CN109061176A - A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide - Google Patents
A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide Download PDFInfo
- Publication number
- CN109061176A CN109061176A CN201810699124.1A CN201810699124A CN109061176A CN 109061176 A CN109061176 A CN 109061176A CN 201810699124 A CN201810699124 A CN 201810699124A CN 109061176 A CN109061176 A CN 109061176A
- Authority
- CN
- China
- Prior art keywords
- extends
- total type
- peptide
- collagen
- aminoterminal
- 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
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
- 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
- G01N33/543—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
- 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
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/108—Osteoporosis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Nanotechnology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention provides the kit that a kind of total Type I collagen aminoterminal of detection serum extends peptide, detection sensitivity height, good reliability.A kind of total Type I collagen aminoterminal of detection serum extends the kit of peptide, it is characterised in that it includes total Type I collagen aminoterminal of calibration object, quality-control product, biotin labeling extends the solution of peptide antibody, total Type I collagen aminoterminal of horseradish peroxidase-labeled extends the nano magnetic microparticle suspending liquid and substrate of the solution of peptide antibody, marked by streptavidin；The concentration that total Type I collagen aminoterminal of biotin labeling extends the solution of peptide antibody is 1.5 μ g/ml；The concentration of the nano magnetic microparticle suspending liquid of marked by streptavidin is 0.5mg/ml；The concentration that total Type I collagen aminoterminal of horseradish peroxidase-labeled extends peptide antibody solution is 1.5 μ g/ml.
Description
Technical field
The invention belongs to diagnostic fields outside medical instrument biological species immune body, and in particular to a kind of total I type glue of detection serum
Former aminoterminal extends the kit and application method of peptide.
Background technique
Osteoporosis is a kind of static disease: some nonspecific symptoms before fracturing are usually ignored: such as waist
Backache.Most of cases are diagnosed after the fracture, and relevant complication is mostly fracture, and this point greatly improves
Medical expense.Early diagnosis can help to reduce payment for medical care.More and more hospital departments have opened up osteoporosis detection, example
Such as: division of endocrinology, orthopaedics, gynemetrics, department of traditional Chinese medicine, and gradually start to pay attention to accepting osteoporosis patient for medical treatment.China is each at present
Grade hospital is mainly auxiliary diagnostic criteria with bone density, not yet uses Bone markers index comprehensively.Basic reason is China
Lack five perfect biological markers diagnostic reagents all the time, leads to the immunology diagnosis one of five markers of bone metabolism
Directly clinical cannot generally it carry out.If can accomplish early stage Accurate Diagnosis, bone loss disorders should be that one kind can treat, control
System, the chronic disease even cured.
The single chain molecule amount 139KDa of I-type collagen, is made of 1460 amino acid.Its space structure is three peptide chains
Helical region and both ends unspiralized region composition.Pro-alpha1 (I) chain corresponds to gene C OL1A1, pro-alpha 2 (I) chain
Corresponding gene C OL1A2.Type I collagen aminoterminal extends the heterotrimer segment that peptide (PINP) is N-terminal, molecular weight about 100KDa.
P1NP reflection is the deposition of type i collagen, therefore is as a bon e formation marker.In type i collagen
In forming process, P1NP is released into extracellular space and eventually enters into blood.P1NP is that trimeric form (is turned by tripolymer collagen
Change), but monomeric form can be become under thermal degradation quickly.The detection of P1NP reagent is P1NP shape all in blood
Formula.The P1NP antigen of structural integrity obtains difficult.
At home, the total Type I collagen aminoterminal of serum extends peptide (tP1NP) detection clinically mainly with the examination of external Roche Holding Ag
Based on agent application, external import reagent price is very expensive, and very big financial burden is brought to patient, is unfavorable for general in base
And.Therefore, a kind of total Type I collagen aminoterminal of serum to be developed extends the kit of peptide detection sensitivity height, good reliability, with
Patient's use cost is reduced, and improves popularization and use.
Summary of the invention
In view of the above-mentioned problems, the present invention provides the kit that a kind of total Type I collagen aminoterminal of detection serum extends peptide,
Detection sensitivity height, good reliability；In addition, the present invention also provides use the kit to detect the total Type I collagen aminoterminal of serum
Extend the method for peptide.
A kind of total Type I collagen aminoterminal of detection serum extends the kit of peptide, which is characterized in that including calibration object, Quality Control
Product, biotin labeling total Type I collagen aminoterminal extend total Type I collagen of the solution of peptide antibody, horseradish peroxidase-labeled
Aminoterminal extends the nano magnetic microparticle suspending liquid and substrate of the solution of peptide antibody, marked by streptavidin；
The calibration object includes that concentration is 0 ng/mL, 10 ng/mL, 25 ng/mL, 100 ng/mL, 400 ng/mL, 1200
Total Type I collagen aminoterminal of ng/mL extends peptide antigenic solution；
The quality-control product include concentration be 25 ng/mL, 400 ng/mL total Type I collagen aminoterminal extend peptide antigenic solution；
The concentration that total Type I collagen aminoterminal of the biotin labeling extends the solution of peptide antibody is 1.5 μ g/ml；
The concentration of the nano magnetic microparticle suspending liquid of the marked by streptavidin is 0.5mg/ml；
The concentration that total Type I collagen aminoterminal of the horseradish peroxidase-labeled extends peptide antibody solution is 1.5 μ g/ml；
The substrate solution includes solution A and solution B, and solution A is containing 0.4% luminol, the aqueous solution of pH9.0, and solution B is
Contain 0.06%Na2B4O7, the aqueous solution of pH5.0.
Further, the calibration object, the quality-control product extend peptide antigen and 0.1M, pH by total Type I collagen aminoterminal
The Tris-HCl buffer that value is 7.2 obtains；Total Type I collagen aminoterminal extends peptide antigen purchased from many gloomy source biotechnologys
(Jiangsu) Co., Ltd, article No. 8001.
Further, total Type I collagen aminoterminal of the biotin labeling extends the preparation method of peptide antibody are as follows: by concentration
Extend peptide antibody for the total Type I collagen aminoterminal of 1mg/ml and be added to the biotin solution that concentration is 10mg/ml by 1:20 volume ratio)
In, it is then added into the phosphate buffer that 0.01M pH value is 7.2, during which liquid 3- is changed in 4 DEG C of dialysis 18 hours or more
4 times, change liquid interval 2 hours or more for the first time, be spaced 4 hours thereafter, dialysis complete after again with 0.01M, the phosphorus that pH value is 7.2
Concentration is adjusted after phthalate buffer dialysis to 1.5 μ g/ml；Total Type I collagen aminoterminal extends peptide antibody purchased from many gloomy source biology skills
Art (Jiangsu) Co., Ltd, article No. 8010.
Further, total Type I collagen aminoterminal of the horseradish peroxidase-labeled extends the preparation method of peptide antibody
Are as follows: it be that the total Type I collagen aminoterminal of 1mg/ml extends peptide antibody to be added to concentration by 1:20 volume ratio by concentration is 5.0mg/mL's
It in horseradish peroxidase solution, after mixing and purifies, the total Type I collagen aminoterminal for obtaining horseradish peroxidase-labeled extends
Peptide antibody, the purifying are that the bicarbonate buffer for being 8-9 with pH is balanced and eluted, then ultraviolet detection and record purifying are schemed
Spectrum, then peptide is extended to total Type I collagen aminoterminal of horseradish peroxidase-labeled with 0.05M, the MES buffer that pH value is 6.0
Antibody is diluted to 1.5 μ g/m；Total Type I collagen aminoterminal extends peptide antibody and is purchased from many gloomy source biotechnology (Jiangsu) Co., Ltds,
Article No. is 8011.
Further, the preparation process of the nano magnetic microparticle suspending liquid of the marked by streptavidin are as follows: first by strepto- parent
It is precipitated with the nanometer magnetic particle of element label with magnetic separator；Then in the case where removing magnetic field, precipitating is with 0.01M, pH value
7.3 phosphate buffer is resuspended, and mixes Magneto separate；Secondly, magnetic field is added, so that the nanometer magnetic particle of marked by streptavidin
It settles out；Again, cleaning precipitating；Finally, the nanometer magnetic particle of marked by streptavidin is scattered in 0.01M, pH value 7.3 after cleaning
Phosphate buffer；The nanometer magnetic particle of the marked by streptavidin is purchased from GE company, article No. 21152104010350.
Further, the kit further includes washing lotion, and the washing lotion is containing 1% tween, 0.1% Proclin300
Tris-HCl buffer, pH7.8.
Mentioned reagent box detects the method that the total Type I collagen aminoterminal of serum extends peptide, comprising the following steps:
(1) total Type I collagen aminoterminal of the nanometer magnetic particle of 50 μ l marked by streptavidin, 50 μ l biotin labelings is extended into peptide
Antibody, total Type I collagen aminoterminal extension peptide antibody of 50 μ l horseradish peroxidase-labeleds and 40 μ l serum sample to be checked mix
And react, reaction, which is placed in magnetic field, stands and goes supernatant, obtains the first solution；
(2) 200 μ l luminous substrates are added into first solution and react, survey luminous intensity.
(3) luminous intensity standard curve, the luminous intensity obtained according to step (3) are drawn using the calibration object of known concentration
The standard curve is compareed, the content that total Type I collagen aminoterminal in test serum sample extends peptide is calculated.
Further, the condition of reaction described in step (1) is to react 10 minutes at 37 DEG C, and the time of the standing is 2
Minute；Further include being cleaned to first solution with cleaning solution in step (1), is placed in magnetic field and stands and go supernatant, clearly
It washes and is repeated 3 times；
The condition of reaction described in step (2) is to react 5 minutes at 37 DEG C, measures and shines in chemiluminescence analysis/analyzer
Intensity.
Further, the intensity in magnetic field is 10000 Gausses (G) in the step (1).
The kit detection sensitivity that the total Type I collagen aminoterminal of detection serum of the invention extends peptide is high, specific performance is good,
It makes a variation small, each reagent component has good stability, and validity period can be to 1 year or more；The kit in clinical studies with external import
The correlation that meets of reagent is up to 90% or more, and expense be only its 1/2, can reduce patient's use cost, improving its popularization makes
With.
Detailed description of the invention
Fig. 1 is the canonical plotting that the kit that total Type I collagen aminoterminal extends peptide is detected in the embodiment of the present invention 1.
Fig. 2 is that the total Type I collagen aminoterminal of detection of the embodiment of the present invention 1 extends the kit of peptide and the property of import reagent
It can comparison diagram.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiments of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technical staff's all other embodiment obtained without making creative work belongs to the model that the present invention protects
It encloses.
Embodiment 1
A kind of total Type I collagen aminoterminal of detection serum extends the kit of peptide, which is characterized in that including calibration object, quality-control product, life
Total Type I collagen aminoterminal of object element label extends total Type I collagen aminoterminal of the solution of peptide antibody, horseradish peroxidase-labeled
Extend the solution of peptide antibody, the nano magnetic microparticle suspending liquid of marked by streptavidin；
The calibration object includes that concentration is 0 ng/mL, 10 ng/mL, 25 ng/mL, 100 ng/mL, 400 ng/mL, 1200
Total Type I collagen aminoterminal of ng/mL extends peptide antigenic solution, and total Type I collagen aminoterminal extends peptide antigen purchased from many gloomy source biologies
Technology (Jiangsu) Co., Ltd, article No. 8001；
The quality-control product includes total Type I collagen aminoterminal extension peptide antigenic solution of the concentration for 25 ng/mL, 400 ng/mL, institute
Calibration object is stated to be obtained by total Type I collagen aminoterminal extension peptide antigen and 0.1M, the Tris-HCl buffer that pH value is 7.2,
Total Type I collagen aminoterminal extends peptide antigen purchased from many gloomy source biotechnology (Jiangsu) Co., Ltds, article No. 8001；
The concentration that total Type I collagen aminoterminal of the biotin labeling extends peptide antibody solution is 1.5 μ g/ml, the biotin
Total Type I collagen aminoterminal of label extends the preparation method of peptide antibody are as follows: extends concentration for the total Type I collagen aminoterminal of 1mg/ml
Peptide antibody is added in the biotin solution (solvent is dimethyl formamide solution) that concentration is 10mg/ml by 1:20 volume ratio,
It is then added into the phosphate buffer that 0.01M pH value is 7.2, during which liquid 3-4 is changed in 4 DEG C of dialysis 18 hours or more
It is secondary, change liquid interval 2 hours or more for the first time, be spaced 4 hours thereafter, dialysis complete after again with 0.01M, the phosphorus that pH value is 7.2
Concentration is adjusted after phthalate buffer dialysis to 1.5 μ g/ml；Wherein, it is raw purchased from many gloomy sources to extend peptide antibody for total Type I collagen aminoterminal
Object technology (Jiangsu) Co., Ltd, article No. 8010, concentration 1mg/ml；
The concentration of the nano magnetic microparticle suspending liquid of the marked by streptavidin is 0.5mg/ml, the marked by streptavidin
The preparation process of nano magnetic microparticle suspending liquid are as follows: the nanometer magnetic particle of marked by streptavidin is precipitated with magnetic separator, then is used
0.01M, the phosphate buffer that pH value is 7.2 are resuspended, and mix Magneto separate and settle out, and repeated washing, the nano magnetic after cleaning are micro-
The nanometer magnetic particle of the phosphate buffer that grain is scattered in 0.01M, pH value is 7.2, the marked by streptavidin is public purchased from GE
Department, article No. 21152104010350；
The concentration that total Type I collagen aminoterminal of the horseradish peroxidase-labeled extends peptide antibody solution is 1.5 μ g/ml, institute
It is that can extend the anti-of peptide specific bond with Type I collagen aminoterminal in human body that the total Type I collagen aminoterminal for stating label, which extends peptide antibody,
Body, total Type I collagen aminoterminal of the horseradish peroxidase-labeled extend the preparation method of peptide antibody are as follows: by concentration are 1mg/
The total Type I collagen aminoterminal of ml extends peptide antibody, and by 1:20 volume ratio to be added to the horseradish peroxidase that concentration is 5.0mg/mL molten
It in liquid, after mixing and purifies, the total Type I collagen aminoterminal for obtaining horseradish peroxidase-labeled extends peptide antibody, purification process
Middle selection Sephadexg200 column, the preferred bicarbonate buffer for being 8-9 with pH balance cylinder, then use labelled antibody and add
Sample needle slowly injects cylinder (bubble is avoided to generate), finally elutes cylinder, ultraviolet inspection with the bicarbonate buffer that pH is 8-9 again
Survey and record purifying map, then with 0.05M, pH value for 6.0 MES buffer to total I type glue of horseradish peroxidase-labeled
Former aminoterminal extends peptide antibody and is diluted to 1.5 μ g/m；Total Type I collagen aminoterminal extends peptide antibody purchased from many gloomy source biotechnologys
(Jiangsu) Co., Ltd, article No. 8011, concentration are 1.5 μ g/ml.
Embodiment 2
The antibody of the biotin labeling of various concentration and the antibody of horseradish peroxidase-labeled are prepared respectively, by biotin labeling
Antibody and horseradish peroxidase-labeled antibody by concentration than grouping, measure respectively its P1NP concentration be 0,100,1200
Luminous value when ng/ml, the results are shown in Table 1.
Table 1: influence of the anti-reagent concentration of various concentration to luminous value.
Evaluation of result: the antibody of biotin labeling and the antibody concentration of horseradish peroxidase-labeled are respectively 1.5 μ g/
When ml, 1.5 μ g/ml, high concentration and low concentration calibration product luminous value ratio are maximum, therefore select this concentration than the work for subsequent experimental
Make concentration.
Different sample-adding amounts and different warm bath times are further detected to reaction system according to the best anti-reagent concentration of screening
Influence, as a result as shown in table 2, table 3.
Influence of the different anti-reagent volumes of table 2 to luminous value.
Influence of the different anti-reagent incubation times of table 3 to luminous value.
Evaluation of result: when anti-reagent pipetting volume amount is 50 μ l, high concentration and low concentration calibration product luminous value ratio are maximum, choose
50 μ l are as the anti-reagent pipetting volume amount of subsequent experimental；When the anti-reagent warm bath time is 10 min, high concentration and low concentration calibration product are sent out
Light value ratio is maximum, and 10 min of selection are the subsequent experimental warm bath time.
Embodiment 3
The total Type I collagen aminoterminal of kit detection serum of above-described embodiment 1 extends the method for peptide, comprising the following steps:
(1) 40 μ l test serums are sequentially added in detection pipe or total Type I collagen aminoterminal extends peptide calibration object, 50 μ l biologies
Total Type I collagen aminoterminal of element label extends the nanometer magnetic particle and 50 μ l horseradish mistakes of peptide antibody, 50 μ l marked by streptavidin
Total Type I collagen aminoterminal of oxide enzyme label extends peptide antibody, after mixing, is incubated for altogether at 37 DEG C 10 minutes, I type in sample
It is captured that collagenous amino end extends peptide, and anchors at nano magnetic microparticle surfaces；Then under the action of an external magnetic field, the intensity in magnetic field
For 10000 Gausses, 2 minutes are stood, magnetic particle settles down, and removes supernatant, 500 μ l cleaning solutions are added, after removing magnetic field, concussion
Magnetic particle is resuspended；This cleaning step is repeated 3 times to remove unbonded enzyme labelled antibody and other impurity, obtains the first solution.
(2) 200 μ l enzyme-catalyzed chemical luminescence substrates are added, removes magnetic field, is incubated for 5 minutes at 37 DEG C, after mixing well, measurement
Luminous value.
(3) processing of data: by concentration value and the luminous value detected of calibration object, pass through four parametrical nonlinearities and be fitted
Standard curve is obtained, the luminous value of sample is substituted into standard curve shown in FIG. 1 and obtains corresponding concentration value.
The working principle of the invention: total Type I collagen aminoterminal of test serum sample and biotin labeling extension peptide antibody,
The nanometer magnetic particle of marked by streptavidin and total Type I collagen aminoterminal of horseradish peroxidase-labeled extend peptide antibody and incubate altogether
It educates, Type I collagen aminoterminal extends peptide in test serum sample and total Type I collagen aminoterminal of biotin labeling extends peptide antibody spy
The opposite sex combines, while by biotin-Streptavidin amplification system, and it is special to extend peptide for total Type I collagen aminoterminal in serum sample
The opposite sex is integrated on solid phase carrier nanometer magnetic particle, and the total Type I collagen aminoterminal for forming antibiotin label extends peptide antibody-I
Collagen Type VI aminoterminal extends the immunocomplex formation that the total Type I collagen aminoterminal of peptide-extends peptide antibody；Under externally-applied magnetic field effect,
Total Type I collagen aminoterminal that Magneto separate washing removes the biotin labeling of extra biotin labeling extends peptide antibody；Additional magnetic
Under field action, multiple Magneto separate washing removal unbonded enzyme labelled antibody and other impurity；Enzyme-catalyzed chemical luminescence substrate is added, surveys
Determine luminous intensity；It is calculated to be measured using the calibration object standard curve of known concentration according to luminous intensity reference standard curve
Total Type I collagen aminoterminal extends the content of peptide in sample；
Embodiment 4
The Performance Evaluation of the kit:
1.1 appearance
It is visually inspected under available light, the magnetic particle reagent in kit should be uniform suspension after shaking up, without obvious agglutination；
Other liquid reagent solution components should be clarified, foreign, sediment and floccule.
1.2 minimum detection limit
Detection 20 times is repeated to " 0 " concentration calibration product (S0), the average value M and standard deviation SD of 20 luminous values are calculated, according to the school A
Two o'clock regression fit is carried out between adjacent B calibration point on schedule and goes out linear function, then brings M+2SD into above-mentioned equation, institute
Corresponding concentration value is minimum detection limit.
1.3 accuracy
Detection 3 times is respectively repeated to P1NP enterprise reference material 1 and P1NP enterprise reference material 2, the average value for measuring concentration results is denoted as
(Mi), relative deviation (Bi) is calculated separately according to formula (1)
Formula (1) Bi=(Mi- T)/T × 100%
In formula: Bi- relative deviation；
MiThe average value of-measurement concentration；
T-calibration concentration.
Formula (1) Bi=(Mi- T)/T × 100%
In formula: Bi- relative deviation；
MiThe average value of-measurement concentration；
T-calibration concentration.
1.4 repeated (withinrun precisions)
Peptide antigenic solution is extended to total Type I collagen aminoterminal of P1NP enterprise reference material 1(25 ng/mL) and the reference of P1NP enterprise
Total Type I collagen aminoterminal of product 2(400ng/mL extends peptide antigenic solution) it is repetition detection 10 times each, calculate 10 measurement concentration knots
The average value M and standard deviation SD of fruit calculate the coefficient of variation according to formula CV=SD/M × 100%,
1.5 difference between batch (betweenrun precision)
Detection 10 times is repeated to same portion P1NP enterprise reference material 2 respectively with three batch kits, calculates 30 measurement concentration knots
The average value M and standard deviation SD of fruit calculate the coefficient of variation according to formula CV=SD/M × 100%.
1.6 37 DEG C of thermal stability
It takes and is placed in 37 DEG C of insulating boxs with a collection of 3 box of kit, respectively on day 1, the 4th day, taken out within the 7th day, the examination of taking-up
It is temporary that agent box is placed on 2-8 DEG C of refrigerator.37 DEG C destroy 1 day, 4 days, 7 days reagents measure each 3 parallel pipes of calibration object.Control group
The same lot number reagent saved for 4 DEG C.Each calibration point replication 2 times when test.Calculate signal retention rate.
1.7 linear
The high level sample close to the range of linearity upper limit is taken to be diluted to 5 kinds of concentration by a certain percentage, wherein the sample palpus of low value concentration
Close to the lower limit of the range of linearity, the sample standard deviation of each concentration is repeated to detect 3 times, calculates its average value, by result average value and
Dilution ratio carries out straight line fitting with least square method, and calculates linearly dependent coefficient r
1.8 anti-interference ability
Using the unconjugated bilirubin, hemoglobin, triglycerides of various concentration, it is added in serum specimen and forms interference sample,
Each run needs to carry out the measurement of calibration curve twice, each replication of every part of sample 3 times.Calculated result deviation, according to
Influence of the result judgement to osteocalcin levels.
1.9 specific
To β-CrossLaps, N-MID calcium element, parathormone (PTH) and 25-OH- vitamin D are diluted to calibration object 1 respectively
1 μ g/mL or so.Then 3 high concentrations are detected with 3 batch kits and recombinates sample.Testing result is not higher than 1 ng/mL
Detect cross reaction.
1, total Type I collagen aminoterminal extends peptide detection kit analysis performance and stability, and the results are shown in Table 4.
Table 4 is analysis performance and stability result.
2, referring to Fig. 2, showing total Type I collagen aminoterminal extension peptide (tP1NP) detection kit, (nanometer magnetic particle is complete
Robotics luminescence method) with the performance comparison of import reagent.
Peptide detection kit is extended using the kit of above-described embodiment 1 and total Type I collagen aminoterminal of Roche company
(article No. is 03,141,071 190) is respectively measured 79 parts of clinical serums of clinical laboratory of Wuxi City the People's Hospital sampling, instrument
Measurement result for Elecsys E601, the two compares and analyzes, as a result as schemed: the two dependent equation are as follows: y=1.0166x
- 1.8106, coefficient R=0.99, P < 0.001.Show that this detection system and import correlation-detection system correlation are good.
The invention has the benefit that
1, with horseradish peroxidase (HRP) for marker enzyme, by chemically reacting labelled antibody, and not using gel chromatography separation
Enzyme, antibody or the antigen of reaction, improve the sensitivity of reaction；
2, using immune magnetic particle as solid phase, with Streptavidin conjugated magnetic microballoon, as general separation agent, not only make to exempt from
Epidemic disease reaction is easier to mix and separate, and substantially increases reaction speed；
3, using chemiluminescent substrate HRP as substrate, which is aura substrate, and is rapidly achieved plateau, is conducive to letter
Number detection, improve the sensitivity and specificity performance of final kit；And chemiluminescent enhancement system has been advanced optimized,
It ensure that signal sensitivity height and the stability of finished product are good, variation is small；
4, the advantages of HRP substrate, is that high sensitivity and platform stationary phase are long.
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright description is applied directly or indirectly in other relevant technology necks
Domain is included within the scope of the present invention.
Claims (8)
1. the kit that a kind of total Type I collagen aminoterminal of detection serum extends peptide, which is characterized in that including calibration object, quality-control product,
Total Type I collagen aminoterminal of biotin labeling extends total Type I collagen amino of the solution of peptide antibody, horseradish peroxidase-labeled
End extends the nano magnetic microparticle suspending liquid and substrate of the solution of peptide antibody, marked by streptavidin；
The calibration object includes that concentration is 0 ng/mL, 10 ng/mL, 25 ng/mL, 100 ng/mL, 400 ng/mL, 1200
Total Type I collagen aminoterminal of ng/mL extends peptide antigenic solution；
The quality-control product include concentration be 25 ng/mL, 400 ng/mL total Type I collagen aminoterminal extend peptide antigenic solution；
The concentration that total Type I collagen aminoterminal of the biotin labeling extends the solution of peptide antibody is 1.5 μ g/ml；
The concentration of the nano magnetic microparticle suspending liquid of the marked by streptavidin is 0.5mg/ml；
The concentration that total Type I collagen aminoterminal of the horseradish peroxidase-labeled extends peptide antibody solution is 1.5 μ g/ml；
The substrate solution includes solution A and solution B, and solution A is containing 0.4% luminol, the aqueous solution of pH9.0, and solution B is
Contain 0.06%Na2B4O7, the aqueous solution of pH5.0.
2. a kind of total Type I collagen aminoterminal of detection serum according to claim 1 extends the kit of peptide, feature exists
In: the calibration object, the quality-control product extend peptide antigen and 0.1M, the Tris- that pH value is 7.2 by total Type I collagen aminoterminal
HCl buffer obtains；Total Type I collagen aminoterminal extends peptide antigen and is purchased from many gloomy source biotechnology (Jiangsu) Co., Ltds,
Article No. is 8001.
3. a kind of total Type I collagen aminoterminal of detection serum according to claim 1 extends the kit of peptide, feature exists
Extend the preparation method of peptide antibody in total Type I collagen aminoterminal of: the biotin labeling are as follows: by concentration be total I type of 1mg/ml
Collagenous amino end extends peptide antibody and is added to the biotin solution that concentration is 10mg/ml by 1:20 volume ratio) in, then added
Enter in the phosphate buffer for being 7.2 to 0.01M pH value, during which 4 DEG C of dialysis 18 hours or more are changed liquid 3-4 times, changed for the first time
Liquid interval 2 hours or more is spaced 4 hours thereafter, and dialysis is saturating with 0.01M, phosphate buffer that pH value is 7.2 again after completing
Concentration is adjusted after analysis to 1.5 μ g/ml；It is limited purchased from many gloomy source biotechnologys (Jiangsu) that total Type I collagen aminoterminal extends peptide antibody
Company, article No. 8010.
4. a kind of total Type I collagen aminoterminal of detection serum according to claim 1 extends the kit of peptide, feature exists
Extend the preparation method of peptide antibody in total Type I collagen aminoterminal of: the horseradish peroxidase-labeled are as follows: by concentration be 1mg/
The total Type I collagen aminoterminal of ml extends peptide antibody, and by 1:20 volume ratio to be added to the horseradish peroxidase that concentration is 5.0mg/mL molten
It in liquid, after mixing and purifies, the total Type I collagen aminoterminal for obtaining horseradish peroxidase-labeled extends peptide antibody, the purifying
It is that the bicarbonate buffer for being 8-9 with pH is balanced and eluted, then ultraviolet detection and record purifying map, then with 0.05M, pH value
1.5 μ g/m are diluted to total Type I collagen aminoterminal extension peptide antibody of horseradish peroxidase-labeled for 6.0 MES buffer；
Total Type I collagen aminoterminal extends peptide antibody purchased from many gloomy source biotechnology (Jiangsu) Co., Ltds, article No. 8011.
5. a kind of total Type I collagen aminoterminal of detection serum according to claim 1 extends the kit of peptide, feature exists
In: the preparation process of the nano magnetic microparticle suspending liquid of the marked by streptavidin are as follows: first by the nanometer of marked by streptavidin
Magnetic particle is precipitated with magnetic separator；Then in the case where removing magnetic field, precipitating 0.01M, the phosphate-buffered that pH value is 7.3
Liquid is resuspended, and mixes Magneto separate；Secondly, magnetic field is added, so that the nanometer magnetic particle of marked by streptavidin settles out；Again, it cleans
Precipitating；Finally, the phosphate-buffered that the nanometer magnetic particle of marked by streptavidin is scattered in 0.01M after cleaning, pH value is 7.3
Liquid；The nanometer magnetic particle of the marked by streptavidin is purchased from GE company, article No. 21152104010350.
6. a kind of total Type I collagen aminoterminal of detection serum according to claim 1 extends the kit of peptide, feature exists
In: the kit further includes washing lotion, and the washing lotion is the Tris-HCl buffer containing 1% tween, 0.1% Proclin300,
pH7.8。
7. extend the method for peptide using the total Type I collagen aminoterminal of any kit detection serum in claim 1 ~ 6, including
Following steps:
(1) total Type I collagen aminoterminal of the nanometer magnetic particle of 50 μ l marked by streptavidin, 50 μ l biotin labelings is extended into peptide
Antibody, total Type I collagen aminoterminal extension peptide antibody of 50 μ l horseradish peroxidase-labeleds and 40 μ l serum sample to be checked mix
And react, reaction, which is placed in magnetic field, stands and goes supernatant, obtains the first solution；
(2) 200 μ l luminous substrates are added into first solution and react, survey luminous intensity；
(3) luminous intensity standard curve is drawn using the calibration object of known concentration, is compareed according to the luminous intensity that step (3) obtains
The content that total Type I collagen aminoterminal in test serum sample extends peptide is calculated in the standard curve.
8. the method that the total Type I collagen aminoterminal of detection serum according to claim 7 extends peptide, it is characterised in that: step
(1) condition of reaction described in is to react 10 minutes at 37 DEG C, and the time of the standing is 2 minutes；Step further includes pair in (1)
First solution is cleaned with cleaning solution, is placed in magnetic field and is stood and go supernatant, and cleaning is repeated 3 times；
The condition of reaction described in step (2) is to react 5 minutes at 37 DEG C, measures and shines in chemiluminescence analysis/analyzer
Intensity.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810699124.1A CN109061176A (en) | 2018-06-29 | 2018-06-29 | A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810699124.1A CN109061176A (en) | 2018-06-29 | 2018-06-29 | A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109061176A true CN109061176A (en) | 2018-12-21 |
Family
ID=64818516
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810699124.1A Pending CN109061176A (en) | 2018-06-29 | 2018-06-29 | A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109061176A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111007269A (en) * | 2019-11-15 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Total I type collagen amino-terminal extension peptide chemiluminescence immunoassay kit and preparation method thereof |
| CN112326972A (en) * | 2020-10-30 | 2021-02-05 | 安徽理工大学 | Chemiluminescence quantitative detection kit for detecting complete PINP in serum |
| CN113933497A (en) * | 2021-11-03 | 2022-01-14 | 辽宁中医药大学附属医院 | I type collagen amino-terminal extension peptide magnetic particle chemiluminescence immunoassay kit and detection method thereof |
| CN116203243A (en) * | 2022-09-13 | 2023-06-02 | 美康生物科技股份有限公司 | PINP monoclonal antibody, kit containing PINP monoclonal antibody and application of PINP monoclonal antibody |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100317008A1 (en) * | 2006-05-09 | 2010-12-16 | Beckman Coulter, Inc. | Nonseparation Assay Methods |
| CN103278623A (en) * | 2013-06-14 | 2013-09-04 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemiluminescence immunity quantitative detection of CK-MB (creatine kinase- isoenzyme) nano magnetic particle and preparation method of kit |
| WO2013169890A1 (en) * | 2012-05-08 | 2013-11-14 | Bg Medicine, Inc. | Systems and methods for assessing disease risk, status, and prognosis |
| CN105759055A (en) * | 2016-03-31 | 2016-07-13 | 广州菲康生物技术有限公司 | P1NP detection kit and preparation method thereof |
-
2018
- 2018-06-29 CN CN201810699124.1A patent/CN109061176A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100317008A1 (en) * | 2006-05-09 | 2010-12-16 | Beckman Coulter, Inc. | Nonseparation Assay Methods |
| WO2013169890A1 (en) * | 2012-05-08 | 2013-11-14 | Bg Medicine, Inc. | Systems and methods for assessing disease risk, status, and prognosis |
| CN103278623A (en) * | 2013-06-14 | 2013-09-04 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemiluminescence immunity quantitative detection of CK-MB (creatine kinase- isoenzyme) nano magnetic particle and preparation method of kit |
| CN105759055A (en) * | 2016-03-31 | 2016-07-13 | 广州菲康生物技术有限公司 | P1NP detection kit and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| C H JENSEN ET AL.: "Quantification of the N-terminal propeptide of human procollagen type I (PINP): comparison of ELISA and RIA with respect to different molecular forms", 《CLINICA CHIMICA ACTA》 * |
| 李丽: "血清总Ⅰ型胶原氨基端延长肽和Ⅰ型胶原羧基端肽在骨质疏松症并发骨折检测的临床意义", 《黑龙江医药》 * |
| 汪美先: "《免疫学基础》", 31 May 1981, 陕西科学技术出版社 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111007269A (en) * | 2019-11-15 | 2020-04-14 | 迪瑞医疗科技股份有限公司 | Total I type collagen amino-terminal extension peptide chemiluminescence immunoassay kit and preparation method thereof |
| CN112326972A (en) * | 2020-10-30 | 2021-02-05 | 安徽理工大学 | Chemiluminescence quantitative detection kit for detecting complete PINP in serum |
| CN113933497A (en) * | 2021-11-03 | 2022-01-14 | 辽宁中医药大学附属医院 | I type collagen amino-terminal extension peptide magnetic particle chemiluminescence immunoassay kit and detection method thereof |
| CN116203243A (en) * | 2022-09-13 | 2023-06-02 | 美康生物科技股份有限公司 | PINP monoclonal antibody, kit containing PINP monoclonal antibody and application of PINP monoclonal antibody |
| CN116203243B (en) * | 2022-09-13 | 2024-07-02 | 美康生物科技股份有限公司 | PINP monoclonal antibody, kit containing PINP monoclonal antibody and application of PINP monoclonal antibody |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103364568B (en) | Laminin nano-magnetic particle chemiluminescent immunity quantitative detection kit and preparation method thereof | |
| CN109061176A (en) | A kind of total Type I collagen aminoterminal of detection serum extends the kit and application method of peptide | |
| CN104634980B (en) | The super quick detection kit of cardiac muscle troponin I and super quick detection method | |
| CN105190311B (en) | Assay with increased dynamic range | |
| CN102998467B (en) | β human chorionic gonadotrophin magnetic microparticle chemiluminescence immune quantitative detection reagent box and preparation method thereof | |
| CN104849469A (en) | Kit for detecting NGAL content and preparation method thereof | |
| CN109061175A (en) | A kind of kit and application method detecting osteocalcin | |
| CN104730247A (en) | Kit suitable for rapidly detecting AMH and INHB by using double-tagging time resolution fluorescence immunoassay method and use method of kit | |
| CN102998465B (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for angiotensin (Ang) I, and preparation method of kit | |
| CN109521004A (en) | For detecting the magnetic microparticle separating chemiluminescence immunoassay of glial fibrillary acid protein (GFAP) | |
| CN106771147B (en) | A kind of quick diagnosis platelet-activating factor acetylhydro-lase kit and its application method | |
| CN108445222A (en) | A kind of kit and preparation method quantitatively detecting cardic fatty acid binding protein | |
| CN101363860A (en) | Syphilis helicoid antibody chemiluminescence immune assay determination kit and method for preparing same | |
| CN101178405B (en) | Tumor-associated antigen 50 chemiluminescence immune analyze quantitative determination reagent box and method of producing the same | |
| CN108445230B (en) | Procalcitonin chemiluminescence detection reagent based on nano antibody and detection method | |
| CN106918708A (en) | A kind of competition law turbid kit of latex enhancing immune transmittance for detecting insulin | |
| CN105277690A (en) | Reagent kit and method for full-automatically measuring antiprotease 3 antibody IgG | |
| CN104237513A (en) | Thyroid peroxidase antibody magnetic-particle chemiluminescence immune quantitative testing kit | |
| CN103033624A (en) | Human myeloperoxidase chemiluminescent immunodetection kit | |
| CN103698535A (en) | Quantitative detection kit of lipoprotein associated phospholipase A2, as well as preparation and operation method of quantitative detection kit | |
| CN108152507A (en) | A kind of I type precollagen amino terminal peptides detection kit and its detection method | |
| CN102998466A (en) | Quantitative detection kit combining magnetic particles with chemiluminescence immunoassay for growth hormone (GH), and preparation method of kit | |
| CN109187971A (en) | Neuronspecific enolase chemiluminescence immune detection reagent kit and preparation method thereof | |
| CN107044977A (en) | A kind of tyrosine phosphatase antibody chemical luminescence immunity detection reagent and preparation method thereof | |
| CN107957495A (en) | A kind of CK-MB detection kits and its application method |
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: 20181221 |