CN108613977A - A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit and its detection method - Google Patents
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit and its detection method Download PDFInfo
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- 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
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/82—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a precipitate or turbidity
- G01N2021/825—Agglutination
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
Abstract
The invention discloses a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit and its detection methods；Kit, including：Reagent one：Phosphate buffer, preservative, polysorbas20, Macrogol 6000；Reagent two：Phosphate buffer, disodium hydrogen phosphate, 2 5g/L sodium dihydrogen phosphates, sodium chloride, preservative, the sensitization latex particle of b-type natriuretic peptide antibody before goat-anti people's N-terminal；Calibration object：Phosphate buffer, bovine serum albumin(BSA), sodium chloride, preservative, b-type natriuretic peptide antigen before N-terminal；Method application latex immunoturbidimetry improves testability, finds the sensitization latex particle for being suitble to detection N-terminal plasma pro-brain natriuretic peptide levels, improves detection sensitivity；Coordinate different reagent sample ratios by different automatic clinical chemistry analyzers, it can simultaneous quantitative detection high-volume sample；Calibration object is included using kit, achievees the purpose that quantitative detection.
Description
Technical field
The present invention relates to in-vitro diagnosis field, especially a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit and its detection side
Method.
Background technology
External diagnosis reagent refers to：It can be used alone or used with instrument, utensil, equipment or system in combination, in disease
During prevention, diagnosis, Treatment monitoring, Observation On The Prognosis, health status evaluation and the prediction of genetic disease, for human body
Sample (various body fluid, cell, tissue samples etc.) carries out reagent, kit, calibration object (object), the quality-control product (object) of vitro detection
Deng.
According to the height of product risks degree, external diagnosis reagent is divided into third class, the second class, first kind product.
Third class product：With the relevant reagent of the detection such as pathogenic pathogen antigen, antibody and nucleic acid；With blood group, tissue matching's phase
The reagent of pass；Relevant reagent is detected with human gene；With the relevant reagent of genetic disease；With narcotics, psychotropic substances,
Medical toxicant detects relevant reagent；Relevant reagent is detected with drug targets for therapy；With tumor-marker quality testing
Survey relevant reagent；With allergy (anaphylactogen) relevant reagent.Second class product：Except clearly be third class, the first kind
Product, other be the second class product, include mainly：Reagent for protein detection；Reagent for carbohydrate detection；For
The reagent of hormone test；Reagent for enzyme detection；Reagent for esters detection；Reagent for vitamin detection；With
In the reagent of inorganic ions detection；For drug and the reagent of drug metabolism analyte detection；Reagent for autoantibody detection；With
In the reagent of microorganism discriminating or drug sensitive test；Reagent for the detection of other physiology, biochemistry or immune function.The first kind
Product：Microbiological culture media (is not used in microorganism discriminating and drug sensitive test)；Sample process product, as hemolytic agent, dilution,
Dyeing liquor etc..
NT-proBNP is inactive N-terminal segment after BNP prohormone (proBNP) division, mainly cardiac muscle cell by
Volume load and pressure load are secreted when increasing by left ventricle.NT-proBNP can be used for evaluate cardiac systolic function it is incomplete and
Diastolic Heart failure and ventricle wall segmentation movement harmony, there is higher sensibility and negative predictive values；It is sent out applied to early stage
The effect of showing heart failure (HF) patient, and carry out risk stratification, monitoring heart failure medications is assessed, and judges the prognosis of Patients with Cardiac Failure,
Distinguish expiratory dyspnea caused by heart failure and other reasons；It can be used as the risk assessment index of acute coronary syndrome (ACS).
NT-proBNP is a kind of molecule that can facilitate processing in the lab, possesses advantage with when analyzing before analysis, than
As all quite stable, blood sample require not tight at different temperatures.At present related NT-proBNP detection methods have colloidal gold method,
On turn luminescence method, immunofluorescence technique etc..
Colloidal gold method be by gold chloride (HAuCl4) reducing agent such as white phosphorus, ascorbic acid, sodium citrate, the tannic acid the effects that
Under, a certain size gold particle is can be grouped to, and since electrostatic interaction becomes a kind of colloidal state of stabilization, formed electronegative
Hydrophobic sol solution becomes stable colloidal state due to electrostatic interaction, therefore claims colloidal gold.Method detection is easier, and disadvantage exists
It, can only single sample detection in that can only qualitatively judge.
On to turn luminescence method be the above forwarding luminescent material (up-converting phosphor, UCP) particle as tracer
A kind of novel detection technique.After it uses the detection immune response that immunochromatography principle carries out immunological marker object, pass through
Sensor quantifies wipe paper item detection band and the UCP markers on quality control band, is realized with detecting band and accusing the quantitative ratio of band
The quantitative analysis of object to be checked.It is divided into dynamic scan and static detection two types according to its operation principle.Disadvantage is to lack at present
Few matched automatic detection system can not achieve the demand of batch pattern detection using the fields POCT are only limitted to.
Immunofluorescence technique carries out the technology of antigen positioning with fluorescent material labelled antibody.With fluorescence antibody tracer or inspection
The method of corresponding antigens claims fluorescence anti-body method；Claimed with known fluorescent antigen marker tracer or the method for checking corresponding antibodies glimmering
Light antigen method.Both methods general name immunofluorescence technique.The method unspecific staining problem is not yet fully solved, result judgement
Objectivity it is insufficient, technical program is yet more complicated.
Three kinds of methods is complicated for operation above, and time-consuming, and instrument and equipment requires high, and the limited sample size of detection now removes
It is measured in certain special laboratories outer, is clinically difficult to realize the automatic detection of extensive sample.
Latex immunoturbidimetry is that the corresponding antibody of test substance is coated on latex particle, and antigen-antibody is made to combine
The volume of object increases, and after light passes through, the Strength Changes of transmitted light and scattering light are more notable, to improve the sensitivity of experiment
Property.
But detection N-terminal plasma pro-brain natriuretic peptide levels find the ideal sensitization latex particle of effect not yet, and the present invention solves in this way
The problem of；Clinically latex immunoturbidimetry is difficult to realize at present matches with automatic clinical chemistry analyzer, and detection is large quantities of
Sample is measured, and result is mostly qualitative；The present invention solves this problem.
Invention content
To solve the deficiencies in the prior art, the purpose of the present invention is to provide a kind of N-terminal plasma pro-brain natriuretic peptide levels detection reagents
Box and its detection method；Present invention application latex immunoturbidimetry improves testability, finds and is suitble to detection N-terminal brain natriuretic peptide
The sensitization latex particle of precursor improves detection sensitivity；Coordinate different reagent sample ratios by different automatic clinical chemistry analyzers,
It can simultaneous quantitative detection high-volume sample；Calibration object is included using kit, achievees the purpose that quantitative detection.
In order to realize that above-mentioned target, the present invention adopt the following technical scheme that：
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit, including：
Reagent one：
Phosphate buffer 1 00-150mmol/L,
Preservative 0.005%-0.06%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer 1 00-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer 1 00-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned,
Reagent one：
Phosphate buffer (PH=6.5) 100-150mmol/L,
Biological preservative Proclin300 0.005%-0.06%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.005%-0.06%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.005%-0.06%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned,
Reagent one：
Phosphate buffer (PH=6.5) 100-150mmol/L,
Biological preservative Proclin300 0.02%-0.05%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.02%-0.05%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.02%-0.05%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned, the sensitization of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody
The diameter range of latex particle is 20-60nm.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned, the sensitization of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody
The diameter range of latex particle is 40nm.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned, reagent one, reagent two, calibration object metered proportions be
8:2:1。
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned, according to Beckman series automatic clinical chemistry analyzer,
The group of reagent sample is divided into：One 160 μ l of reagent, 2 40 μ l of reagent, 20 μ l of calibration object.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned, according to the remarkable serial full-automatic biochemical analysis of China of section
The group of instrument, reagent sample is divided into：One 240 μ l of reagent, 2 60 μ l of reagent, 30 μ l of calibration object.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit above-mentioned, according to Hitachi's series automatic clinical chemistry analyzer, examination
The group of agent sample is divided into：One 128 μ l of reagent：2 32 μ l of reagent：16 μ l of calibration object.
A kind of detection method of N-terminal plasma pro-brain natriuretic peptide levels detection kit, which is characterized in that include the following steps：
Step 1：Parameter list is corresponded to according to automatic clinical chemistry analyzer, in instrument parameter interface arrange parameter；
Step 2：Blood samples of patients is acquired, is centrifuged in 30 minutes, takes supernatant as detection sample, according to parameter setting sample
Grade is put into sample disc；
Step 3：According to parameter setting reagent position, in reagent disc, it is respectively and correspondingly placed into reagent one and reagent two, simultaneously
Counter sample position is put into calibration object in sample disc；
Reagent one：
Phosphate buffer (PH=6.5) 100-150mmol/L,
Preservative 0.005%-0.06%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-；
Step 4：Start calibration test, passes through calibration；
Step 5：Start pattern detection, by reagent needle and sample needle, draws reagent one and sample respectively, be put into reaction
Cup stirs reagent I and sample, reacts 5 minutes；
Step 6：By reagent needle and sample needle, reagent two and sample are drawn respectively, is put into reaction cup, stirred, be incubated 5
Minute, allow NT-proBNP (antigen) and the anti-NT-proBNP (antibody) of specificity in sample to meet in the liquid phase, formation is insoluble
Property antigen-antibody complex, generate turbidity；
Step 7：According to specific absorbance changes of the NT-proBNP at 570nm, the suction of NT-proBNP in sample is measured
Light varience value, by compared with kit internal calibration product concentration and calibration object absorbance change value, you can be calculate by the following formula
Go out the content of NT-proBNP in sample；
The positive of NT-proBNP defines value：Age<50 years old patients and serum ＜ 450ng/L；Age is at 50-75 Sui
Patient and serum ＜ 900ng/L；75 years old patient of age ＞ and serum ＜ 1800ng/L.
The invention has the beneficial effects that：
N-terminal plasma pro-brain natriuretic peptide levels (NT-proBNP) detection kit enhances immunoassay using particulate:In sample
NT-proBNP (antigen) and the anti-NT-proBNP (antibody) of specificity meet in the liquid phase, and it is anti-to form insoluble antigen-immediately
Nanocrystal composition generates certain turbidity.The height of turbidity is directly proportional to the content of NT-proBNP in sample, by locating with same
The calibration object of reason compares, you can calculates the content of NT-proBNP in sample；
This kit uses 15-60nm latexes, mostly anti-in conjunction with sheep, forms sensitization latex particle, detection sensitivity is made to reach
1ng/L；
This kit improves the stabilization of kit using the Proclin300 biological preservatives of 0.02-0.05 concentration ranges
Property；
The present invention coordinates different reagent sample ratios by different automatic clinical chemistry analyzers, can simultaneous quantitative detection high-volume
Sample；
The kit that the present invention uses includes calibration object, achievees the purpose that quantitative detection.
Description of the drawings
Fig. 1 is the scatter plot of the testing result of sample of the present invention patients serum；
Fig. 2 is the testing result of sample of the present invention patients serum according to tactic scatter plot from small to large；
Fig. 3 is the ends N--preceding b-type natriuretic peptide distribution of results figure of sample of the present invention patients serum.
Specific implementation mode
Specific introduce is made to the present invention below in conjunction with the drawings and specific embodiments.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit, including：
Reagent one：
Phosphate buffer (PH=6.5) 100-150mmol/L,
Preservative 0.005%-0.06%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer (PH=8.0) 100-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-.
It should be noted that：The raw material that instant component uses is purchased by outside and is obtained, specific such as table one without preparing
It is shown：
Table one
A kind of detection method of N-terminal plasma pro-brain natriuretic peptide levels detection kit, includes the following steps：
Step 1：Parameter list is corresponded to according to automatic clinical chemistry analyzer, in instrument parameter interface arrange parameter.
Step 2：Blood samples of patients is acquired, is centrifuged in 30 minutes, takes supernatant as detection sample, according to parameter setting sample
Grade is put into sample disc.
Step 3：According to parameter setting reagent position, in reagent disc, it is respectively and correspondingly placed into reagent one and reagent two, simultaneously
Counter sample position is put into calibration object in sample disc.
Step 4：Start calibration test, passes through calibration.
Step 5：Start pattern detection, by reagent needle and sample needle, draws reagent one and sample respectively, be put into reaction
Cup stirs reagent I and sample, reacts 5 minutes；It should be noted that such operation can reduce interfering substance pair in sample
The influence of NT-proBNP detections.
Step 6：By reagent needle and sample needle, reagent two and sample are drawn respectively, is put into reaction cup, stirred, be incubated 5
Minute, allow NT-proBNP (antigen) and the anti-NT-proBNP (antibody) of specificity in sample to meet in the liquid phase, formation is insoluble
Property antigen-antibody complex, generate turbidity；It should be noted that：The height of turbidity is directly proportional to the content of NT-BNP in sample.
Step 7：According to specific absorbance changes of the NT-proBNP at 570nm, the suction of NT-proBNP in sample is measured
Light varience value, by compared with kit internal calibration product concentration and calibration object absorbance change value, you can be calculate by the following formula
Go out the content of NT-proBNP in sample；
The content of N-terminal plasma pro-brain natriuretic peptide levels (NT-proBNP) is related with patient age, passes through 200 healthy individuals samples
Verification,
Verification process is as follows：
One, it is selected in sample requirement and experimental method
Because the content of N-terminal brain natriuretic peptide (NT-PROBNP) is related with patient age, this experiment carries out sample packet acquisition
Test.It is divided into three groups according to the age：<50 years old patients serums；50-75 Sui patients serum；75 years old patients serum of ＞.
Healthy Human Serum sample standard deviation picks up from physical examination normal person's sample of hospital laboratory, and sample requires fresh no haemolysis, nothing
It is jaundice, turbid without fat.
Sample size：<Patient men and women tests 200 parts of samples within 50 years old；50-75 Sui patient men and women tests 200 parts of samples；＞ 75
Year, patient men and women tested 200 parts of samples.
Laboratory apparatus：Hitachi 7060 ensures that instrument state is normal before testing.
Experimenter requires：Laboratory worker is familiar with experiment content, and energy testing instruments energy skilled operation
Experimental data is analyzed and counted.
According to kit operation instructions, machine location parameter in setting measures collected serum sample, then to measuring
As a result for statistical analysis.
Two, the requirement and analysis of data
1, it is the reliability for ensuring with reference to Value Data, chooses 200 sample datas.
2, in data doubtful outlier judgement, by the difference D of doubtful outlier and its consecutive points and data range R phases
It removes, if D/R≤1/3 is thought of as non-outlier.If outlier there are two or more can do minimum doubtful outlier as above
Processing, if all ＞ 1/3, must all remove all the points；If all≤1/3, retaining all data.
If should be to fill other data after 3, thering is outlier to be removed.
4, distribution map is drawn, the distribution characteristics of data is understood.As data are converted rear in normal state in normal distribution or data
Distribution, can be according toIndicate 95% data distribution range.If data are not in normal distribution, nonparametric method can be used
Processing determines that the reference of 2.5% and 97.5% digit limits with method of percentiles, 95% reference interval is determined with this.
Three, experimental data and statistic processes
1, test sample tables of data is as follows：(unit ng/L)
<50 years old patients serums:
50-75 Sui patient
75 years old patient of ＞
2, sample scatter plot is as shown in Figure 1；
Outlier judges：
2.1, first by all data according to being ranked sequentially from small to large, it is as shown in Figure 2 to do scatter plot：2.2 range estimations, data
Point is without doubtful outlier.
3, sample data distribution map
The ends 3-1N--preceding b-type natriuretic peptide distribution frequency table
<50 years old patients serums:
The ends 3-2N--preceding b-type natriuretic peptide distribution of results figure, as shown in Fig. 3 (a)；
According to normal distribution rule, there is 95% observed value to existIn range.That is 34.08-479.37ng/L.
Obviously, the distribution of the entire data of this experiment belongs to partial velocities, and 95% distribution cannot be withIt calculates true
It is fixed.It needs to use method of percentiles.The reference limit for determining 2.5% and 97.5% digit, 95% reference interval is determined with this.
In formula：
Pr：R percentiles；
L：The lower limit organized where r percentiles；
W：The width organized where r percentiles；
f：The frequency organized where r percentiles
n：Total frequency；
C：The cumulative frequencies for the previous group organized where r percentiles.
It calculates：
1, the numerical value of the 95th percentile, statistics is 200, its 95th percentile should be 200 × 95%=190
A, this percentile thereby determines that each value in formula at the 7th group known to from frequency distribution table：P95=is calculated
433.33ng/L
2. the numerical value of the 2.5th percentile, statistics is 200, its 2.5th percentile should be 200 × 2.5%=
5, this percentile thereby determines that each value in formula, P2.5 is calculated at the 1st group known to from frequency distribution table
=74.74ng/L
3, the 97.5th percentile, the numerical value of statistics are 200, its 97.5th percentile should be the 200th ×
97.5%=195, this percentile thereby determines that each value in formula at the 7th group known to from frequency distribution table：
P97.5=442.67ng/L is calculated
50-75 Sui patients serum
The ends 3-2N--preceding b-type natriuretic peptide distribution of results figure；Such as Fig. 3 (b)
According to normal distribution rule, there is 95% observed value to existIn range.That is 158.57-952.55ng/L.
Obviously, the distribution of the entire data of this experiment belongs to partial velocities, and 95% distribution cannot be withIt calculates and determines.
It needs to use method of percentiles.The reference limit for determining 2.5% and 97.5% digit, 95% reference interval is determined with this.
In formula：
Pr：R percentiles；
L：The lower limit organized where r percentiles；
W：The width organized where r percentiles；
f：The frequency organized where r percentiles
n：Total frequency；
C：The cumulative frequencies for the previous group organized where r percentiles.
It calculates：
1, the numerical value of the 95th percentile, statistics is 200, its 95th percentile should be 200 × 95%=190
A, this percentile thereby determines that each value in formula at the 7th group known to from frequency distribution table：P95=is calculated
864.29ng/L
2. the numerical value of the 2.5th percentile, statistics is 200, its 2.5th percentile should be 200 × 2.5%=
5, this percentile thereby determines that each value in formula, P2.5 is calculated at the 1st group known to from frequency distribution table
=217.24ng/L
3, the 97.5th percentile, the numerical value of statistics are 200, its 97.5th percentile should be the 200th ×
97.5%=195, this percentile thereby determines that each value in formula at the 7th group known to from frequency distribution table：
P97.5=882.14ng/L is calculated
75 years old patient of ＞
The ends 3-2N--preceding b-type natriuretic peptide distribution of results figure, as shown in Fig. 3 (c)；
According to normal distribution rule, there is 95% observed value to existIn range.That is 82.20-1890.55ng/
L.Obviously, the distribution of the entire data of this experiment belongs to partial velocities, and 95% distribution cannot be withIt calculates true
It is fixed.It needs to use method of percentiles.The reference limit for determining 2.5% and 97.5% digit, 95% reference interval is determined with this.
In formula：
Pr：R percentiles；
L：The lower limit organized where r percentiles；
W：The width organized where r percentiles；
f：The frequency organized where r percentiles
n：Total frequency；
C：The cumulative frequencies for the previous group organized where r percentiles.
It calculates：
1, the numerical value of the 95th percentile, statistics is 200, its 95th percentile should be 200 × 95%=190
A, this percentile thereby determines that each value in formula at the 7th group known to from frequency distribution table：P95=is calculated
1695.65ng/L
2. the numerical value of the 2.5th percentile, statistics is 200, its 2.5th percentile should be 200 × 2.5%=
5, this percentile thereby determines that each value in formula, P2.5 is calculated at the 1st group known to from frequency distribution table
=164.44ng/L
3, the 97.5th percentile, the numerical value of statistics are 200, its 97.5th percentile should be the 200th ×
97.5%=195, this percentile thereby determines that each value in formula at the 7th group known to from frequency distribution table：
P97.5=1747.83ng/L is calculated
Experiment finally gets normal healthy people serum sample<50 years old patients；50-75 Sui patient；75 years old patient of ＞ each 200
The test data of part, test data is without outlier.Sample distribution is in partial velocities, true using objective and easy method of percentiles
It is fixed:The distribution of 2.5%-97.5% is respectively：74.74—442.67ng/L；217.24—882.14ng/L；164.44—
1747.83ng/L。
In conjunction with domestic and foreign literature reference, after being adjusted according to the age, the positive of NT-proBNP defines value and is：Age<50
Year patient and serum ＜ 450ng/L；Patient and serum ＜ 900ng/L of the age at 50-75 Sui；75 years old patient of age ＞ and blood
Clear ＜ 1800ng/L.
In order to verify the present invention it is each preferably and effect, do it is following experiments have shown that：
Experiment one, the Selection experiment of latex diameter；
As a preferred embodiment, the diameter range of the sensitization latex particle of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody is 20-
60nm。
Experiment 1.1：Purchase goat-anti people N-terminal-preceding b-type natriuretic peptide that same antibody combination different-diameter latex particle is formed
The preceding b-type natriuretic peptide antigen of same N-terminal-is added in the sensitization latex particle of antibody, and mixing 1-2 minutes, observation is solidifying in 5 minutes
Collection is as a result, test result is shown in Table two.
Table two
As shown in Table 2, a diameter of 20,40,60nm latex combination goat-anti people N-terminal-preceding b-type natriuretic peptide antibody is formed
Goat-anti people N-terminal-preceding b-type natriuretic peptide antibody sensitization latex particle, it is as a result ideal.
Experiment 2.2 includes the following steps：
Step 1 detects a diameter of 20,40,60,80,100,200nm latex respectively with ultraviolet-uisible spectrophotometer
In conjunction with the sensitization latex for goat-anti people N-terminal-preceding b-type natriuretic peptide antibody that goat-anti people N-terminal-preceding b-type natriuretic peptide antibody is formed
Grain absorbance, target call absorbance A 1 >=1.2.
Same antigen is added after completing step 1 in step 2, measures absorbance A 2 again.It is required that A2-A1 >=0.05；See
Table three.
Table three
It can be derived that when latex diameter is within the scope of 20-60nm, agglutinating reaction is full and uniform, absorbance from the above experiment
Test variation is apparent, high sensitivity；Consider R＆D costs problem, while meeting performance indicator requirement, subsequent experimental selects latex
The sensitization latex particle of a diameter of 40nm carries out.
Experiment two：The selection of ratio of reagents；
As a preferred embodiment, the metered proportions of reagent one and reagent two are 4:When 1, reagent surplus is minimum, and reagent utilizes
Rate highest.
By prepared reagent one (R1), reagent two (R2), according to 5:Isosorbide-5-Nitrae:1,3:1 ratio is divided into 3 groups；Reagent one,
Two reacting dose of reagent is respectively set to according to ratio of reagents：250,50；240,60；225；75, i.e. reagent total amount is consistent.Same
The analysis of one full-automatic biochemical is above (Hitachi 7080), while detecting the sample of same sample size, until the alarm of reagent needle cannot inhale
Until taking amount of reagent, reagent one, two surplus of reagent, and detection sample number are observed.Reagent utilization rate is calculated according to result.As a result
It is shown in Table four and table five.
Table four
Table five
From table four, five it is found that 4:1,3:1 ratio of reagents, actually detected sample number are not much different, and observation reagent is remaining
Amount, with 4:1 ratio be it is minimum, and 4:1 ratio, reagent utilization rate highest, therefore subsequent experimental selection 4:1 ratio of reagents
It carries out.
Experiment three：The selection of reagent sample calibration product metering；
As a preferred embodiment, reagent metering and the metered proportions of calibration object are：10 reagents：1 calibration object；
The concentration of potent antibodies in how much decision reaction volumes of amount of reagent, and reagent sample proportion can influence the line of reagent
Property range.According to reference value situation and clinical reagent pattern detection linear demand, to N-terminal plasma pro-brain natriuretic peptide levels (NT-proBNP)
The detection kit range of linearity is set as 50-35000ng/L, while should meet claimed below:
A) linearly dependent coefficient r >=0.990；
B) deviation from linearity：[100-5000) absolute deviation should be no more than ± 500ng/L within the scope of ng/L；[5000-
32000] relative deviation should be no more than ± 10% within the scope of ng/L.
Fixed R1, R2 amount of reagent draw different sample sizes, the setting of sample application amount respectively to 7 samples of linear segmented
As shown in Table 6；Using regression equation calculation deviation and related coefficient, linearity test experimental result is as shown in Table 7；
Table six
Table seven
From above 5 groups of test results it is found that under amount of reagent fixing situation, sample size is 30 μ l, and reagent linear effects are most
Good, related coefficient and deviation can meet test requirements document.There is no false positive situation (antibody excess, the detection when sample size deficiency
As a result it is linear to exceed reagent), also without sample size surplus when false negative situation (antigen excess, reagent reaction is incomplete, does not examine
Measure actual content in sample.)
Known to Comprehensive Experiment two, three：Reagent one, reagent two, calibration object optimal metered proportions be 8:2:1.
Experiment four：The selection of stabilizer and dosage experiment；
As a preferred embodiment, it is 0.02%-0.05% that preservative, which selects biological preservative Proclin300, optimal dosage,.
Experiment 4.1：Stabilizing agent dosage selects；
Same batch N-terminal plasma pro-brain natriuretic peptide levels (NT-proBNP) detection kit (latex immunoturbidimetry) is divided into phase
Deng several pieces, it is every it is a in the Proclin300 of different amounts is added, the dosage of stabilizer is not to influence having for kit
Composition and performance indicator are imitated as premise, detection respectively the results are shown in Table eight per the property indices of a kit：
Table eight
Above-mentioned experimental data shows that within the scope of 0.005%-0.06%, NT-proBNP is measured the dosage of Proclin300
The all technical of kit is in tolerance interval, but the indices measurement result within the scope of 0.02%-0.05%
For most perfect condition.
Experiment 4.2：Stabilizer stablizing effect is tested
Experiment 4.1 shows Proclin300 dosages kit indices measurement result within the scope of 0.02%-0.05%
Ideal state, therefore prepare and contain 0.20 ‰, 0.25 ‰, 0.30 ‰, 0.35 ‰, 0.40 ‰, 0.45 ‰, 0.50 ‰ respectively；
The NT-proBNP assay kits of Proclin300 do stabilizing effect experiment, while preparing the NT- of the Sodium azide containing 1g/L
The two stablizing effect is compared by proBNP assay kits in the stable effect end of term.External diagnosis reagent case, the term of validity 12
A month, therefore to the above reagent preparation box, respectively in November, December, 13 months, detection reagent performance, and carry out paired observation；
Proclin300 stablizing effect experimental results are shown in Table nine：
Table nine
13 months, Proclin300 stablizing effects were shown in Table ten with 1g/L Sodium azide stablizing effect comparison results：
Table ten
After 13 months, to same reagent box, the result observation of different stabilizers is added it is found that 0.02%-0.05%
Proclin300 stabilizers, effect are more outstanding than common Sodium azide stabilizer.
According to the above different experiments obtain as a result, best is selected as：Select 40nm latex combination NT-proBNT antibody
Sensitization latex particle raw material, select 0.03%proclin300 dosage stabilizers, prepare 4:1 reagent；In parameter setting, reagent
The ratio of metering and calibration object is 10:1.According to the total sample-adding amount of different instruments, overall consumption, but ratio of reagents can be suitably adjusted,
And reagent sample proportion is constant.
Concrete case is as follows：
△ is according to Beckman DXC800 automatic clinical chemistry analyzers, the ratio of reagent sample：160 reagents one：40 examinations
Agent two：20 calibration objects.
It is matched with Beckman DXC800 series automatic clinical chemistry analyzers, reagent sample ratio is adopted according to test result
With 160/40/20 ratio, reach the requirement of 350 person-portion of one-time detection.
△ is according to 330 serial automatic clinical chemistry analyzer of China of section brilliance, the ratio of reagent sample：240 reagents one：60
Reagent two：30 calibration objects.
It is matched with China of section remarkable 330 automatic clinical chemistry analyzers, reagent sample ratio is according to test result, using 240/
60/30 ratio reaches the requirement of 230 person-portion of one-time detection.
△ is according to 7060 automatic clinical chemistry analyzer of Hitachi, the ratio of reagent sample：128 reagents one：32 reagents two：
16 calibration objects.
It is matched with Hitachi 7060 serial automatic clinical chemistry analyzer, reagent sample ratio is used according to test result
128/32/16 ratio reaches the requirement of 450 person-portion of one-time detection.
A kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit of present invention offer and its detection method；Kit uses particulate
Enhancing immunoassay, NT-proBNP (antigen) and the anti-NT-proBNP (antibody) of specificity meets in the liquid phase in sample,
Insoluble antigen-antibody complex is formed immediately, generates certain turbidity；The height of turbidity contains with NT-proBNP in sample
Measure it is directly proportional, by compared with the calibration object equally handled, you can calculate the content of NT-proBNP in sample；This kit
It is mostly anti-in conjunction with sheep using 15-60nm latexes, sensitization latex particle is formed, detection sensitivity is made to reach 1ng/L；This kit is adopted
The stability of kit is improved with the Proclin300 biological preservatives of 0.02-0.05 concentration ranges；The present invention passes through different complete
Automatic biochemistry analyzer coordinates different reagent sample ratios, can simultaneous quantitative detection high-volume sample；The kit that the present invention uses
Calibration object is included, to achieve the purpose that quantitative detection.
The basic principles, main features and advantages of the invention have been shown and described above.The technical staff of the industry should
Understand, the invention is not limited in any way above-described embodiment, all to be obtained by the way of equivalent substitution or equivalent transformation
Technical solution is all fallen in protection scope of the present invention.
Claims (10)
1. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit, which is characterized in that including：
Reagent one：
Phosphate buffer 1 00-150mmol/L,
Preservative 0.005%-0.06%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer 1 00-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer 1 00-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-.
2. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 1, which is characterized in that
Reagent one：
Phosphate buffer PH=6.5 100-150mmol/L,
Biological preservative Proclin300 0.005%-0.06%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer PH=8.0 100-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.005%-0.06%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer PH=8.0 100-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.005%-0.06%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-.
3. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 2, which is characterized in that
Reagent one：
Phosphate buffer PH=6.5 100-150mmol/L,
Biological preservative Proclin300 0.02%-0.05%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer PH=8.0 100-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.02%-0.05%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer PH=8.0 100-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Biological preservative Proclin300 0.02%-0.05%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-.
4. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 1, which is characterized in that the goat-anti people N
The diameter range of the sensitization latex particle of the preceding b-type natriuretic peptide antibody in end-is 20-60nm.
5. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 4, which is characterized in that the goat-anti people N
The diameter range of the sensitization latex particle of the preceding b-type natriuretic peptide antibody in end-is 40nm.
6. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 1, which is characterized in that reagent one, reagent
Two, the metered proportions of calibration object are 8:2:1.
7. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 6, which is characterized in that according to Bake
Graceful series automatic clinical chemistry analyzer, the group of reagent sample are divided into：One 160 μ l of reagent, 2 40 μ l of reagent, 20 μ l of calibration object.
8. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 6, which is characterized in that according to China of section
Remarkable series automatic clinical chemistry analyzer, the group of reagent sample are divided into：One 240 μ l of reagent, 2 60 μ l of reagent, 30 μ l of calibration object.
9. a kind of N-terminal plasma pro-brain natriuretic peptide levels detection kit according to claim 6, which is characterized in that according to Hitachi
The group of serial automatic clinical chemistry analyzer, reagent sample is divided into：One 128 μ l of reagent：2 32 μ l of reagent：16 μ l of calibration object.
10. a kind of detection method of N-terminal plasma pro-brain natriuretic peptide levels detection kit, which is characterized in that include the following steps：
Step 1：Parameter list is corresponded to according to automatic clinical chemistry analyzer, in instrument parameter interface arrange parameter；
Step 2：Blood samples of patients is acquired, is centrifuged in 30 minutes, takes supernatant as detection sample, according to parameter setting sample position,
It is put into sample disc；
Step 3：According to parameter setting reagent position, in reagent disc, it is respectively and correspondingly placed into reagent one and reagent two, while in sample
Counter sample position is put into calibration object in product disk；
Reagent one：
Phosphate buffer 1 00-150mmol/L,
Preservative 0.005%-0.06%,
Polysorbas20 0.5-2.0mL/L,
Macrogol 6000 25-60g/L；
Reagent two：
Phosphate buffer 1 00-150mmol/L,
Disodium hydrogen phosphate 20-60g/L,
Sodium dihydrogen phosphate 2-5g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The sensitization latex particle 1-5ml/L of goat-anti people N-terminal-preceding b-type natriuretic peptide antibody；
Calibration object：
Phosphate buffer 1 00-150mmol/L,
Bovine serum albumin(BSA) 10-50g/L,
Sodium chloride 10-50g/L,
Preservative 0.005%-0.06%,
The preceding b-type natriuretic peptide antigen 1-5ml/L of N-terminal-；
Step 4：Start calibration test, passes through calibration；
Step 5：Start pattern detection, by reagent needle and sample needle, draws reagent one and sample respectively, be put into reaction cup, stir
Reagent I and sample are mixed, is reacted 5 minutes；
Step 6：By reagent needles and sample needle, reagent two and sample are drawn respectively, is put into reaction cup, stirred, be incubated 5 minutes,
It allows NT-proBNP (antigen) and the anti-NT-proBNP (antibody) of specificity in sample to meet in the liquid phase, is formed insoluble anti-
Antigen-antibody complex generates turbidity；
Step 7：According to specific absorbance changes of the NT-proBNP at 570nm, the absorbance of NT-proBNP in sample is measured
Changing value, by compared with kit internal calibration product concentration and calibration object absorbance change value, you can be calculate by the following formula out sample
The content of NT-proBNP in product；
The positive of NT-proBNP defines value：Age<50 years old patients and serum ＜ 450ng/L；Patient of the age at 50-75 Sui
And serum ＜ 900ng/L；75 years old patient of age ＞ and serum ＜ 1800ng/L.
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