CN108490192A - Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method - Google Patents

Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method Download PDF

Info

Publication number
CN108490192A
CN108490192A CN201810260079.XA CN201810260079A CN108490192A CN 108490192 A CN108490192 A CN 108490192A CN 201810260079 A CN201810260079 A CN 201810260079A CN 108490192 A CN108490192 A CN 108490192A
Authority
CN
China
Prior art keywords
mgf
antibody
ct24e
solution
preparation
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201810260079.XA
Other languages
Chinese (zh)
Inventor
李大军
蔡淑娟
徐霞飞
涂策
张金林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Suzhou Hybiome Biomedical Engineering Co Ltd
Original Assignee
Suzhou Hybiome Biomedical Engineering Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Suzhou Hybiome Biomedical Engineering Co Ltd filed Critical Suzhou Hybiome Biomedical Engineering Co Ltd
Priority to CN201810260079.XA priority Critical patent/CN108490192A/en
Publication of CN108490192A publication Critical patent/CN108490192A/en
Priority to PCT/CN2018/104107 priority patent/WO2019184249A1/en
Pending legal-status Critical Current

Links

Classifications

    • 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

Abstract

The present invention relates to a kind of detection Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method, the kit of preparation includes：The magnetic particle working solution of Streptavidin coupling, MGF the and/or MGF Ct24E antibody working solutions of biotin labeling, MGF the and/or MGF Ct24E antibody working solutions of chemiluminescent labels label, MGF and MGF Ct24E calibration objects and/or quality-control product working solution, Chemoluminescent substrate, cleaning solution, the luminescence system of kit is direct chemiluminescence, utilize Streptavidin biotin signal amplification system, detection sensitivity is high, the range of linearity is wide, testing result is reproducible, and the accurate quantitative analysis of Mechano growth factor and/or its E peptide may be implemented.

Description

Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method
Technical field
The present invention relates to a kind of detection Mechano growth factor and/or the direct chemical luminescence reagent kits and usage of its E peptide, belong to Technical field of immunoassay.
Background technology
Mechano growth factor (MGF) is initially found in the skeletal muscle for stretching stimulation, is muscle under power stimulation induction, by The important Isoforms that insulin-like growth factor-Ⅰ (IGF- I) gene selectable montage generates, alternative splicing are happened at I bases of IGF- Because of the site before exon 6, and MGF is in this region of exon 5, the mankind and rodent class insert respectively 49bp and 52bp causes the translation of exon 6 that frameshift mutation occurs, generates the E peptide sequences (MGF- of 24 amino acid of special C-terminal Ct24E)。
MGF and MGF-Ct24E can activate muscle satellite cell, promote regeneration, promote osteoblastic proliferation, at Osteocyte or bone tissue to stress stimulation and nerve, musculature to playing a role in the response of damage, to maturation movement god Through first maintenance also important role, have the function of promoting various kinds of cell proliferation and survival.Also the study found that MGF and MGF-Ct24, which helps to improve, to be remembered.
By being chemically modified to the ends C- and/or the ends N-, such as Pegylation, L-D types are amino acid converting, glycosyl MGF is modified in the methods of change, sulphation, amidation, acetylation, can increase the stability of MGF, the MGF after modification and MGF-Ct24E Can be made into pharmaceutical preparation, such as aqueous and non-aqueous sterile injection liquid, aqueous and non-aqueous sterile suspensions, preparation can by skin, The modes such as parenteral, muscle, subcutaneous or transdermal skin are administered, or by being injected directly into blood or being applied directly to mucous membrane group Administration is knitted, for treating a variety of diseases such as skeletal muscle disorder, myocardium obstacle, neurological disorder.
MGF and MGF-Ct24E can be applied to field of tissue engineering technology, pass through suitable mode and modified poly-lactic acid material Connection prepares biological activity bionic material, for osteanagenesis reparation, revascularization reparation etc..In addition, MGF and MGF-Ct24E can By by loading to Mg alloy surface in a manner of suitable, the release in time that implants comes gram to stimulate the growth of osteoblast Take the problem of understrressing causes.
The application of MGF and MGF-Ct24E is so extensive, how to detect osteocyte or bone tissue is stressed stimulation or god Whether there is MGF and MGF-Ct24E to generate when being damaged through, musculature, how to monitor preparation in vivo extent of metabolism so as to In formulating treatment/dosage regimen, the rate of release of MGF and MGF-Ct24E in biomimetic material how is monitored, these are all to need to compel It cuts and solves the problems, such as.However, only have protein immunoblotting method detection MGF and MGF-Ct24E in the prior art, and the protein Immune-blotting method only qualitatively analysis, cannot quantify, and speed is slower, there has been no quickly and accurately quantitatively detect at present The method or kit of MGF and MGF-Ct24E.
Invention content
The technical problem to be solved by the present invention is to：To solve, there has been no quickly and accurately quantitatively detect MGF in the prior art And MGF-Ct24E method or kit the technical issues of, provide it is a kind of detection Mechano growth factor, its E peptide it is direct chemistry hair Light kit and preparation method.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of direct chemical luminescence reagent kit detecting Mechano growth factor, its E peptide, including：The magnetic of Streptavidin coupling Property particle working solution, MGF the and/or MGF-Ct24E antibody working solutions of biotin labeling, chemiluminescent labels label MGF And/or MGF-Ct24E antibody working solutions, Chemoluminescent substrate, cleaning solution.
Preferably, MGF the and/or MGF-Ct24E antibody is monoclonal antibody, polyclonal antibody, monoclonal antibody Fab Segment or polyclonal antibody Fab segments, are preferably derived from mouse, rabbit, sheep.
Preferably, the chemiluminescent labels be acridinium ester, acridine ester derivant, acridine sulfonamide, different luminol or Different luminol derivative, the acridine ester derivant be acridinium ester acid-NHS esters, acridinic acid propane sulfonic acid sodium salt or 9- azetidinecarboxylic acids, The different luminol derivative is different luminol (4- amino phthalyl hydrazines), the different Rumi of N- (4- aminobutyls)-N- ethyls Promise, N- (6- amino base)-N- ethyls different luminol, isothiocyanic acid different luminol or N- (4- isothiocyanos butyl)-N- ethyls Different luminol.
Preferably, the magnetic particle working solution of the Streptavidin coupling is 6.8~7.6 by pH, a concentration of One kind in the phosphate buffer of 0.01mol/L~0.2mol/L, Tris buffer solutions, HEPES buffer solution, MOPSO buffer solutions Be formulated, the buffer solution contain 0.05~2wt% bovine serum albumin(BSA) and/or casein, 0.02~1wt% 12 Alkyl polyglycol ether (Brij35) and/or polyethylene glycol are to isooctyl phenyl ether (Triton X-100) and/or polyoxyethylene Sorbitan mono-laurate (Tween 20) and/or ethoxylated dodecyl alcohol (paregal O -20), 0.05~0.5wt% Proclin 300 and/or Sodium azide.
Preferably, MGF the and/or MGF-Ct24E antibody of the biotin labeling and chemiluminescent labels mark MGF and/or MGF-Ct24E antibody working solution is 6.0~7.6 by pH, and the phosphate of a concentration of 0.01mol/L~0.2mol/L is slow One kind in fliud flushing, Tris buffer solutions, HEPES buffer solution, MOPSO buffer solutions is formulated, and the buffer solution contains 0.5~ The bovine serum albumin(BSA) of 5wt%, the mouse IgG of 1mg/L~100mg/L, the sodium chloride of 0.5~5wt%, 1~5wt% sucrose, Brij35 the and/or Triton X- of the calf serum of 0.05~2wt%, the casein of 0.02~1wt%, 0.02~1wt% The proclin 300 and/or Sodium azide of 100 and/or Tween 20 and/or paregal O -20,0.05~0.5wt%.
Preferably, it is described detection Mechano growth factor and/or its E peptide direct chemical luminescence reagent kit further include MGF and/or MGF-Ct24E calibration objects working solution and/or quality-control product working solution, MGF the and/or MGF-Ct24E calibration objects and/or quality-control product Working solution is 6.0~7.6 by pH, phosphate buffer, Tris buffer solutions, the HEPES of a concentration of 0.01mol/L~0.2mol/L One kind in buffer solution, MES buffer solutions is formulated, the bovine serum albumin(BSA) containing 0.5~5wt%, 10 in the buffer solution The human serum of~50wt%, the sodium chloride of 0.5~3wt%, the ethylene glycol of 2~20wt%, 0.02~1wt% Brij35 and/ Or Triton X-100 and/or Tween 20 and/or paregal O -20, the proclin 300 of 0.05~0.5wt% and/or folded Nitrogen sodium.
Preferably, it is 6.8~7.6 that the cleaning solution, which is pH, the phosphate-buffered of a concentration of 0.1mol/L~0.5mol/L Liquid or Tris buffer solutions, the buffer solution contain Triton X-100 and/or Tween 20, the 5~20wt% of 0.2~10wt% Sodium chloride, 0.5~15wt% proclin 300 and/or Sodium azide.
Preferably, the Chemoluminescent substrate includes substrate solution 1 and substrate solution 2, the substrate solution 1 containing 0.1~ The dimethylformamide and/or ethyl alcohol of 1.5wt%, the Triton X-100 and/or Tween 20,0.05 of 0.1~1.5wt%~ The hydrogen peroxide of 5wt% and the EDTA of 1mmol/L-5mmol/L or its salt；The substrate solution 2 containing 0.1~1.5wt% two Methylformamide and/or ethyl alcohol, the Triton X-100 and/or Tween 20 of 0.1-1.5wt%, 0.05~0.5wt% The EDTA or its salt of proclin 300, the sodium hydroxide of 0.01mol/L-1mol/L, 1mmol/L-5mmol/L.
The present invention also provides the preparation method of a kind of above-mentioned detection Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, packets It includes：The preparation of the magnetic particle working solution of Streptavidin coupling, MGF the and/or MGF-Ct24E antibody work of biotin labeling The preparation of liquid, the preparation of the MGF and/or MGF-Ct24E antibody working solutions of chemiluminescent labels label, the preparation of cleaning solution, The reagent of above-mentioned preparation is dispensed and is assembled by the preparation of Chemoluminescent substrate.
Preferably, the preparation method of the MGF and/or MGF-Ct24E antibody of the biotin labeling is：By N- hydroxysuccinimidyls The biotin of acid imide activation is with antibody according to 1:The molar ratio of 10-100 rotates mixing reaction at 2-8 DEG C or under room temperature 0.5-2 hours, dialysis removed extra biotin, obtains MGF the and/or MGF-Ct24E antibody of biotin labeling.
Preferably, when the chemiluminescent labels are acridinium ester, acridine ester derivant, acridine sulfonamide, chemiluminescence The preparation method of MGF and/or MGF-Ct24E antibody of substance markers is marked to be：By chemiluminescent labels and antibody according to 1:10- 80 molar ratio rotates mixing at 2-8 DEG C or under room temperature and reacts 0.5-2 hours, and dialysis removes extra chemiluminescent labeling Object obtains chemiluminescent labels label MGF and/or MGF-Ct24E antibody.
Preferably, when the chemiluminescent labels are different luminol, different luminol derivative, chemiluminescent labels mark The preparation method of the MGF and/or MGF-Ct24E antibody of note is：Solid EDC is added in antibody-solutions by several times, then is added dropwise Enter N- (4- ammonia butyl)-N- ethyls different luminol (ABEI) solution, makes antibody:ABEI:The molar ratio of EDC is 1:30-70:250- 350, the pH of reaction system is controlled between 3.5-4.0, is reacted at room temperature 2-6 hours, is terminated and is reacted with Acetate Solution, is removed more Remaining chemiluminescent labels and free ABEI obtain chemiluminescent labels label MGF and/or MGF-Ct24E antibody.
The beneficial effects of the invention are as follows：
The present invention provides a kind of detection Mechano growth factor, the chemical luminescence reagent kit and preparation method of its E peptide, the reagents of preparation The luminescence system of box is direct chemiluminescence, utilizes Streptavidin-biotin signal amplification system, detection sensitivity height, line Property range is wide, and testing result is reproducible, and the accurate quantitative analysis of Mechano growth factor and/or its E peptide may be implemented；Especially by the examination Agent box be used for Full-automatic chemiluminescence system, sample-adding, be incubated, cleaning and detection and etc. can be achieved automation, avoid people To operate the result error brought, improves work efficiency, by built-in standard curve to test software, only need test sample i.e. Can quantify detection sample in Mechano growth factor and/or Mechano growth factor E peptides, make detection more rapidly, it is more reliable, more stable.
Specific implementation mode
The present invention is described in further detail now.
Embodiment 1 detects the preparation method of Mechano growth factor and/or the chemical luminescence reagent kit of its E peptide
1) preparation of the magnetic particle working solution of Streptavidin coupling
The magnetic particle suspension that Streptavidin is coupled, Magneto separate remove supernatant, are 6.8 with pH, a concentration of 0.1mol/ The Tris buffer solutions of L are resuspended to a concentration of 0.5mg/mL, the buffer solution contain 0.5wt% bovine serum albumin(BSA), Triton X-100,300 0.05%proclin and 0.05% Sodium azide of 0.05wt%.
2) preparation of the MGF antibody of biotin labeling
1mg MGF mouse monoclonal antibodies are taken, it is always dense that appropriate PBS buffer solution (0.05M, pH7.0-7.6) adjustment antibody is added Degree is added in bag filter and dialyses to 1mg/ml, and a dialyzate was changed every 3-4 hours, replaces 3-4 times, will after dialysis Antibody is transferred in centrifuge tube or cryopreservation tube；
The accurate biotin (NHS-Biotin) for weighing the activation of 1mg n-hydroxysuccinimides is added suitable quantity of water and adjusts it A concentration of 2mg/ml obtains NHS-Biotin aqueous solutions；
NHS-Biotin aqueous solutions are added in antibody, make NHS-Biotin with antibody according to 1:50 molar ratio is in 2-8 DEG C rotation mixing react 1 hour, obtain the MGF antibody-solutions of biotin labeling；
The MGF antibody-solutions of biotin labeling are transferred to bag filter to dialyse, primary dialysis was changed every 3-4 hours Liquid is replaced 3-4 times, in the MGF antibody to centrifuge tube or cryopreservation tube that biotin labeling is collected after dialysis, 1/10 volume is added and contains The PBS-BSA buffer solutions (0.01M, pH7.0-7.6) of 5wt%BSA add glycerine after mixing, and institute's glycerol adding volume is is received Collection labelled antibody volume adds the volume of PBS-BSA buffer solutions, mixing to be placed on≤- 15 DEG C and save backup.
3) preparation of the MGF antibody of acridinium ester label
1mg MGF mouse monoclonal antibodies are taken, it is always dense that appropriate CBS buffer solutions (0.05M, pH8.0-9.6) adjustment antibody is added Degree is added to 1mg/ml in bag filter, dialyses, and a dialyzate was changed every 3-4 hours, replaces 3-4 times, will after dialysis Antibody-solutions are transferred in centrifuge tube or cryopreservation tube；
It is accurate to weigh 1mg acridinium esters, dimethylformamide is added and adjusts its a concentration of 2mg/ml, obtains acridine ester solution；
Acridine ester solution is added in antibody, makes acridinium ester with antibody according to 1:50 molar ratio rotates mixed at 2-8 DEG C Even reaction 1 hour, obtains the MGF antibody-solutions of acridinium ester label；
The MGF antibody-solutions of acridinium ester label are transferred to bag filter to dialyse, primary dialysis was changed every 3-4 hours Liquid is replaced 3-4 times, is collected in acridinium ester label MGF antibody to centrifuge tube or cryopreservation tube after dialysis, and 1/10 volume is added and contains The PBS-BSA buffer solutions (0.01M, pH7.0-7.6) of 5wt%BSA add glycerine after mixing, and institute's glycerol adding volume is is received Collection labelled antibody volume adds the volume of PBS-BSA buffer solutions, mixing to be placed on≤- 15 DEG C and save backup.
4) preparation of the MGF antibody working solutions of the MGF antibody working solution of biotin labeling and acridinium ester label
It is 6.0 to prepare pH, and the phosphate buffer of a concentration of 0.05mol/L, the buffer solution contains the cow's serum of 3wt% The calf of albumin, the casein of 0.5wt%, the sodium chloride of mouse IgG, 0.5wt% of 50mg/L, the sucrose of 1wt%, 1wt% Serum, 0.5wt% Tween-20,0.05wt% proclin 300；Then it is with the buffer antibody concentration The MGF antibody working solution and antibody concentration of the biotin labeling of 0.5mg/L are the MGF antibody of 0.25mg/L acridinium ester labels Working solution.
5) preparation of calibration object and quality-control product working solution
It is 7.4 to prepare pH, and N- (2- ethoxys) piperazine-N '-(2-ethanesulfonic acid) (HEPES) of a concentration of 0.1mol/L is buffered Liquid, the buffer solution contain the bovine serum albumin(BSA) of 3wt%, the human serum of 10wt%, the sodium chloride of 0.5wt%, 10wt% Ethylene glycol, 0.5wt% Tween-20,0.2wt% proclin 300；Then with buffer MGF calibration objects and matter Control product working solution, calibration object totally 6 points, the concentration of MGF be respectively 0pmol/L, 0.8pmol/L, 4pmol/L, 16pmol/L, 80pmol/L, 200pmol/L, quality-control product point high, normal, basic three concentration, the concentration of MGF be respectively 4pmol/L, 16pmol/L, 80pmol/L。
6) preparation of cleaning solution
It is 7.4 to prepare pH, and the phosphate buffer of a concentration of 0.1mol/L, the buffer solution contains the Tween of 10wt% 20, the proclin 300 of the sodium chloride of 10wt%, 10wt%.
7) preparation of Chemoluminescent substrate
Substrate solution 1 is prepared, the Tween 20 for the dimethylformamide, 1wt% that it contains 1wt%, the EDTA of 3mmol/L are made Disodium salt, 1.5wt% hydrogen peroxide, are kept in dark place；
Substrate solution 2 is prepared, the sodium hydroxide of the dimethylformamide, 0.5mol/L that it contains 1wt%, 0.2wt% are made The EDETATE DISODIUM of proclin 300, the Tween 20 of 1wt%, 2mmol/L.
8) reagent packing and assembling, by 3.0ml/ bottles of the magnetic particle working solution of Streptavidin coupling, biotin labeling 12ml/ bottles of MGF antibody working solutions, acridinium ester label 12ml/ bottles of MGF antibody working solutions, calibration object/quality-control product working solution After 1.0ml/ bottles of packing, fits together, be stored in 2-8 DEG C, by 500ml/ bottles of cleaning solution, 500ml/ bottles of Chemoluminescent substrate Individually packaging, is stored in 20 DEG C -25 DEG C.
Embodiment 2 detects the preparation method of Mechano growth factor and/or the chemical luminescence reagent kit of its E peptide
1) preparation of the magnetic particle working solution of Streptavidin coupling
The magnetic particle suspension that Streptavidin is coupled, Magneto separate remove supernatant, are 6.8 with pH, a concentration of 0.2mol/ The HEPES buffer solution of L is resuspended to a concentration of 1mg/mL, and the buffer solution contains the bovine serum albumin(BSA) of 2wt%, 0.02wt% Triton X-100,0.05% Sodium azide.
2) preparation of the MGF antibody of biotin labeling
1mg MGF sheep monoclonal antibodies are taken, appropriate PBS buffer solution (0.05M, pH7.0-7.6) adjustment antibody concentration is added It to 1mg/ml, and is added in bag filter and dialyses, a dialyzate was changed every 3-4 hours, replace 3-4 times, will resist after dialysis Body is transferred in centrifuge tube or cryopreservation tube；
The accurate biotin (NHS-Biotin) for weighing the activation of 1mg n-hydroxysuccinimides is added suitable quantity of water and adjusts it A concentration of 2mg/ml obtains NHS-Biotin aqueous solutions；
NHS-Biotin aqueous solutions are added in antibody, make NHS-Biotin with antibody according to 1:100 molar ratio is in 2-8 DEG C rotation mixing react 2 hours, obtain the MGF antibody-solutions of biotin labeling；
The MGF antibody-solutions of biotin labeling are transferred to bag filter to dialyse, primary dialysis was changed every 3-4 hours Liquid is replaced 3-4 times, in the MGF antibody to centrifuge tube or cryopreservation tube that biotin labeling is collected after dialysis, 1/10 volume is added and contains The PBS-BSA buffer solutions (0.01M, pH7.0-7.6) of 5wt%BSA add glycerine after mixing, and institute's glycerol adding volume is is received Collection labelled antibody volume adds the volume of PBS-BSA buffer solutions, mixing to be placed on≤- 15 DEG C and save backup.
3) preparation of the MGF antibody of acridinium ester label
1mg MGF rabbit polyclonal antibodies are taken, appropriate CBS buffer solutions (0.05M, pH8.0-9.6) adjustment antibody concentration is added It to 1mg/ml, and is added in bag filter, dialyses, a dialyzate was changed every 3-4 hours, replace 3-4 times, will resist after dialysis Liquid solution is transferred in centrifuge tube or cryopreservation tube；
It is accurate to weigh 1mg acridinium esters, dimethylformamide is added and adjusts its a concentration of 2mg/ml, obtains acridine ester solution；
Acridine ester solution is added in antibody, makes acridinium ester with antibody according to 1:80 molar ratio rotates mixed at 2-8 DEG C Even reaction 1 hour, obtains the MGF antibody-solutions of acridinium ester label；
The MGF antibody-solutions of acridinium ester label are transferred to bag filter to dialyse, primary dialysis was changed every 3-4 hours Liquid is replaced 3-4 times, is collected in acridinium ester label MGF antibody to centrifuge tube or cryopreservation tube after dialysis, and 1/10 volume is added and contains The PBS-BSA buffer solutions (0.01M, pH7.0-7.6) of 5wt%BSA add glycerine after mixing, and institute's glycerol adding volume is is received Collection labelled antibody volume adds the volume of PBS-BSA buffer solutions, mixing to be placed on≤- 15 DEG C and save backup.
4) preparation of the MGF antibody working solutions of the MGF antibody working solution of biotin labeling and acridinium ester label
It is 6.0 to prepare pH, and the phosphate buffer of a concentration of 0.2mol/L, the buffer solution contains the cow's serum of 5wt% The calf of albumin, the casein of 0.0.02wt%, the sodium chloride of mouse IgG, 5wt% of 1mg/L, the sucrose of 1wt%, 2wt% Serum, 1wt% Tween-20,0.05wt% proclin 300；Then with a concentration of 0.5mg/L's of the buffer Biotin labeling MGF antibody working solution and a concentration of 0.25mg/L acridinium ester labels MGF antibody working solutions.
6) preparation of calibration object and quality-control product working solution
It is 7.6 to prepare pH, and N- (2- ethoxys) piperazine-N '-(2-ethanesulfonic acid) (HEPES) of a concentration of 0.2mol/L is buffered Liquid, the buffer solution contain the bovine serum albumin(BSA) of 5wt%, the human serum of 10wt%, the sodium chloride of 0.5wt%, 20wt% Ethylene glycol, 0.02wt% Tween-20,0.5wt% proclin 300；Then with the schools buffer MGF-Ct24E Quasi- product and quality-control product working solution, calibration object working solution totally 6 points, the concentration of MGF-Ct24E is respectively 0pmol/L, 0.8pmol/ L, 4pmol/L, 16pmol/L, 80pmol/L, 200pmol/L, high, normal, basic three concentration of quality-control product point, the concentration of MGF-Ct24E Respectively 4pmol/L, 16pmol/L, 80pmol/L.
7) preparation of cleaning solution
It is 7.6 to prepare pH, and the phosphate buffer of a concentration of 0.2mol/L, the buffer solution contains the Tween of 10wt% 20, the proclin 300 of the sodium chloride of 5wt%, 15wt%.
8) preparation of Chemoluminescent substrate
Substrate solution 1 is prepared, makes Tween20, the 1mmol/L's for the dimethylformamide, 1.5wt% that it contains 1.5wt% EDETATE DISODIUM, 5wt% hydrogen peroxide, are kept in dark place；
Substrate solution 2 is prepared, the sodium hydroxide of the dimethylformamide, 1mol/L that it contains 1.5wt%, 0.5wt% are made The EDETATE DISODIUM of proclin 300, the Tween 20 of 1.5wt%, 5mmol/L.
9) reagent packing and assembling, by 3.0ml/ bottles of the magnetic particle working solution of Streptavidin coupling, biotin labeling 12ml/ bottles of MGF antibody working solutions, acridine sulfonamide label 12ml/ bottle of MGF antibody working solutions, calibration object/quality-control product work After 1.0ml/ bottles of packing of liquid, fits together, be stored in 2-8 DEG C, by 500ml/ bottles of cleaning solution, Chemoluminescent substrate 500ml/ Bottle is individually packed, and is stored in 20 DEG C -25 DEG C.
Embodiment 3 detects the preparation method of Mechano growth factor and/or the chemical luminescence reagent kit of its E peptide
1) preparation of the magnetic particle working solution of Streptavidin coupling
The magnetic particle suspension that Streptavidin is coupled, Magneto separate remove supernatant, are 7.6 with pH, a concentration of The phosphate buffer of 0.01mol/L is resuspended to a concentration of 0.1mg/mL, and the buffer solution contains the casein egg of 0.05wt% In vain, Brij35,0.5%proclin 300 of 1wt%.
2) preparation of the MGF-Ct24E antibody of biotin labeling
1mg MGF-Ct24E sheep monoclonal antibodies are taken, it is anti-that appropriate PBS buffer solution (0.05M, pH7.0-7.6) adjustment is added Bulk concentration is added in bag filter and dialyses to 1mg/ml, and a dialyzate was changed every 3-4 hours, replaces 3-4 times, dialysis Antibody is transferred in centrifuge tube or cryopreservation tube afterwards；
The accurate biotin (NHS-Biotin) for weighing the activation of 1mg n-hydroxysuccinimides is added suitable quantity of water and adjusts it A concentration of 2mg/ml obtains NHS-Biotin aqueous solutions；
NHS-Biotin aqueous solutions are added in MGF-Ct24E sheep monoclonal antibodies, NHS-Biotin and MGF-Ct24E are made Sheep monoclonal antibody is according to 1:10 molar ratio rotates mixing and reacts 0.5 hour at ambient temperature, obtains biotin labeling MGF-Ct24E antibody-solutions；
The MGF-Ct24E antibody-solutions of biotin labeling are transferred to bag filter to dialyse, were changed every 3-4 hours primary Dialyzate is replaced 3-4 times, in the MGF-Ct24E antibody to centrifuge tube or cryopreservation tube that biotin labeling is collected after dialysis, is added 1/ PBS-BSA buffer solution (0.01M, pH7.0-7.6) of 10 volumes containing 5wt%BSA adds glycerine, institute's glycerol adding body after mixing Product is the volume that collected labelled antibody volume adds PBS-BSA buffer solutions, and mixing is placed on≤- 15 DEG C and saves backup.
3) ABEI marks the preparation of MGF-Ct24E antibody
1mg MGF-Ct24E mouse monoclonal antibody Fab segments are taken, appropriate CBS buffer solutions (0.05M, pH8.0-9.6) are added Antibody concentration is adjusted to 1mg/ml, and is added in bag filter, is dialysed, a dialyzate was changed every 3-4 hours, replaces 3-4 It is secondary, antibody-solutions are transferred in centrifuge tube or cryopreservation tube after dialysis；
Solid 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides (EDC) are added to antibody-solutions by several times In, then N- (4- ammonia butyl)-N- ethyls different luminol (ABEI) with 0.1mol/L HCL dissolvings is added dropwise, make antibody: ABEI:The molar ratio of EDC is 1:50:300, pH is between 3.5-4.0 for control, reacts at room temperature 4.5 hours, with 1.0mol/L acetic acid Salting liquid terminates reaction, and reaction solution is dialysed 48 hours with 1mmol/L HCl solutions, after then using 1mmol/L HCl solutions balance Glucan G-25 columns remove free ABEI, Fraction collection identifies quality, and after required aliquots ,≤- 15 DEG C of preservations are standby With.
4) the MGF-Ct24E antibody working solutions of the MGF-Ct24E antibody working solution of biotin labeling and ABEI labels It prepares
It is 7.6 to prepare pH, and the Tris buffer solutions of a concentration of 0.01mol/L, the buffer solution contains the cow's serum of 0.5wt% Calf serum, the 0.02wt% of albumin, the sodium chloride of mouse IgG, 0.5wt% of 100mg/L, the sucrose of 5wt%, 0.05wt% Paregal O -20,0.5wt% Sodium azide；Then with the biotin labeling MGF- of a concentration of 0.5mg/L of the buffer The ABEI of Ct24E antibody working solution and a concentration of 0.25mg/L mark MGF-Ct24E antibody working solutions.
5) preparation of calibration object and quality-control product working solution
It is 6.8 to prepare pH, and the MES buffer solutions of a concentration of 0.01mol/L, the buffer solution contains the cow's serum of 0.5wt% The Triton of albumin, the casein of 1wt%, the human serum of 50wt%, the sodium chloride of 3wt%, the ethylene glycol of 2wt%, 1wt% The proclin 300 of X-100,0.05wt%；Then with buffer MGF calibration objects and quality-control product working solution, calibration object Working solution totally 6 points, the concentration of MGF be respectively 0pmol/L, 0.8pmol/L, 4pmol/L, 16pmol/L, 80pmol/L, 200pmol/L, high, normal, basic three concentration of quality-control product point, the concentration of MGF is respectively 4pmol/L, 16pmol/L, 80pmol/L.
6) preparation of cleaning solution
It is 6.8 to prepare pH, and the phosphate buffer of a concentration of 0.5mol/L, the buffer solution contains 0.2wt%'s The Sodium azide of Triton X-100, the sodium chloride of 20wt%, 0.5wt%.
7) preparation of Chemoluminescent substrate
Substrate solution 1 is prepared, Triton X-100 of the ethyl alcohol, 0.1wt% that it contains 0.1wt%, 5mmol/L are made EDTA, 0.05wt% hydrogen peroxide, are kept in dark place；
Substrate solution 2 is prepared, the sodium hydroxide of the ethyl alcohol, 0.01mol/L that it contains 0.1wt%, 0.05wt% are made The EDTA of proclin 300, Triton X-100 of 0.1wt%, 1mmol/L.
8) reagent packing and assembling, by 3.0ml/ bottles of the magnetic particle working solution of Streptavidin coupling, biotin labeling 12ml/ bottles of MGF-Ct24E antibody working solutions, different luminol label 12ml/ bottles of MGF-Ct24E antibody working solutions, calibration object/ After the 1.0ml/ bottles of packing of quality-control product working solution, fits together, be stored in 2-8 DEG C, by 500ml/ bottles of cleaning solution, chemiluminescence bottom 500ml/ bottles of thing liquid is individually packed, and is stored in 20 DEG C -25 DEG C.
Embodiment 4 is using the method for the kit detection MGF and/or MGF-Ct24E of the present invention with Full-automatic chemiluminescence Immunity analysis instrument (AE-180) is detecting instrument, and kit is loaded on instrument and is detected, steps are as follows：
By sample (or calibration object or quality-control product), MGF the and/or MGF-Ct24E antibody working solution of biotin labeling and change MGF the and/or MGF-Ct24E antibody working solutions for learning luminescent label are added in reaction cup, and 37 DEG C are incubated 15 minutes, shape At antibody-antigen-antibody sandwich complex solution；
The addition coated magnetic particle working solution of Streptavidin in antibody-antigen-antibody sandwich complex solution, 37 DEG C be incubated 15 minutes, formed magnetic composite suspension；
Magnetic composite suspension is placed in magnetic field, after 10-30 times of cleaning solution dilution, washs the magnetic coupling Object；
Substrate solution 1 and substrate solution 2 are successively injected into the magnetic composite after washing, detect its photon intensity.
The Performance Evaluation of the kit of 5 present invention of embodiment
1) measurement of minimum detection limit (sensitivity for analysis)：
Using the kit of embodiment 1, pass through the detection method of embodiment 4, replication S0 calibration objects (0ng/ml) 20 Secondary, RLU values are：3151、3125、3287、3116、3064、3147、2456、2410、2585、2552、2683、2964、3333、 3197,2913,3291,3187,3235,2533,3024, RLU average valuesIt is 2963, standard deviation (s) is 308；It uses again same The method replication S1 calibration objects (0.8pmol/L) of sample 2 times, RLU values are：21028,20015, RLU average values are 20522； 2 regression fits, which are carried out, according to the calibration object concentration-RLU average values between S0 calibration objects and S1 calibration objects obtains linear function, It willIt is worth (3579) to substitute into linear function, finds out corresponding concentration value i.e. minimum detection limit, result 0.028pmol/L.
Using above-mentioned same method, the lowest detection obtained using the kit of embodiment 2 is limited to 0.021pmol/L, The lowest detection obtained using the kit of embodiment 3 is limited to 0.035pmol/L.
2) rate of recovery (accuracy) is tested：
Using the kit of embodiment 1, the high level calibration object of 80pmol/L is added to 0.8pmol/L low value calibration objects In, recycling sample is obtained, the ratio between the high level calibration object being added and the volume of low value calibration object being added into are 1:9, pass through implementation The detection method of example 4 repeats detection 2 times to high level calibration object, low value calibration object, recycling sample, obtains corresponding RLU values respectively, And calculate corresponding concentration value and mean concentration, testing result are shown in Table 1, calculate rate of recovery R according to the following formula, result is 103.2%.
In formula：R --- the rate of recovery；
V0--- the volume of low value calibration object；
V --- the volume of high level calibration object；
After C --- high level calibration object is added, the detectable concentration of sample is recycled；
C0--- the detectable concentration of low value calibration object；
CS--- the detectable concentration of high level calibration object.
1 rate of recovery experimental result of table
Using above-mentioned same method, the use of the rate of recovery that the kit of embodiment 2 obtains is 98.2%, uses embodiment The rate of recovery that 3 kit obtains is 99.5%.
3) linearity test：
Using the kit of embodiment 1, by the detection method of embodiment 4, by concentration be respectively 0.8pmol/L, The calibration object working solution of 4pmol/L, 16pmol/L, 80pmol/L, 200pmol/L, each concentration level repeat detection 3 times, obtain Corresponding RLU values, and corresponding concentration value and mean concentration are calculated to obtain, by mean concentration and theoretical value least square method Fitting a straight line is carried out, linearly dependent coefficient is calculated, obtains R=0.9999 (the results are shown in Table 2).
2 repeated experiment result of table
Using above-mentioned same method, the use of the linearly dependent coefficient that the kit of embodiment 2 obtains is 0.9996, uses The linearly dependent coefficient that the kit of embodiment 3 obtains is 0.9995.
4) repeated experiment：
Using the kit of embodiment 1, by the detection method of embodiment 4, replication concentration be respectively 4pmol/L, Each 10 times of the quality-control product working solution of 16pmol/L, 80pmol/L, obtains corresponding RLU values, and calculates to obtain corresponding concentration value, concentration Average valueWith standard deviation (s), by formulaThe coefficient of variation is calculated, three concentration levels The corresponding coefficient of variation of quality-control product working solution is respectively 3.6%, 4.5%, 3.8% (the results are shown in Table 3).
3 repeated experiment result of table
Using above-mentioned same method, the quality-control product working solution pair of three concentration levels is obtained using the kit of embodiment 2 The coefficient of variation answered is respectively 4.6%, 2.5%, 5.8%, and the quality-control product of three concentration levels is obtained using the kit of embodiment 3 The corresponding coefficient of variation of working solution is respectively 3.1%, 5.5%, 2.8%.
It is enlightenment with above-mentioned desirable embodiment according to the present invention, through the above description, relevant staff is complete Various changes and amendments can be carried out without departing from the scope of the technological thought of the present invention' entirely.The technology of this invention Property range is not limited to the contents of the specification, it is necessary to determine its technical scope according to right.

Claims (10)

1. the direct chemical luminescence reagent kit of a kind of detection Mechano growth factor, its E peptide, which is characterized in that including：Streptavidin The magnetic particle working solution of coupling, MGF the and/or MGF-Ct24E antibody working solutions of biotin labeling, chemiluminescent labels mark MGF the and/or MGF-Ct24E antibody working solutions of note, Chemoluminescent substrate, cleaning solution；
MGF the and/or MGF-Ct24E antibody is monoclonal antibody, polyclonal antibody, monoclonal antibody Fab segments or more grams Grand Fab fragments.
2. the direct chemical luminescence reagent kit of detection Mechano growth factor according to claim 1, its E peptide, which is characterized in that The chemiluminescent labels be acridinium ester, acridine ester derivant, acridine sulfonamide, different luminol or different luminol derivative, The acridine ester derivant is that acridinium ester acid-NHS esters, acridinic acid propane sulfonic acid sodium salt or 9- azetidinecarboxylic acids, the different luminol are spread out Biology is different luminol (4- amino phthalyl hydrazines), N- (4- aminobutyls)-N- ethyls different luminol, (6- amino is by N- Base)-N- ethyls different luminol, isothiocyanic acid different luminol or N- (4- isothiocyanos butyl)-N- ethyl different luminols.
3. the direct chemical luminescence reagent kit of detection Mechano growth factor according to claim 1 or 2, its E peptide, feature exist In the magnetic particle working solution that the Streptavidin is coupled is 6.8~7.6 by pH, a concentration of 0.01mol/L~0.2mol/L Phosphate buffer, Tris buffer solutions, HEPES buffer solution, one kind in MOPSO buffer solutions be formulated, the buffer solution The Brij-35 of bovine serum albumin(BSA) and/or casein, 0.02~1wt% containing 0.05~2wt% and/or Polyethylene glycol to isooctyl phenyl ether and/or polyoxyethylene 20 sorbitan monolaurate and/or ethoxylated dodecyl alcohol, The proclin 300 and/or Sodium azide of 0.05~0.5wt%.
4. the direct chemical luminescence reagent kit of Mechano growth factor, its E peptide is detected according to claim 1-3 any one of them, Be characterized in that, the MGF of the MGF and/or MGF-Ct24E antibody and chemiluminescent labels of the biotin labeling label and/ Or MGF-Ct24E antibody working solution is 6.0~7.6 by pH, the phosphate buffer of a concentration of 0.01mol/L~0.2mol/L, One kind in Tris buffer solutions, HEPES buffer solution, MOPSO buffer solutions is formulated, and the buffer solution contains 0.5~5wt%'s Bovine serum albumin(BSA), the mouse IgG of 1mg/L~100mg/L, the sodium chloride of 0.5~5wt%, the sucrose of 1~5wt%, 0.05~ The calf serum of 2wt%, the casein of 0.02~1wt%, the Brij-35 of 0.02~1wt% and/or poly- second Glycol is to isooctyl phenyl ether and/or polyoxyethylene 20 sorbitan monolaurate and/or ethoxylated dodecyl alcohol, 0.05 The proclin 300 and/or Sodium azide of~0.5wt%.
5. the direct chemical luminescence reagent kit of Mechano growth factor, its E peptide is detected according to claim 1-4 any one of them, It is characterized in that, the direct chemical luminescence reagent kit of the detection Mechano growth factor and/or its E peptide further includes MGF and/or MGF- Ct24E calibration objects working solution and/or quality-control product working solution, MGF the and/or MGF-Ct24E calibration objects and/or quality-control product work Liquid is 6.0~7.6 by pH, the phosphate buffer of a concentration of 0.01mol/L~0.2mol/L, Tris buffer solutions, HEPES bufferings One kind in liquid, MES buffer solutions is formulated, and the bovine serum albumin(BSA) containing 0.5~5wt% in the buffer solution, 10~ The human serum of 50wt%, the sodium chloride of 0.5~3wt%, the ethylene glycol of 2~20wt%, 0.02~1wt% Brij35 and/or The proclin 300 and/or nitrine of Triton X-100 and/or Tween 20 and/or paregal O -20,0.05~0.5wt% Sodium.
6. the direct chemical luminescence reagent kit of Mechano growth factor, its E peptide is detected according to claim 1-5 any one of them, Be characterized in that, the cleaning solution is that pH is 6.8~7.6, the phosphate buffer of a concentration of 0.1mol/L~0.5mol/L or Tris buffer solutions, the buffer solution contain the chlorine of the Triton X-100 and/or Tween 20 of 0.2~10wt%, 5~20wt% Change the proclin 300 and/or Sodium azide of sodium, 0.5~15wt%.
7. the direct chemical luminescence reagent kit of Mechano growth factor, its E peptide is detected according to claim 1-6 any one of them, It is characterized in that, the Chemoluminescent substrate includes substrate solution 1 and substrate solution 2, and the substrate solution 1 contains 0.1~1.5wt%'s Pair of dimethylformamide and/or ethyl alcohol, the Triton X-100 and/or Tween 20 of 0.1~1.5wt%, 0.05~5wt% The EDTA or its salt of oxygen water and 1mmol/L-5mmol/L；Dimethylformamide of the substrate solution 2 containing 0.1~1.5wt% And/or the Triton X-100 and/or Tween 20 of ethyl alcohol, 0.1-1.5wt%, 0.05~0.5wt% proclin 300, The sodium hydroxide of 0.01mol/L-1mol/L, the EDTA of 1mmol/L-5mmol/L or its salt.
8. a kind of preparation method of the direct chemical luminescence reagent kit of detection Mechano growth factor, its E peptide, which is characterized in that including：Strepto- The preparation of the magnetic particle working solution of Avidin coupling, the system of the MGF and/or MGF-Ct24E antibody working solutions of biotin labeling It is standby, the preparation of the MGF and/or MGF-Ct24E antibody working solutions of chemiluminescent labels label, the preparation of cleaning solution, chemistry hair The reagent of above-mentioned preparation is dispensed and is assembled by the preparation of light substrate solution.
9. the preparation method of the direct chemical luminescence reagent kit of detection Mechano growth factor according to claim 8, its E peptide, special Sign is that the preparation method of the MGF and/or MGF-Ct24E antibody of the biotin labeling is：N-hydroxysuccinimide is lived The biotin of change is with antibody according to 1:The molar ratio of 10-100 rotates mixing at 2-8 DEG C or under room temperature and reacts 0.5-2 hours, Dialysis removes extra biotin, obtains MGF the and/or MGF-Ct24E antibody of biotin labeling.
10. the preparation method of the direct chemical luminescence reagent kit of detection Mechano growth factor, its E peptide according to claim 8 or claim 9, It is characterized in that, when the chemiluminescent labels are acridinium ester, acridine ester derivant, acridine sulfonamide, chemiluminescent labeling The preparation method of the MGF and/or MGF-Ct24E antibody of substance markers is：By chemiluminescent labels and antibody according to 1:10-80's Molar ratio rotates mixing at 2-8 DEG C or under room temperature and reacts 0.5-2 hours, and dialysis removes extra chemiluminescent labels, It obtains chemiluminescent labels and marks MGF and/or MGF-Ct24E antibody；
The chemiluminescent labels be different luminol, different luminol derivative when, chemiluminescent labels label MGF and/ Or the preparation method of MGF-Ct24E antibody is：Solid EDC is added in antibody-solutions by several times, then N- (4- ammonia fourths are added dropwise Base)-N- ethyls different luminol (ABEI) solution, make antibody:ABEI:The molar ratio of EDC is 1:30-70:250-350, control are anti- It answers the pH of system between 3.5-4.0, reacts at room temperature 2-6 hours, terminated and reacted with Acetate Solution, remove extra chemistry hair Signal object and free ABEI obtain chemiluminescent labels label MGF and/or MGF-Ct24E antibody.
CN201810260079.XA 2018-03-27 2018-03-27 Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method Pending CN108490192A (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201810260079.XA CN108490192A (en) 2018-03-27 2018-03-27 Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method
PCT/CN2018/104107 WO2019184249A1 (en) 2018-03-27 2018-09-05 Direct chemiluminescence kit for detecting mechano-growth factor and e peptide thereof, and preparation method therefor

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810260079.XA CN108490192A (en) 2018-03-27 2018-03-27 Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method

Publications (1)

Publication Number Publication Date
CN108490192A true CN108490192A (en) 2018-09-04

Family

ID=63316671

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810260079.XA Pending CN108490192A (en) 2018-03-27 2018-03-27 Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method

Country Status (2)

Country Link
CN (1) CN108490192A (en)
WO (1) WO2019184249A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109324038A (en) * 2018-11-13 2019-02-12 迈克生物股份有限公司 A kind of magnetic particle buffer for interstitialcellstimulating hormone (ICSH) assay kit
CN109709336A (en) * 2018-12-26 2019-05-03 张嘉庆 A kind of detection kit and detection method of vascular endothelial growth factor
WO2019184249A1 (en) * 2018-03-27 2019-10-03 苏州长光华医生物医学工程有限公司 Direct chemiluminescence kit for detecting mechano-growth factor and e peptide thereof, and preparation method therefor
CN110850076A (en) * 2019-11-28 2020-02-28 南京迪安医学检验所有限公司 Anti-double-chain DNA antibody IgG chemiluminescence immunoassay kit and preparation method and application thereof
CN111060695A (en) * 2019-12-23 2020-04-24 江苏三联生物工程有限公司 Electrochemiluminescence kit for detecting anti-LRP 4 antibody and preparation method thereof
CN112198309A (en) * 2020-09-29 2021-01-08 上海雅酶生物医药科技有限公司 Chemiluminescence substrate detection kit with high sensitivity and stable signal
CN117405890A (en) * 2023-12-15 2024-01-16 北京美联泰科生物技术有限公司 Chemiluminescent detection kit and method for pepsinogen II

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101713779A (en) * 2009-12-22 2010-05-26 陕西北美基因股份有限公司 Method for performing immunological test on biomolecules by avidin/streptavidin magnetic composite particles
WO2011116432A1 (en) * 2010-03-26 2011-09-29 Bond University Ltd Methods and compositions for detecting banned substances
CN102998466A (en) * 2012-11-20 2013-03-27 博奥赛斯（天津）生物科技有限公司 Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for growth hormone (GH), and preparation method of kit
CN103941021A (en) * 2014-05-09 2014-07-23 深圳市国赛生物技术有限公司 Kit for detecting interleukin-6 and method for detecting interleukin-6 for non-diagnostic purpose
CN105651990A (en) * 2015-12-30 2016-06-08 深圳市新产业生物医学工程股份有限公司 Chemiluminiscence detection kit for 17alpha-hydroxyprogesterone and preparation method and application thereof
CN107543927A (en) * 2017-11-03 2018-01-05 太原瑞盛生物科技有限公司 The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of placenta growth factor

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107247135A (en) * 2017-08-11 2017-10-13 太原瑞盛生物科技有限公司 A kind of chemiluminescence detection kit of sulfamethazine and preparation method thereof
CN107290526A (en) * 2017-08-24 2017-10-24 太原瑞盛生物科技有限公司 A kind of chemiluminescence detection kit of tetracycline and preparation method thereof
CN107807241A (en) * 2017-11-03 2018-03-16 太原瑞盛生物科技有限公司 The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of intravascular ErbB1
CN107817354A (en) * 2017-11-03 2018-03-20 太原瑞盛生物科技有限公司 A kind of chemiluminescence detection kit of interleukin 6 and preparation method thereof
CN108490192A (en) * 2018-03-27 2018-09-04 苏州长光华医生物医学工程有限公司 Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101713779A (en) * 2009-12-22 2010-05-26 陕西北美基因股份有限公司 Method for performing immunological test on biomolecules by avidin/streptavidin magnetic composite particles
WO2011116432A1 (en) * 2010-03-26 2011-09-29 Bond University Ltd Methods and compositions for detecting banned substances
CN102998466A (en) * 2012-11-20 2013-03-27 博奥赛斯（天津）生物科技有限公司 Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for growth hormone (GH), and preparation method of kit
CN103941021A (en) * 2014-05-09 2014-07-23 深圳市国赛生物技术有限公司 Kit for detecting interleukin-6 and method for detecting interleukin-6 for non-diagnostic purpose
CN105651990A (en) * 2015-12-30 2016-06-08 深圳市新产业生物医学工程股份有限公司 Chemiluminiscence detection kit for 17alpha-hydroxyprogesterone and preparation method and application thereof
CN107543927A (en) * 2017-11-03 2018-01-05 太原瑞盛生物科技有限公司 The magnetic microparticle chemiluminescence detection kit and preparation method of a kind of placenta growth factor

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HONGWU DU等: "Generation and evaluation of antibodies against human MGF E-peptide by reverse phase protein microarray and reverse competitive ELISA", 《BIOANALYSIS》 *
IRINA V. KRAVCHENKO等: "Monoclonal Antibodies to Mechano-Growth Factor", 《HYBRIDOMA》 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019184249A1 (en) * 2018-03-27 2019-10-03 苏州长光华医生物医学工程有限公司 Direct chemiluminescence kit for detecting mechano-growth factor and e peptide thereof, and preparation method therefor
CN109324038A (en) * 2018-11-13 2019-02-12 迈克生物股份有限公司 A kind of magnetic particle buffer for interstitialcellstimulating hormone (ICSH) assay kit
CN109709336A (en) * 2018-12-26 2019-05-03 张嘉庆 A kind of detection kit and detection method of vascular endothelial growth factor
CN110850076A (en) * 2019-11-28 2020-02-28 南京迪安医学检验所有限公司 Anti-double-chain DNA antibody IgG chemiluminescence immunoassay kit and preparation method and application thereof
CN111060695A (en) * 2019-12-23 2020-04-24 江苏三联生物工程有限公司 Electrochemiluminescence kit for detecting anti-LRP 4 antibody and preparation method thereof
CN112198309A (en) * 2020-09-29 2021-01-08 上海雅酶生物医药科技有限公司 Chemiluminescence substrate detection kit with high sensitivity and stable signal
CN117405890A (en) * 2023-12-15 2024-01-16 北京美联泰科生物技术有限公司 Chemiluminescent detection kit and method for pepsinogen II
CN117405890B (en) * 2023-12-15 2024-03-29 北京美联泰科生物技术有限公司 Chemiluminescent detection kit and method for pepsinogen II

Also Published As

Publication number Publication date
WO2019184249A1 (en) 2019-10-03

Similar Documents

Publication Publication Date Title
CN108490192A (en) Detect Mechano growth factor, the direct chemical luminescence reagent kit of its E peptide, preparation method
CN108414771A (en) Detect electrochemical luminescence kit, the preparation method of Mechano growth factor, its E peptide
CN108490168A (en) Detect Mechano growth factor, the adamantane chemistry luminescence reagent box of its E peptide, preparation method
CN103901203B (en) Chemical luminescent analysis reagent kid of a kind of Procalcitonin and preparation method thereof and detection method
CN104697988B (en) Detect kit and its detection method and the application of antibody of HCV
CN108700584A (en) Labeled complex and preparation method thereof, kit, application and detecting system
CN102998467B (en) β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof
CN101738473B (en) Treponema pallidum antibody diagnostic kit and preparation method thereof
CN105548565A (en) Kit for detecting trypanosoma cruzi antibody as well as preparation and application thereof
CN112782156B (en) Chitinase 3-like protein 1 kit and preparation method thereof
CN101545913A (en) Chemoluminescence immunoassay measuring kit and preparation method thereof for triiodothyronine magnetic particles
CN106353506A (en) Kit for detecting osteocalcin content and testing method thereof
CN107817354A (en) A kind of chemiluminescence detection kit of interleukin 6 and preparation method thereof
CN111323577B (en) Anti-cardiolipin antibody IgG magnetic particle chemiluminescence immunoassay kit
CN108445230B (en) Procalcitonin chemiluminescence detection reagent based on nano antibody and detection method
CN102998466A (en) Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for growth hormone (GH), and preparation method of kit
CN106918708A (en) A kind of competition law turbid kit of latex enhancing immune transmittance for detecting insulin
CN104698185A (en) Kit for detecting treponema pallidum antibody as well as detection method and application thereof
CN107807240A (en) A kind of chemiluminescence detection kit of Procalcitonin and preparation method thereof
CN106645729A (en) Chemiluminescent kit for quantitatively detecting mycobacterium tuberculosis r interferon as well as preparation method, detection method and evaluation method of chemiluminescent kit
CN105492605A (en) Virus-like particle to be used in immunoassay method, blocking agent to be used therein and kit comprising same
US20240010680A1 (en) Peptide disinfectant for preventing coronaviruses
CN108931652A (en) A kind of kit with Magnetism particulate immuno chemistry luminescence method detection myoglobin content
CN101377496A (en) Chemiluminescence immune analytic reagent kit for detecting thyroid peroxidase autoantibody
CN106405098A (en) An anti-beta2 glycoprotein I antibody chemiluminescence immunodetection kit and a preparing method thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20180904