentration of 0.2 mol/L was prepared, and the buffer contained 5 wt% of bovine serum. Albumin, 10% by weight of human serum, 0.5% by weight of sodium chloride, 20% by weight of ethylene glycol, 0.02% by weight of Tween-20, 0.5% by weight of proclin 300; then the buffer was used to prepare MGF-Ct24E calibrator And the quality control working fluid, calibrator working fluid a total of 6 points, MGF-Ct24E concentration of 0pmol / L, 0.8pmol / L, 4pmol / L, 16pmol / L, 80pmol / L, 200pmol / L, quality control The concentration of MGF-Ct24E was 4 pmol/L, 16 pmol/L and 80 pmol/L, respectively.
7)清洗液的配制7) Preparation of cleaning solution
配制pH为7.6，浓度为0.2mol/L的磷酸盐缓冲液，所述缓冲液含有10wt％的Tween 20、5wt％的氯化钠、15wt％的proclin 300。A phosphate buffer solution having a pH of 7.6 and a concentration of 0.2 mol/L was prepared, and the buffer contained 10% by weight of Tween 20, 5% by weight of sodium chloride, and 15% by weight of proclin 300.
8)化学发光底物液的配制8) Preparation of chemiluminescent substrate solution
配制底物液1，使其含有1.5wt％的二甲基甲酰胺、1.5wt％的Tween 20、1mmol/L的EDTA二钠盐、5wt％双氧水，避光保存；The substrate solution 1 was prepared to contain 1.5 wt% of dimethylformamide, 1.5 wt% of Tween 20, 1 mmol/L of EDTA disodium salt, and 5 wt% of hydrogen peroxide, and stored in the dark;
配制底物液2，使其含有1.5wt％的二甲基甲酰胺、1mol/L的氢氧化钠、0.5wt％的proclin 300、1.5wt％的Tween 20、5mmol/L的EDTA二钠盐。The substrate solution 2 was prepared to contain 1.5% by weight of dimethylformamide, 1 mol/L of sodium hydroxide, 0.5% by weight of proclin 300, 1.5% by weight of Tween 20, and 5 mmol/L of EDTA disodium salt.
9)试剂分装及组装，将链霉亲和素偶联的磁性微粒工作液3.0ml/瓶、生物素标记的MGF抗体工作液12ml/瓶、吖啶磺酰胺标记的MGF抗体工作液12ml/瓶、校准品/质控品工作液1.0ml/瓶分装后，组装在一起，保存于2-8℃，将清洗液500ml/瓶、化学发光底物液500ml/瓶单独包装，保存于20℃-25℃。9) Reagent packaging and assembly, streptavidin-coupled magnetic particle working solution 3.0ml/bottle, biotin-labeled MGF antibody working solution 12ml/vial, acridinesulfonamide-labeled MGF antibody working solution 12ml/ Bottle, calibrator / quality control working fluid 1.0ml / bottle is packed, assembled together, stored at 2-8 ° C, 500ml / bottle of cleaning solution, 500ml / bottle of chemiluminescent substrate liquid, separately stored in 20 °C-25 °C.
实施例3检测力生长因子和/或其E肽的化学发光试剂盒的制法Example 3 Method for Producing Chemiluminescence Kit for Detecting Power Growth Factor and/or Its E-peptide
1)链霉亲和素偶联的磁性微粒工作液的制备1) Preparation of streptavidin-coupled magnetic particle working solution
将链霉亲和素偶联的磁微粒悬浮液，磁分离去除上清，用pH为7.6，浓度为0.01mol/L的磷酸盐缓冲液重悬至浓度为0.1mg/mL，所述缓冲液含有0.05wt％的酪蛋白蛋白、1wt％的Brij35、0.5％proclin 300。The streptavidin-coupled magnetic particle suspension was magnetically separated to remove the supernatant, and resuspended to a concentration of 0.1 mg/mL with a phosphate buffer having a pH of 7.6 and a concentration of 0.01 mol/L. Containing 0.05% by weight of casein protein, 1% by weight of Brij35, 0.5% proclin 300.
2)生物素标记的MGF-Ct24E抗体的制备2) Preparation of biotinylated MGF-Ct24E antibody
取1mg MGF-Ct24E羊单克隆抗体，加入适量PBS缓冲液(0.05M，pH7.0-7.6)调整抗体浓度至1mg/ml，并加入透析袋中进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后将抗体转移至离心管或冻存管内；Take 1mg MGF-Ct24E goat monoclonal antibody, add appropriate amount of PBS buffer (0.05M, pH7.0-7.6) to adjust the antibody concentration to 1mg/ml, and add dialysis bag to dialysis, change dialysis every 3-4 hours Liquid, replace 3-4 times, transfer the antibody to the centrifuge tube or cryotube after dialysis;
准确称取1mg N-羟基琥珀酰亚胺活化的生物素(NHS-Biotin)，加入适量水调整其浓度为2mg/ml，得NHS-Biotin水溶液；Accurately weigh 1 mg of N-hydroxysuccinimide-activated biotin (NHS-Biotin), add appropriate amount of water to adjust the concentration to 2 mg / ml, to obtain NHS-Biotin aqueous solution;
将NHS-Biotin水溶液加入MGF-Ct24E羊单克隆抗体中，使NHS-Biotin与MGF-Ct24E羊单克隆抗体按照1:10的摩尔比在室温条件下旋转混匀反应0.5小时，得生物素标记的MGF-Ct24E抗体溶液；The NHS-Biotin aqueous solution was added to the MGF-Ct24E goat monoclonal antibody, and the NHS-Biotin and MGF-Ct24E goat monoclonal antibody were vortexed and mixed at a molar ratio of 1:10 at room temperature for 0.5 hour to obtain biotin-labeled MGF-Ct24E antibody solution;
将生物素标记的MGF-Ct24E抗体溶液转移至透析袋进行透析，每隔3-4小时 换一次透析液，更换3-4次，透析后收集生物素标记的MGF-Ct24E抗体至离心管或冻存管内，加入1/10体积含5wt％BSA的PBS-BSA缓冲液(0.01M，pH7.0-7.6)，混匀后再加入甘油，所加甘油体积为所收集标记抗体体积加PBS-BSA缓冲液的体积，混匀后置于≤-15℃保存备用。Transfer biotin-labeled MGF-Ct24E antibody solution to dialysis bag for dialysis, change dialysate every 3-4 hours, replace 3-4 times, collect biotin-labeled MGF-Ct24E antibody to centrifuge tube or freeze after dialysis In the storage tube, add 1/10 volume of PBS-BSA buffer (0.01M, pH7.0-7.6) containing 5wt% BSA, mix and add glycerol, the volume of glycerol added is the volume of the collected labeled antibody plus PBS-BSA The volume of the buffer is mixed and stored at ≤ -15 ° C for later use.
3)ABEI标记MGF-Ct24E抗体的制备3) Preparation of ABEI-labeled MGF-Ct24E antibody
取1mg MGF-Ct24E鼠单克隆抗体Fab片段，加入适量CBS缓冲液(0.05M，pH8.0-9.6)调整抗体浓度至1mg/ml，并加入透析袋中，进行透析，每隔3-4小时换一次透析液，更换3-4次，透析后将抗体溶液转移至离心管或冻存管内；Take 1 mg of MGF-Ct24E murine monoclonal antibody Fab fragment, add appropriate CBS buffer (0.05M, pH 8.0-9.6) to adjust the antibody concentration to 1mg/ml, and add to the dialysis bag for dialysis every 3-4 hours. Change the dialysate once, replace 3-4 times, transfer the antibody solution to the centrifuge tube or cryotube after dialysis;
将固体1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)分次加入到抗体溶液中，再逐滴加入用0.1mol/L HCL溶解的N-(4-氨丁基)-N-乙基异鲁米诺(ABEI)，使抗体:ABEI:EDC的摩尔比为1:50:300，控制pH在3.5-4.0之间，室温反应4.5小时，用1.0mol/L醋酸盐溶液终止反应，反应液用1mmol/L HCl溶液透析48小时，然后用1mmol/L HCl溶液平衡后的葡聚糖G-25柱除去游离的ABEI，分步收集，鉴定质量，所需部分分装后，≤-15℃保存备用。The solid 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) was added to the antibody solution in portions, and N-dissolved with 0.1 mol/L HCL was added dropwise. (4-aminobutyl)-N-ethylisoluminol (ABEI), the antibody: ABEI: EDC molar ratio is 1:50:300, the pH is controlled between 3.5-4.0, and the reaction is carried out at room temperature for 4.5 hours. The reaction was stopped with a 1.0 mol/L acetate solution, and the reaction solution was dialyzed against a 1 mmol/L HCl solution for 48 hours, and then the free ABEI was removed by a dextran G-25 column equilibrated with 1 mmol/L HCl solution, and collected stepwise. Identification quality, after the required part of the package, ≤ -15 ° C for storage.
4)生物素标记的MGF-Ct24E抗体工作液以及ABEI标记的MGF-Ct24E抗体工作液的制法4) Preparation method of biotin-labeled MGF-Ct24E antibody working solution and ABEI-labeled MGF-Ct24E antibody working solution
配制pH为7.6，浓度为0.01mol/L的Tris缓冲液，所述缓冲液含有0.5wt％的牛血清白蛋白、100mg/L的鼠IgG、0.5wt％的氯化钠、5wt％的蔗糖、0.05wt％的小牛血清、0.02wt％的平平加O-20、0.5wt％的叠氮钠；然后用该缓冲液配制浓度为0.5mg/L的生物素标记MGF-Ct24E抗体工作液，以及浓度为0.25mg/L的ABEI标记MGF-Ct24E抗体工作液。A Tris buffer having a pH of 7.6 and a concentration of 0.01 mol/L was prepared, and the buffer contained 0.5 wt% of bovine serum albumin, 100 mg/L of mouse IgG, 0.5 wt% of sodium chloride, and 5% by weight of sucrose. 0.05 wt% of calf serum, 0.02 wt% of flattened plus O-20, 0.5 wt% of sodium azide; then, the buffer was used to prepare a biotin-labeled MGF-Ct24E antibody working solution at a concentration of 0.5 mg/L, and ABEI-labeled MGF-Ct24E antibody working solution at a concentration of 0.25 mg/L.
5)校准品和质控品工作液的配制5) Preparation of calibrator and quality control working fluid
配制pH为6.8，浓度为0.01mol/L的MES缓冲液，所述缓冲液含有0.5wt％的牛血清白蛋白、1wt％的酪蛋白、50wt％的人血清、3wt％的氯化钠、2wt％的乙二醇、1wt％的Triton X-100、0.05wt％的proclin 300；然后用该缓冲液配制MGF校准品和质控品工作液，校准品工作液共6个点，MGF的浓度分别为0pmol/L、0.8pmol/L、4pmol/L、16pmol/L、80pmol/L、200pmol/L，质控品分高中低三个浓度，MGF的浓度分别为4pmol/L、16pmol/L、80pmol/L。A MES buffer having a pH of 6.8 and a concentration of 0.01 mol/L was prepared. The buffer contained 0.5 wt% of bovine serum albumin, 1 wt% of casein, 50 wt% of human serum, 3 wt% of sodium chloride, 2 wt. % ethylene glycol, 1wt% Triton X-100, 0.05wt% proclin 300; then use this buffer to prepare MGF calibrator and quality control working fluid, calibrator working solution a total of 6 points, MGF concentration The concentration of MGF was 4pmol/L, 16pmol/L and 80pmol, respectively, at 0pmol/L, 0.8pmol/L, 4pmol/L, 16pmol/L, 80pmol/L, and 200pmol/L. /L.
6)清洗液的配制6) Preparation of cleaning solution
配制pH为6.8，浓度为0.5mol/L的磷酸盐缓冲液，所述缓冲液含有0.2wt％的Triton X-100、20wt％的氯化钠、0.5wt％的叠氮钠。A phosphate buffer solution having a pH of 6.8 and a concentration of 0.5 mol/L was prepared, and the buffer contained 0.2 wt% of Triton X-100, 20 wt% of sodium chloride, and 0.5 wt% of sodium azide.
7)化学发光底物液的配制7) Preparation of chemiluminescent substrate solution
配制底物液1，使其含有0.1wt％的乙醇、0.1wt％的Triton X-100、5mmol/L的EDTA、0.05wt％双氧水，避光保存；The substrate solution 1 was prepared to contain 0.1 wt% ethanol, 0.1 wt% Triton X-100, 5 mmol/L EDTA, 0.05 wt% hydrogen peroxide, and stored in the dark;
配制底物液2，使其含有0.1wt％的乙醇、0.01mol/L的氢氧化钠、0.05wt％的proclin 300、0.1wt％的Triton X-100、1mmol/L的EDTA。The substrate liquid 2 was prepared to contain 0.1 wt% of ethanol, 0.01 mol/L of sodium hydroxide, 0.05 wt% of proclin 300, 0.1 wt% of Triton X-100, and 1 mmol/L of EDTA.
8)试剂分装及组装，将链霉亲和素偶联的磁性微粒工作液3.0ml/瓶、生物素标记的MGF-Ct24E抗体工作液12ml/瓶、异鲁米诺标记的MGF-Ct24E抗体工作液12ml/瓶、校准品/质控品工作液1.0ml/瓶分装后，组装在一起，保存于2-8℃，将清洗液500ml/瓶、化学发光底物液500ml/瓶单独包装，保存于20℃-25℃。8) Reagent packaging and assembly, streptavidin-coupled magnetic microparticle working solution 3.0ml/vial, biotinylated MGF-Ct24E antibody working solution 12ml/vial, isoluminol-labeled MGF-Ct24E antibody Working solution 12ml/bottle, calibrator/quality control working fluid 1.0ml/bottle, assembled, stored at 2-8°C, and separately package 500ml/bottle of washing solution and 500ml/bottle of chemiluminescent substrate Store at 20 ° C - 25 ° C.
实施例4使用本申请的试剂盒检测MGF和/或MGF-Ct24E的方法以全自动化学发光免疫分析仪(AE-180)为检测仪器，将试剂盒装载到仪器上进行检测，步骤如下：Example 4 Method for Detecting MGF and/or MGF-Ct24E Using the Kit of the Present Application A fully automated chemiluminescence immunoassay analyzer (AE-180) was used as a detection instrument, and the kit was loaded onto the instrument for detection. The steps are as follows:
将样本(或校准品或质控品)、生物素标记的MGF和/或MGF-Ct24E抗体工作液和化学发光标记物标记的MGF和/或MGF-Ct24E抗体工作液加入到反应杯中，37℃孵育15分钟，形成抗体-抗原-抗体夹心复合物溶液；Add a sample (or calibrator or control), biotinylated MGF and / or MGF-Ct24E antibody working solution and chemiluminescent label labeled MGF and / or MGF-Ct24E antibody working solution to the reaction cup, 37 Incubate at °C for 15 minutes to form an antibody-antigen-antibody sandwich complex solution;
于抗体-抗原-抗体夹心复合物溶液中加入链霉亲和素包被的磁性微粒工作液，37℃孵育15分钟，形成磁性复合物悬浮液；Adding streptavidin-coated magnetic microparticle working solution to the antibody-antigen-antibody sandwich complex solution, incubating at 37 ° C for 15 minutes to form a magnetic composite suspension;
将磁性复合物悬浮液置于磁场内，将清洗液稀释10-30倍后，洗涤所述磁性复合物；After the magnetic composite suspension is placed in a magnetic field, and the cleaning solution is diluted 10-30 times, the magnetic composite is washed;
向洗涤后的磁性复合物中先后注入底物液1及底物液2，检测其光子强度。The substrate solution 1 and the substrate solution 2 were successively injected into the washed magnetic composite to measure the photon intensity.
实施例5本申请的试剂盒的性能评估Example 5 Performance Evaluation of the Kit of the Present Application
1)最低检测限(分析灵敏度)的测定：1) Determination of the minimum detection limit (analytical sensitivity):
使用实施例1的试剂盒，通过实施例4的检测方法，重复测定S0校准品(0ng/ml)20次，RLU值为：3151、3125、3287、3116、3064、3147、2456、2410、2585、2552、2683、2964、3333、3197、2913、3291、3187、3235、2533、3024，RLU平均值(x)为2963，标准差(s)为308；再用同样的方法重复测定S1校准品(0.8pmol/L)2次，RLU值为：21028、20015，RLU平均值为20522； 根据S0校准品与S1校准品之间的校准品浓度-RLU平均值进行两点回归拟合得出一次方程，将x+2s值(3579)代入一次方程中，求出对应的浓度值即最低检测限，结果为0.028pmol/L。Using the kit of Example 1, the S0 calibrator (0 ng/ml) was repeatedly measured 20 times by the detection method of Example 4, and the RLU values were: 3151, 3125, 3287, 3116, 3064, 3147, 2456, 2410, 2585. , 2552, 2683, 2964, 3333, 3197, 2913, 3291, 3187, 3235, 2533, 3024, RLU average (x) is 2963, standard deviation (s) is 308; repeat the same method to determine S1 calibrator (0.8pmol/L) 2 times, RLU value: 21028, 20015, RLU average value is 20522; According to the calibrator concentration-RLU average between S0 calibrator and S1 calibrator, two-point regression fitting is used to obtain one time. For the equation, the x+2s value (3579) is substituted into the first equation, and the corresponding concentration value, that is, the lowest detection limit, is obtained, and the result is 0.028 pmol/L.
采用上述同样的方法，使用实施例2的试剂盒得到的最低检测限为0.021pmol/L，使用实施例3的试剂盒得到的最低检测限为0.035pmol/L。Using the same method as above, the minimum detection limit obtained using the kit of Example 2 was 0.021 pmol/L, and the minimum detection limit obtained using the kit of Example 3 was 0.035 pmol/L.
2)回收率(准确度)实验：2) Recovery rate (accuracy) experiment:
使用实施例1的试剂盒，将80pmol/L的高值校准品加入到0.8pmol/L低值校准品中，得回收样本，所加入的高值校准品与被加入的低值校准品的体积之比为1:9，通过实施例4的检测方法，对高值校准品、低值校准品、回收样本分别重复检测2次，得相应的RLU值，并计算得相应的浓度值和浓度平均值，检测结果见表1，按照下列公式计算回收率R，结果为103.2％。Using the kit of Example 1, a high value calibrator of 80 pmol/L was added to a 0.8 pmol/L low value calibrator to recover the sample, the high value calibrator added and the volume of the low value calibrator added. The ratio is 1:9. By the detection method of the fourth embodiment, the high-value calibrator, the low-value calibrator, and the recovered sample are repeatedly detected twice, and the corresponding RLU value is obtained, and the corresponding concentration value and concentration average are calculated. The value, the test results are shown in Table 1, and the recovery rate R was calculated according to the following formula, and the result was 103.2%.
Figure PCTCN2018104107-appb-000001
Figure PCTCN2018104107-appb-000001
式中：R——回收率；Where: R - recovery rate;
V 0——低值校准品的体积； V 0 - the volume of the low value calibrator;
V——高值校准品的体积；V——the volume of the high value calibrator;
C——高值校准品加入后，回收样本的检测浓度；C—— After the high-value calibrator is added, the detected concentration of the sample is recovered;
C 0——低值校准品的检测浓度； C 0 - the detected concentration of the low value calibrator;
C S——高值校准品的检测浓度。 C S - The detected concentration of the high value calibrator.
表1回收率实验结果Table 1 recovery rate experiment results
Figure PCTCN2018104107-appb-000002
Figure PCTCN2018104107-appb-000002
采用上述同样的方法，使用实施例2的试剂盒得到的回收率为98.2％，使用实施例3的试剂盒得到的回收率为99.5％。Using the same method as above, the recovery obtained by using the kit of Example 2 was 98.2%, and the recovery obtained by using the kit of Example 3 was 99.5%.
3)线性检测：3) Linear detection:
使用实施例1的试剂盒，通过实施例4的检测方法，将浓度分别为0.8pmol/L、4pmol/L、16pmol/L、80pmol/L、200pmol/L的校准品工作液，每一浓度水平重复检测3次，得相应的RLU值，并计算得相应的浓度值和浓度平均值，将浓度平均值和理论值用最小二乘法进行直线拟合，计算线性相关系数，得R＝0.9999(结果见表2)。Using the kit of Example 1, by using the detection method of Example 4, calibrator working solutions having concentrations of 0.8 pmol/L, 4 pmol/L, 16 pmol/L, 80 pmol/L, and 200 pmol/L, respectively, at each concentration level Repeat the test 3 times, the corresponding RLU value is obtained, and the corresponding concentration value and concentration average value are calculated. The average value and the theoretical value are straight-line fitted by the least squares method, and the linear correlation coefficient is calculated to obtain R=0.9999 (result See Table 2).
表2重复性实验结果Table 2 repeatability test results
Figure PCTCN2018104107-appb-000003
Figure PCTCN2018104107-appb-000003
采用上述同样的方法，使用实施例2的试剂盒得到的线性相关系数为0.9996，使用实施例3的试剂盒得到的线性相关系数为0.9995。Using the same method as above, the linear correlation coefficient obtained using the kit of Example 2 was 0.9996, and the linear correlation coefficient obtained using the kit of Example 3 was 0.9995.
4)重复性实验：4) Repeatability experiment:
使用实施例1的试剂盒，通过实施例4的检测方法，重复测定浓度分别为4pmol/L、16pmol/L、80pmol/L的质控品工作液各10次，得相应的RLU值，并计算得相应的浓度值、浓度平均值
Figure PCTCN2018104107-appb-000004
和标准差(s)，按公式 Using the kit of Example 1, the test solution having the concentrations of 4 pmol/L, 16 pmol/L, and 80 pmol/L was repeatedly measured 10 times each time, and the corresponding RLU value was obtained and calculated. Corresponding concentration value and concentration average
Figure PCTCN2018104107-appb-000004
And standard deviation (s), according to the formula
Figure PCTCN2018104107-appb-000005
计算变异系数，三个浓度水平的质控品工作液对应的变异系数分别为3.6％、4.5％、3.8％(结果见表3)。
Figure PCTCN2018104107-appb-000005
The coefficient of variation was calculated. The corresponding coefficients of variation for the control fluids at the three concentration levels were 3.6%, 4.5%, and 3.8%, respectively (see Table 3 for the results).
表3重复性实验结果Table 3 results of repetitive experiments
Figure PCTCN2018104107-appb-000006
Figure PCTCN2018104107-appb-000006
Figure PCTCN2018104107-appb-000007
Figure PCTCN2018104107-appb-000007
采用上述同样的方法，使用实施例2的试剂盒得三个浓度水平的质控品工作液对应的变异系数分别为4.6％、2.5％、5.8％，使用实施例3的试剂盒得三个浓度水平的质控品工作液对应的变异系数分别为3.1％、5.5％、2.8％。Using the same method as above, using the kit of Example 2, the coefficient of variation corresponding to the working fluid of the three control levels was 4.6%, 2.5%, and 5.8%, respectively, and three concentrations were obtained using the kit of Example 3. The corresponding coefficients of variation for the horizontal control solution were 3.1%, 5.5%, and 2.8%, respectively.
以上述依据本申请的理想实施例为启示，通过上述的说明内容，相关工作人员完全可以在不偏离本项申请技术思想的范围内，进行多样的变更以及修改。本项申请的技术性范围并不局限于说明书上的内容，必须要根据权利要求范围来确定其技术性范围。In view of the above-described embodiments of the present application, various changes and modifications may be made by those skilled in the art without departing from the scope of the present invention. The technical scope of the application is not limited to the contents of the specification, and the technical scope thereof must be determined according to the scope of the claims.

Claims (10)

  1. 一种检测力生长因子、其E肽的直接化学发光试剂盒，其特征在于，包括：链霉亲和素偶联的磁性微粒工作液，生物素标记的MGF和/或MGF-Ct24E抗体工作液，化学发光标记物标记的MGF和/或MGF-Ct24E抗体工作液，化学发光底物液，清洗液；A direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof, comprising: streptavidin-coupled magnetic particle working solution, biotin-labeled MGF and/or MGF-Ct24E antibody working solution , a chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody working solution, a chemiluminescent substrate solution, a cleaning solution;
    所述MGF和/或MGF-Ct24E抗体为单克隆抗体、多克隆抗体、单克隆抗体Fab片段或多克隆抗体Fab片段。The MGF and/or MGF-Ct24E antibody is a monoclonal antibody, a polyclonal antibody, a monoclonal antibody Fab fragment or a polyclonal antibody Fab fragment.
  2. 根据权利要求1所述的检测力生长因子、其E肽的直接化学发光试剂盒，其特征在于，所述化学发光标记物为吖啶酯、吖啶酯衍生物、吖啶磺酰胺、异鲁米诺或异鲁米诺衍生物，所述吖啶酯衍生物为吖啶酯酸-NHS酯、吖啶酸丙磺酸钠盐或9-吖啶羧酸，所述异鲁米诺衍生物为异鲁米诺(4-氨基邻苯二甲酰肼)、N-(4-氨基丁基)-N-乙基异鲁米诺、N-(6-氨基已基)-N-乙基异鲁米诺、异硫氰酸异鲁米诺或N-(4-异硫氰基丁基)-N-乙基异鲁米诺。The direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof according to claim 1, wherein the chemiluminescent label is an acridinium ester, an acridinium ester derivative, an acridinesulfonamide, or an isorule. a minoin or isoluminol derivative, the acridinium ester derivative being an acridinium-NHS ester, an acridinium propane sulfonate or a 9-acridine carboxylic acid, the isoluminol derivative Is isoluminol (4-aminophthalic acid hydrazide), N-(4-aminobutyl)-N-ethylisoluminol, N-(6-aminohexyl)-N-ethyl Isoluminol, isoluminol isothiocyanate or N-(4-isothiocyanatobutyl)-N-ethylisoluminol.
  3. 根据权利要求1或2所述的检测力生长因子、其E肽的直接化学发光试剂盒，其特征在于，所述链霉亲和素偶联的磁性微粒工作液由pH为6.8～7.6，浓度为0.01mol/L～0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPSO缓冲液中的一种配制而成，所述缓冲液含有0.05～2wt％的牛血清白蛋白和/或酪蛋白、0.02～1wt％的十二烷基聚乙二醇醚和/或聚乙二醇对异辛基苯基醚和/或聚氧乙烯失水山梨醇单月桂酸酯和/或月桂醇聚氧乙烯醚、0.05～0.5wt％的proclin 300和/或叠氮钠。The direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof according to claim 1 or 2, wherein the streptavidin-coupled magnetic microparticle working solution has a pH of 6.8 to 7.6, a concentration It is prepared by mixing one of 0.01 mol/L to 0.2 mol/L of phosphate buffer, Tris buffer, HEPES buffer, and MOPSO buffer, and the buffer contains 0.05 to 2 wt% of bovine serum albumin and / or casein, 0.02 to 1 wt% of dodecyl polyglycol ether and / or polyethylene glycol to isooctyl phenyl ether and / or polyoxyethylene sorbitan monolaurate and / or Lauryl ethoxylate, 0.05 to 0.5% by weight of proclin 300 and/or sodium azide.
  4. 根据权利要求1-3任一项所述的检测力生长因子、其E肽的直接化学发光试剂盒，其特征在于，所述生物素标记的MGF和/或MGF-Ct24E抗体以及化学发光标记物标记的MGF和/或MGF-Ct24E抗体工作液由pH为6.0～7.6，浓度为0.01mol/L～0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MOPSO缓冲液中的一种配制而成，所述缓冲液含有0.5～5wt％的牛血清白蛋白、1mg/L～100mg/L的鼠IgG、0.5～5wt％的氯化钠、1～5wt％的蔗糖、0.05～2wt％的小牛血清、0.02～1wt％的酪蛋白、0.02～1wt％的十二烷基聚乙二醇醚和/或聚乙二醇对异辛基苯基醚和/或聚氧乙烯失水山梨醇单月桂酸酯和/或月桂醇聚氧乙烯醚、0.05～0.5wt％的proclin 300和/或叠氮钠。The direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof according to any one of claims 1 to 3, wherein the biotin-labeled MGF and/or MGF-Ct24E antibody and a chemiluminescent label The labeled MGF and/or MGF-Ct24E antibody working solution is one of phosphate buffer solution, Tris buffer solution, HEPES buffer solution and MOPSO buffer solution having a pH of 6.0-7.6 and a concentration of 0.01 mol/L to 0.2 mol/L. The buffer solution comprises 0.5 to 5 wt% of bovine serum albumin, 1 mg/L to 100 mg/L of mouse IgG, 0.5 to 5 wt% of sodium chloride, 1 to 5 wt% of sucrose, 0.05 to 2 wt. % calf serum, 0.02 to 1 wt% casein, 0.02 to 1 wt% of dodecyl polyethylene glycol ether and/or polyethylene glycol dehydrated isooctylphenyl ether and/or polyoxyethylene Sorbitol monolaurate and/or lauryl polyoxyethylene ether, 0.05 to 0.5% by weight of proclin 300 and/or sodium azide.
  5. 根据权利要求1-4任一项所述的检测力生长因子、其E 肽的直接化学发光试剂盒，其特征在于，所述检测力生长因子和/或其E肽的直接化学发光试剂盒还包括MGF和/或MGF-Ct24E校准品工作液和/或质控品工作液，所述MGF和/或MGF-Ct24E校准品和/或质控品工作液由pH为6.0～7.6，浓度为0.01mol/L～0.2mol/L的磷酸盐缓冲液、Tris缓冲液、HEPES缓冲液、MES缓冲液中的一种配制而成，所述缓冲液中含有0.5～5wt％的牛血清白蛋白、10～50wt％的人血清、0.5～3wt％的氯化钠、2～20wt％的乙二醇、0.02～1wt％的Brij35和/或Triton X-100和/或Tween 20和/或平平加O-20、0.05～0.5wt％的proclin 300和/或叠氮钠。The direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof according to any one of claims 1 to 4, wherein the direct chemiluminescence kit for detecting a force growth factor and/or its E peptide is further Including MGF and / or MGF-Ct24E calibrator working fluid and / or control working fluid, the MGF and / or MGF-Ct24E calibrator and / or control working fluid from pH 6.0 ~ 7.6, concentration of 0.01 It is prepared by mixing one of phosphate buffer, Tris buffer, HEPES buffer and MES buffer with mol/L to 0.2 mol/L, and the buffer contains 0.5 to 5 wt% of bovine serum albumin, 10 ～50% by weight of human serum, 0.5 to 3% by weight of sodium chloride, 2 to 20% by weight of ethylene glycol, 0.02 to 1% by weight of Brij35 and/or Triton X-100 and/or Tween 20 and/or flat plus O- 20. 0.05 to 0.5% by weight of proclin 300 and/or sodium azide.
  6. 根据权利要求1-5任一项所述的检测力生长因子、其E肽的直接化学发光试剂盒，其特征在于，所述清洗液为pH为6.8～7.6，浓度为0.1mol/L～0.5mol/L的磷酸盐缓冲液或Tris缓冲液，所述缓冲液含有0.2～10wt％的Triton X-100和/或Tween 20、5～20wt％的氯化钠、0.5～15wt％的proclin 300和/或叠氮钠。The direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof according to any one of claims 1 to 5, wherein the cleaning solution has a pH of 6.8 to 7.6 and a concentration of 0.1 mol/L to 0.5. a mol/L phosphate buffer or a Tris buffer containing 0.2 to 10% by weight of Triton X-100 and/or Tween 20, 5 to 20% by weight of sodium chloride, 0.5 to 15% by weight of proclin 300, and / or sodium azide.
  7. 根据权利要求1-6任一项所述的检测力生长因子、其E肽的直接化学发光试剂盒，其特征在于，所述化学发光底物液包括底物液1和底物液2，所述底物液1含有0.1～1.5wt％的二甲基甲酰胺和/或乙醇、0.1～1.5wt％的Triton X-100和/或Tween 20、0.05～5wt％的双氧水以及1mmol/L-5mmol/L的EDTA或其盐；所述底物液2含有0.1～1.5wt％的二甲基甲酰胺和/或乙醇、0.1-1.5wt％的Triton X-100和/或Tween 20、0.05～0.5wt％的proclin 300、0.01mol/L-1mol/L的氢氧化钠、1mmol/L-5mmol/L的EDTA或其盐。The direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof according to any one of claims 1 to 6, wherein the chemiluminescent substrate liquid comprises a substrate liquid 1 and a substrate liquid 2, The substrate solution 1 contains 0.1 to 1.5% by weight of dimethylformamide and/or ethanol, 0.1 to 1.5% by weight of Triton X-100 and/or Tween 20, 0.05 to 5% by weight of hydrogen peroxide, and 1 mmol/L to 5 mmol. /L of EDTA or a salt thereof; the substrate solution 2 contains 0.1 to 1.5% by weight of dimethylformamide and/or ethanol, 0.1 to 1.5% by weight of Triton X-100 and/or Tween 20, 0.05 to 0.5 Wt% of proclin 300, 0.01 mol/L to 1 mol/L of sodium hydroxide, 1 mmol/L to 5 mmol/L of EDTA or a salt thereof.
  8. 一种检测力生长因子、其E肽的直接化学发光试剂盒的制法，其特征在于，包括：链霉亲和素偶联的磁性微粒工作液的制备，生物素标记的MGF和/或MGF-Ct24E抗体工作液的制备，化学发光标记物标记的MGF和/或MGF-Ct24E抗体工作液的制备，清洗液的配制，化学发光底物液的配制，将上述制备的试剂进行分装及组装。A method for preparing a direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof, comprising: preparation of a streptavidin-coupled magnetic particle working solution, biotin-labeled MGF and/or MGF - Preparation of Ct24E antibody working solution, preparation of chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody working solution, preparation of cleaning solution, preparation of chemiluminescent substrate solution, dispensing and assembly of the above prepared reagent .
  9. 根据权利要求8所述的检测力生长因子、其E肽的直接化学发光试剂盒的制法，其特征在于，所述生物素标记的MGF和/或MGF-Ct24E抗体的制备方法为：将N-羟基琥珀酰亚胺活化的生物素与抗体按照1:10-100的摩尔比在2-8℃或室温条件下旋转混匀反应0.5-2小时，透析除去多余的生物素，得生 物素标记的MGF和/或MGF-Ct24E抗体。The method of claim 8, wherein the biotin-labeled MGF and/or MGF-Ct24E antibody is prepared by: N - Hydroxysuccinimide-activated biotin and antibody are rotated and mixed at a molar ratio of 1:10-100 at 2-8 ° C or room temperature for 0.5-2 hours, and dialysis is performed to remove excess biotin to obtain biotin labeling. MGF and / or MGF-Ct24E antibodies.
  10. 根据权利要求8或9所述的检测力生长因子、其E肽的直接化学发光试剂盒的制法，其特征在于，所述化学发光标记物为吖啶酯、吖啶酯衍生物、吖啶磺酰胺时，化学发光标记物标记的MGF和/或MGF-Ct24E抗体的制备方法为：将化学发光标记物与抗体按照1:10-80的摩尔比在2-8℃或室温条件下旋转混匀反应0.5-2小时，透析除去多余的化学发光标记物，得化学发光标记物标记MGF和/或MGF-Ct24E抗体；The method for producing a direct chemiluminescence kit for detecting a force growth factor and an E peptide thereof according to claim 8 or 9, wherein the chemiluminescent label is an acridinium ester, an acridinium ester derivative, or an acridine In the case of a sulfonamide, the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody is prepared by rotating a chemiluminescent label with an antibody at a molar ratio of 1:10-80 at 2-8 ° C or room temperature. The reaction is carried out for 0.5-2 hours, and the excess chemiluminescent label is removed by dialysis to obtain a chemiluminescent label labeled MGF and/or MGF-Ct24E antibody;
    所述化学发光标记物为异鲁米诺、异鲁米诺衍生物时，化学发光标记物标记的MGF和/或MGF-Ct24E抗体的制备方法为：将固体EDC分次加入到抗体溶液中，再逐滴加入N-(4-氨丁基)-N-乙基异鲁米诺(ABEI)溶液，使抗体:ABEI:EDC的摩尔比为1:30-70:250-350，控制反应体系的pH在3.5-4.0之间，室温反应2-6小时，用醋酸盐溶液终止反应，除去多余的化学发光标记物和游离的ABEI，得化学发光标记物标记MGF和/或MGF-Ct24E抗体。When the chemiluminescent label is an isoluminol or an isoluminol derivative, the chemiluminescent label-labeled MGF and/or MGF-Ct24E antibody is prepared by adding the solid EDC to the antibody solution in portions. The N-(4-aminobutyl)-N-ethylisoluminol (ABEI) solution was added dropwise, and the molar ratio of antibody:ABEI:EDC was 1:30-70:250-350, and the reaction system was controlled. The pH is between 3.5 and 4.0, and the reaction is carried out at room temperature for 2-6 hours. The reaction is terminated with an acetate solution to remove excess chemiluminescent label and free ABEI to obtain a chemiluminescent label labeled MGF and/or MGF-Ct24E antibody. .
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