CN106442355A - Determination reagent for heart-type fatty acid binding protein and preparation method of determination reagent - Google Patents
Determination reagent for heart-type fatty acid binding protein and preparation method of determination reagent Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
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Abstract
The invention relates to a determination reagent for heart-type fatty acid binding protein and a preparation method, and belongs to the technical field of methods for testing or analyzing materials by means of chemical or physical properties of determination materials. The determination reagent is prepared from a reagent R1 and a reagent R2, wherein the reagent R1 is a solution formed by dissolving bovine serum protein into purified water added with a buffer solution, and the reagent R2 is a solution formed by dissolving anti-human heart-type fatty acid binding protein monoclonal antibody (mouse) latex into purified water. When the determination reagent is applied to determination of the heart-type fatty acid binding protein, the determination reagent has the advantages of being rapid to determine, sensitive to determination, good in accuracy, high in specificity, good in stability and the like.
Description
Technical field
The present invention relates to mensure reagent of a kind of cardic fatty acid binding protein and preparation method thereof, belong to by means of mensure
The chemically or physically property of material is testing or analysis of material technical field.
Background technology
Cardic fatty acid binding protein (hFABP) is the new little cytoplasmic protein of the one kind being rich in heart.It has height
Heartspecific (is namely mainly expressed) in heart tissue, but also has low concentration to express in the tissue beyond heart.The heart
Myocardial ischemia damages after occurring, and hFABP 1-3 hour can be found after episode in blood, and 6-8 hour reaches peak
Value and blood plasma level recovers normal in 24-30 hour.Heart fat acid is made up of 132 aminoacid with reference to cytoplasmic protein,
Molecular weight is 15kDa.Cardic fatty acid binding protein (hFABP) gene is located on chromosome I.It is heart albumen the abundantest
One of matter.HFABP with reference to two fatty acid molecules and participates in the transport of fatty acyl group coenzyme A, is active in oxidizing process, thus
Energy is produced in mitochondrion.
Clinical proof：In terms of myocardial damage, cardic fatty acid binding protein has more clinical valency than Myoglobin, CK-MB
Value, specific as follows：
(1) in the early diagnosiss of myocardial damage, h-FABP is sensitiveer, be due to：In myocardium middle and high concentration；In Cytoplasm
Middle restriction；Low-molecular-weight and area are little；Relative organization's specificity；Similar to the distribution of CK-MB in tissue beyond heart, Yi Ji
After myocardial damage, premature disconnection enters blood plasma and urine.
(2) h-FABP can be promptly released in blood in ACS morbidity early stage, and the diagnosis for ACS has jump, with
When possess high specific, high sensitivity, the feature of high coincidence rate to myocardial damage；H-FABP concentration, can in ACS long-term prognosis
Effectively identify the high-risk patient of the adverse events such as AMI, heart failure and unstable angina；
(3) h-FABP and troponin joint-detection can improve diagnostic sensitivity, have more diagnostic value to ACS.
(4) early application of hFABP can overcome the trap of acute chest pain early stage cardiac troponin detection.Myocardium myo calcium
Albumen is more lasting in the circulating cycle, may more contribute to the later stage diagnosis of acute myocardial injury.
The domestic at present method measuring cardic fatty acid binding protein mainly has：
(1) mass spectrography, the method operating procedure is complicated, expensive, is unsuitable in routine clinical development.
(2) non-competing sandwich ELISA, the method dosing accuracy is poor, and the operating time is long, and automaticity is low, general
For qualitative detection.
Based on this, make the application.
Content of the invention
For existing cardic fatty acid binding protein drawbacks described above present in test process, present invention firstly provides one
Plant rapid sensitive, accuracy is good, specificity is high, good stability, liquid double reagent are easy and simple to handle, using latex advanced both at home and abroad
Strengthen immunoturbidimetry it is adaptable to clinical full-automatic or semiautomatic biochemistry analysis (manual sample-adding product, reagent) cardioid fatty acid
Associated proteins measure reagent.
For achieving the above object, the technical scheme that the application takes is as follows：
A kind of mensure reagent of cardic fatty acid binding protein, is made up of R1 reagent and R2 reagent, and described R1 reagent is
It is dissolved in the solution formed in the purified water being added with buffer by bovine serum albumin；Described R2 reagent is by anti-human cardioid
Fatty acid binding protein monoclonal antibody (Mus) latex is dissolved in the solution formed in purified water.
Further, as preferred：
In described R1 reagent, described buffer is dipotassium hydrogen phosphate-potassium phosphate buffer, it is furthermore preferred that institute
The pH of the dipotassium hydrogen phosphate-potassium phosphate buffer stated is 7.2, and concentration is 40mmol/L；Bovine serum albumin, its concentration is
50g/L.
In described R2 reagent, anti-human cardic fatty acid binding protein monoclonal antibody (Mus) latex concentration is 5ml/L.
Meanwhile, present invention also provides a kind of cardic fatty acid binding protein as characterized above measures the preparation side of reagent
Method, comprises the steps：
(1) R1 preparation of reagents：Add buffer in purified water, stir to being completely dissolved；Add bovine serum albumin stirring
To being completely dissolved, continue constant volume after stirring；
(2) R2 preparation of reagents：Anti-human cardic fatty acid binding protein monoclonal antibody (Mus) latex is added in purified water,
Stir to being completely dissolved rear constant volume；
(3) the R1 reagent to above-mentioned preparation and R2 reagent enter line sensitivity, the range of linearity, preci-sion and accuracy are surveyed
Fixed.
Further, as preferred：
Described mixing speed is 450 revs/min.Mixing speed is too high to lead to shearing force excessive, affect prepared solution
Surface tension and bonding force, mixing speed is too low, affects its mixture effect, controls when 450 revs/min, is guaranteeing that stirring cuts
While cutting moderate, also promote the fusion of institute's substance in it.
In step (1), described purified water initial volume is 800ml, and constant volume is 1000ml.Before and after constant volume, volume becomes
Change in 20-30%, can be very good to ensure in it, to be added the good amalgamation of other materials it is ensured that the R1 reagent being configured is dense
Degree, stability of solution are optimal.
In step (2), described purified water initial volume is 800ml, and constant volume is 1000ml.Before and after constant volume, volume becomes
Change in 20-30%, can be very good to ensure in it, to be added the good amalgamation of other materials it is ensured that the R2 reagent being configured is dense
Degree, stability of solution are optimal.
The operation principle of the present invention is as follows：
H-FABP in specimen can produce the aggregation of antigen-antibody with antibody on latex particle for the absorption, is formed anti-
Original antibody complex, the change of its turbidity is by measuring the absorbance of specific wavelength, you can calculate containing of H-FABP in specimen
Amount.
(1) sensitivity evaluation test
According to GB/T26124-2011《Clinical chemistry external diagnosis reagent (box)》In " sensitivity for analysis " is detected will
Ask, sensitivity for analysis evaluation is carried out with sample detecting that the absorbance producing changes under regulation parameter with test kit, be scaled n
The difference (Δ A) of the absorbance of unit is as sensitivity for analysis.Sensitivity for analysis evaluation test is to sensitivity for analysis evaluation sample
This replication 20 times.Assessment result：Sensitivity for analysis 26ng/mL absorbance difference (Δ A) >=0.0500ABS.
(2) range of linearity evaluation test
According to GB/T26124-2011《Clinical chemistry external diagnosis reagent (box)》With《External diagnosis reagent analytical performance is commented
Estimate the guideline range of linearity (exposure draft)》In suggestion, when the reagent range of linearity is verified, to be verified
5 concentration levels, each concentration level replication 3 times is selected in the range of linearity.
(3) precision evaluation test
Precision evaluation includes repeatability and difference between batch, and at least assesses the precision of two concentration level samples, wherein
There is a concentration about medical science decision level.Therefore, reproducibility adopts under the conditions of repeatability, with two concentration levels
Control material (one of concentration is close to medical science decision level) test, each concentration retest 10 times；Difference between batch is evaluated
3 different lot numbers are tested respectively using the control material (one of concentration is close to medical science decision level) of two concentration levels
Test kit, each lot number is tested 3 times.
(4) accuracy estimating test
With the people source sample no less than 40 variable concentrations in the range of detectable concentration, using specified analysis system as
Comparison method, every part of sample is detected respectively by test agent operational approach and comparison method.Calculate two groups with linear regression method
The correlation coefficient (r) of result and the relative deviation of each concentration point.
Assessment result：Repeated coefficient of variation CV≤8% of three batches of reagent；Relative deviation≤10%；Reagent linearly up to
160ng/mL, sensitivity for analysis 26ng/mL absorbance difference (Δ A) >=0.0500ABS.
The method that the present invention utilizes latex enhancing immune turbidimetry for Determination cardic fatty acid binding protein, its advantage is quick
Sensitive, accuracy is good, and specificity is high, and good stability is easy and simple to handle, is applicable to clinical full-automatic or semi-automatic biochemical analyzer
Support the use.
Brief description
Fig. 1 is antigen-absorbance curve figure in the application；
Fig. 2 is antibody-absorbance curve figure in the application；
Fig. 3 is the accuracy block diagram measuring reagent in the application.
Specific embodiment
Embodiment 1
Main agents used by the present embodiment：
R1 reagent：Phosphate buffer：PH7.2,40mmol/L；Bovine serum albumin：50g/L.
R2 reagent：Anti-human cardic fatty acid binding protein monoclonal antibody (Mus) latex：5ml/L.
1) preparation of R1 reagent
1. 800ml purified water is added in appropriate containers.
2. open motor stirrer, mixing speed is 450 revs/min.
3. weigh 3.58g dipotassium hydrogen phosphate to add in above-mentioned purified water, stir to being completely dissolved.
4. weigh 2.76g potassium dihydrogen phosphate to add in above-mentioned solution, stir to being completely dissolved.
5. weigh 6.68g bovine serum albumin to add in above-mentioned solution, stir to being completely dissolved.
7. stir more than ten minutes
8. it is settled to 1000ml.
2) preparation of R2 reagent
1. 800ml purified water is added in appropriate containers.
2. open motor stirrer, mixing speed is 450 revs/min.
3. weigh 2.5g anti-human cardic fatty acid binding protein monoclonal antibody (Mus) latex to add in above-mentioned purified water, stir
Mix to being completely dissolved.
4. stir more than ten minutes.
5. it is settled to 1000ml.
3) reagent test
1. instrument configuration:Instrument uses Hitachi 7100 automatic clinical chemistry analyzer.
Location parameter sets in instrument in strict accordance with product description, and basic location parameter is as follows：
A. method：End-point method, the Direction of Reaction：Upwards；Wavelength：700nm, 37 DEG C of temperature.
B. cuvette optical path：1cm；R1 reagent:150 μ l, R2 reagent:150 μ l, sample：8μl.
C. the first light-metering point：Add read within latter 0.5 minute in R2.
D. the second light-metering point：Add read within latter 5 minutes in R2.
2. reagent visual examination：Visual inspection.
3. loading quantity inspection：Use general gage measuring.
4. reagent blank measures:
With distilled water or deionized water as blank sample test kit, under test dominant wavelength, record test starting
When absorbance (A1 reagent) and the absorbance (A2 reagent) after about 5 minutes, A2 is the blank absorbency of working reagent.
5. the range of linearity measures:
The high level sample normal saline doubling dilution close to the range of linearity upper limit is taken to be configured to the sample of variable concentrations gradient
This series.Desired value is calculated with H-number and dilution ratio.Dilution process (is accordingly adjusted according to the big I of high level with reference to shown in table 1
Whole dilution gradient).
Table 1 Sample Dilution table
| Embodiment | 1 | 2 | 3 | 4 | 5 |
| Normal saline (ml) | 0 | 0.5 | 0.5 | 0.5 | 0.5 |
| High level specimen H (ml) | 1 | 0.5 | 0.5 | 0.5 | 0.5 |
| Dilution ratio | Former times | 1/2 | 1/4 | 1/8 | 1/16 |
The concentration of embodiment 1 sample is known definite value CH, and embodiment 2-5 concentration of specimens presses formula：Concentration of specimens=CH ×
Dilution ratio, calculates as desired value, the series of samples having diluted fully is mixed, and measurement system after calibration is pressed from low
It is worth high level sequential parallel to measure 2 times, as corresponding embodiment sample measured value, (as 2 times, measurement result has obvious deviation to average
Should reject and resurvey).
Statistical analysis：With desired value as abscissa, sample measured value is vertical coordinate mapping and linear regression analysis, determines
Linearly interval calculates linear equation y=a+bx and correlation coefficient, is linearly good when correlation coefficient r >=0.990.
Data Analysis Services adopt Microsoft Excel software.
Correlation formula is as follows：
In formula, C：Concentration；V：Volume；b：The slope of the regression line；∣a∣：The absolute value of regression line intercept；r：Correlation coefficient；
Xi：Each pipe desired value；Yi：Each pipe measured value；i：1、2、3.……、n；n：Measure sample number.
6. precision measures:
A. repeatability measures:
With clinical samples or quality controlled serum test kit, retest 10 times, the meansigma methodss of computation and measurement valueWith
Standard deviation (s).It is calculated as follows the coefficient of variation (CV).
With coefficient of variation CV (%)：
In formula：The average of test serum sample；
Xi measures the measurement result of serum sample；
N measures number of times；
The CV coefficient of variation.
B. difference between batch measures
Take three lot number censorship reagent, each lot number takes 3 bottles, measure 1 part of clinical sample or quality controlled serum respectively, can be selected for
Roche quality-control product, calculates 9 parts of reagent respectively and measures averageMensure average with each 3 parts of reagent of lot numberAnd the coefficient of variation with three lot number reagent mensure of Microsoft Excel software statistics.
Correlation formula is as follows:
In formula：
——In maximum；
——In minima；
Grand mean.
7. accuracy measures:
Correlation coefficient r >=0.990.Relative deviation≤15%；
8. sensitivity for analysis:
With the sample test test kit of concentration known or activity, record the absorbance producing under test kit specifies parameter and change
Become, be scaled the absorbance difference (Δ A) of n unit.
According to the calculated desired value of table 1, measured value, coefficient of variation CV, batch between extreme difference, relative deviation B and extinction
Degree difference DELTA A collects into table 2.
Table 2 test result summary sheet
Table 3 sensitivity for analysis synopsis (1-20 is parallel laboratory test sequence number)
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
| 0.0524 | 0.0568 | 0.0533 | 0.0527 | 0.0611 | 0.0602 | 0.0523 | 0.0555 | 0.0548 | 0.0542 |
| 11 | 12 | 13 | 14 | 15 | 16 | 17 | 18 | 19 | 20 |
| 0.0611 | 0.0601 | 0.0635 | 0.0631 | 0.0524 | 0.0536 | 0.0552 | 0.0557 | 0.0548 | 0.0539 |
Repeated synopsis in 4 batches, table
Table 5 betweenrun precision synopsis
Assessment result and table 2-5 result show：Repeated coefficient of variation CV≤8% of the application reagent；Relative deviation≤
10%；Reagent is linearly up to 160ng/mL, sensitivity for analysis 26ng/mL absorbance difference (Δ A) >=0.0500ABS.
The method that the application utilizes latex enhancing immune turbidimetry for Determination cardic fatty acid binding protein, its advantage is quick
Sensitive, accuracy is good, and specificity is high, and good stability is easy and simple to handle, is applicable to clinical full-automatic or semi-automatic biochemical analyzer
Support the use.
Above content be preferred implementation with reference to the invention provided technical scheme is made detailed further
Describe in detail bright it is impossible to assert that the invention is embodied as being confined to these explanations above-mentioned, the affiliated technology for the invention
For the those of ordinary skill in field, without departing from the concept of the premise of the invention, some simple deductions can also be made
Or replace, all should be considered as belonging to the protection domain of the invention.
Claims (8)
1. cardic fatty acid binding protein mensure reagent it is characterised in that：It is made up of R1 reagent and R2 reagent, described R1 tries
Agent is the solution being dissolved in by bovine serum albumin formed in the purified water being added with buffer；Described R2 reagent is by anti-human
Cardic fatty acid binding protein monoclonal antibody（Mus）Latex is dissolved in the solution formed in purified water.
2. cardic fatty acid binding protein as claimed in claim 1 mensure reagent it is characterised in that：Described R1 reagent
In, described buffer is dipotassium hydrogen phosphate-potassium phosphate buffer.
3. cardic fatty acid binding protein as claimed in claim 1 or 2 mensure reagent it is characterised in that：Described buffering
The pH of liquid is 7.2, and concentration is 40mmol/L；Bovine serum albumin, its concentration is 50g/L.
4. cardic fatty acid binding protein as claimed in claim 1 mensure reagent it is characterised in that：Described R2 reagent
In, anti-human cardic fatty acid binding protein monoclonal antibody（Mus）Latex concentration is 5ml/L.
5. cardic fatty acid binding protein as claimed in claim 1 measures the preparation method of reagent it is characterised in that including as follows
Step：
（1）R1 preparation of reagents：Add buffer in purified water, stir to being completely dissolved；Bovine serum albumin is added to stir to complete
CL, continues constant volume after stirring；
（2）R2 preparation of reagents：Anti-human cardic fatty acid binding protein monoclonal antibody is added in purified water（Mus）Latex, stirring
To being completely dissolved rear constant volume；
（3）R1 reagent to above-mentioned preparation and R2 reagent enter line sensitivity, the range of linearity, preci-sion and accuracy are measured.
6. cardic fatty acid binding protein as claimed in claim 5 measure reagent preparation method it is characterised in that：Described
Mixing speed is 450 revs/min.
7. cardic fatty acid binding protein as claimed in claim 5 measure reagent preparation method it is characterised in that：Step
（1）In, described purified water initial volume is 800ml, and constant volume is 1000ml.
8. cardic fatty acid binding protein as claimed in claim 5 measure reagent preparation method it is characterised in that：Step
（2）In, described purified water initial volume is 800ml, and constant volume is 1000ml.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363036A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363035A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111487413A (en) * | 2019-01-29 | 2020-08-04 | 艾维可生物科技有限公司 | Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method |
| CN112881370A (en) * | 2021-03-19 | 2021-06-01 | 浙江达美生物技术有限公司 | Glycocholic acid determination reagent and preparation method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111363036A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363035A (en) * | 2018-12-25 | 2020-07-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363036B (en) * | 2018-12-25 | 2021-10-12 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111363035B (en) * | 2018-12-25 | 2021-12-03 | 东莞市朋志生物科技有限公司 | Recombinant antibody of anti-heart fatty acid binding protein |
| CN111487413A (en) * | 2019-01-29 | 2020-08-04 | 艾维可生物科技有限公司 | Detection kit for quantitatively detecting heart-type fatty acid binding protein by E L ISA method |
| CN112881370A (en) * | 2021-03-19 | 2021-06-01 | 浙江达美生物技术有限公司 | Glycocholic acid determination reagent and preparation method thereof |
| CN112881370B (en) * | 2021-03-19 | 2021-11-26 | 浙江达美生物技术有限公司 | Glycocholic acid determination reagent and preparation method thereof |
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Application publication date: 20170222 |