CN108613977B - N-terminal brain natriuretic peptide precursor detection kit - Google Patents
N-terminal brain natriuretic peptide precursor detection kit Download PDFInfo
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- G—PHYSICS
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- 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
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- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/32—Cardiovascular disorders
Abstract
The invention discloses an N-terminal brain natriuretic peptide precursor detection kit and a detection method thereof; a kit, comprising: a first reagent: phosphate buffer, preservative, tween 20 and polyethylene glycol 6000; and a second reagent: phosphate buffer solution, disodium hydrogen phosphate, 2-5g/L sodium dihydrogen phosphate, sodium chloride, preservative, sensitized emulsion particles of goat anti-human N-terminal-pro B-type natriuretic peptide antibody; calibration products: phosphate buffer solution, bovine serum albumin, sodium chloride, preservative, N-terminal-pre-B type natriuretic peptide antigen; the method adopts a latex immunoturbidimetry method to improve test sensitivity, finds sensitized latex particles suitable for detecting the N-terminal pro-brain natriuretic peptide and improves detection sensitivity; by matching different full-automatic biochemical analyzers with different reagent sample ratios, a large batch of samples can be simultaneously and quantitatively detected; the kit is used for containing a calibrator to achieve the purpose of quantitative detection.
Description
Technical Field
The invention relates to the field of in-vitro diagnosis, in particular to an N-terminal brain natriuretic peptide precursor detection kit and a detection method thereof.
Background
The in vitro diagnostic reagent refers to: reagents, kits, calibrators, quality control materials and the like which can be used alone or in combination with instruments, equipment or systems for in vitro detection of human samples (various body fluids, cells, tissue samples and the like) in the processes of prevention, diagnosis, treatment monitoring, prognosis observation, health state evaluation and prediction of genetic diseases.
According to the degree of risk of the product, the in vitro diagnostic reagent is divided into a third class, a second class and a first class of products in turn. The third product category: reagents related to detection of antigens, antibodies and nucleic acids of pathogenic pathogens; reagents related to blood type, tissue matching; reagents related to human gene detection; an agent associated with a genetic disease; reagents related to the detection of narcotics, psychopharmaceuticals, and toxic medical drugs; reagents related to the detection of the target of action of the therapeutic drug; reagents associated with the detection of tumor markers; agents associated with allergies (allergens). The second type of products: besides the products which are already defined as the third class and the first class, the other products are the second class and mainly comprise: reagents for protein detection; reagents for detection of saccharides; reagents for hormone detection; reagents for enzyme detection; reagents for ester detection; reagents for vitamin detection; reagents for inorganic ion detection; reagents for the detection of drugs and drug metabolites; reagents for autoantibody detection; reagents for microbial identification or susceptibility testing; reagents for detecting other physiological, biochemical or immunological function indexes. The first category of products: microbial culture media (not used for microbial identification and susceptibility testing); products for sample treatment, such as hemolytic agents, diluents, staining solutions, and the like.
NT-proBNP is an N-terminal fragment of BNP prohormone (proBNP) inactive after division and secreted mainly by the left ventricle when the cardiomyocytes are subjected to an increase in volume and pressure load. NT-proBNP can be used for evaluating the systolic dysfunction, the diastolic dysfunction and the ventricular wall segment motion coordination, and has higher sensitivity and negative prediction value; the method is applied to early discovery of patients with Heart Failure (HF), risk stratification, monitoring of curative effect evaluation of heart failure drugs, judgment of prognosis of the patients with heart failure, and differentiation of dyspnea caused by heart failure and other reasons; can be used as risk assessment index of Acute Coronary Syndrome (ACS).
NT-proBNP is a molecule which can be conveniently processed in a laboratory, and has advantages before and during analysis, such as being quite stable at different temperatures and having low blood sample requirement. Currently, the related NT-proBNP detection methods include a colloidal gold method, an up-conversion luminescence method, an immunofluorescence method and the like.
The colloidal gold method is that under the action of reducing agent such as white phosphorus, ascorbic acid, sodium citrate and tannic acid, chloroauric acid (HAuCl4) can be polymerized into gold particles with a certain size, and the gold particles become a stable colloidal state due to electrostatic interaction, so that a negatively charged hydrophobic colloidal solution is formed, and the gold particles become a stable colloidal state due to electrostatic interaction, so that the gold particles are called colloidal gold. The method has the advantages of simple and convenient detection, and the defect that only qualitative judgment can be carried out, and only single sample detection can be carried out.
The up-conversion luminescence method is a novel detection technology using up-converting phosphor (UCP) particles as a tracer. After the immune reaction of the immune marker is detected by adopting the immunochromatography principle, the quantitative analysis of the object to be detected is realized by the quantitative ratio of the detection band and the finger control band through the UCP marker on the quantitative wipe paper strip detection band and the quality control band of the sensor. The method is divided into two types of dynamic scanning and static detection according to the working principle. The method has the defects that a full-automatic detection system matched with the method is lacked at present, the application is limited in the POCT field, and the requirement of batch sample detection cannot be met.
The immunofluorescence method is a technique for labeling an antibody with a fluorescent substance to localize an antigen. The method of tracing or checking the corresponding antigen with a fluorescent antibody is called a fluorescent antibody method; the method of tracing or examining the corresponding antibody with a known fluorescent antigen marker is called fluorescent antigen method. These two methods are collectively referred to as immunofluorescence techniques. The problem of nonspecific staining by the method is not completely solved, the objectivity of result judgment is insufficient, and the technical procedure is also complicated.
The three methods have the disadvantages of complex operation, long time consumption, high requirements on instruments and equipment and limited number of detected samples, and are difficult to realize automatic detection of large-scale samples clinically except for measurement in some special laboratories.
The latex immunoturbidimetry is to coat the antibody corresponding to the substance to be tested on latex particles, so that the volume of the antigen-antibody conjugate is increased, and the intensity change of transmitted light and scattered light is more obvious after light passes through the latex particles, thereby improving the sensitivity of the test.
But the detection of the N-terminal brain natriuretic peptide precursor does not find the sensitized latex particles with ideal effect, and the invention solves the problem; at present, the latex immunoturbidimetry is difficult to realize and is matched with a full-automatic biochemical analyzer for use clinically, a large number of samples are detected, and the result is mostly qualitative; the present invention solves such problems.
Disclosure of Invention
In order to solve the defects of the prior art, the invention aims to provide an N-terminal brain natriuretic peptide precursor detection kit and a detection method thereof; according to the invention, the latex immunoturbidimetry is used for improving the test sensitivity, so that sensitized latex particles suitable for detecting the N-terminal pro-brain natriuretic peptide are found, and the detection sensitivity is improved; by matching different full-automatic biochemical analyzers with different reagent sample ratios, a large batch of samples can be simultaneously and quantitatively detected; the kit is used for containing a calibrator to achieve the purpose of quantitative detection.
In order to achieve the above object, the present invention adopts the following technical solutions:
an N-terminal pro-brain natriuretic peptide detection kit comprising:
a first reagent:
100-150mmol/L of phosphate buffer solution,
0.005 to 0.06 percent of preservative,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
100-150mmol/L of phosphate buffer solution,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody;
calibration products:
100-150mmol/L of phosphate buffer solution,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
n-terminal-pre B-type natriuretic peptide antigen 1-5 ml/L.
The detection kit for the N-terminal pro-brain natriuretic peptide,
a first reagent:
phosphate buffer (pH 6.5) 100-150mmol/L,
3000.005-0.06 percent of biological preservative Proclin,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
phosphate buffer (pH 8.0) 100-150mmol/L,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
3000.005-0.06 percent of biological preservative Proclin,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody;
calibration products:
phosphate buffer (pH 8.0) 100-150mmol/L,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
3000.005-0.06 percent of biological preservative Proclin,
n-terminal-pre B-type natriuretic peptide antigen 1-5 ml/L.
The detection kit for the N-terminal pro-brain natriuretic peptide,
a first reagent:
phosphate buffer (pH 6.5) 100-150mmol/L,
3000.02% -0.05% of biological preservative Proclin,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
phosphate buffer (pH 8.0) 100-150mmol/L,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
3000.02% -0.05% of biological preservative Proclin,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody;
calibration products:
phosphate buffer (pH 8.0) 100-150mmol/L,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
3000.02% -0.05% of biological preservative Proclin,
n-terminal-pre B-type natriuretic peptide antigen 1-5 ml/L.
In the detection kit for the N-terminal pro-brain natriuretic peptide, the diameter range of the sensitized latex particles of the goat anti-human N-terminal-pro-B-type natriuretic peptide antibody is 20-60 nm.
In the detection kit for the N-terminal pro-brain natriuretic peptide, the diameter range of the sensitized latex particles of the goat anti-human N-terminal-pro-B-type natriuretic peptide antibody is 40 nm.
In the detection kit for the N-terminal pro-brain natriuretic peptide, the metering ratio of the first reagent, the second reagent and the calibrator is 8:2: 1.
If the N-terminal pro-brain natriuretic peptide detection kit adopts a beckman series full-automatic biochemical analyzer, the components of a reagent sample are as follows: 160. mu.l of reagent one, 40. mu.l of reagent two and 20. mu.l of calibrator.
If the N-terminal brain natriuretic peptide precursor detection kit adopts a Kowa excellent series full-automatic biochemical analyzer, the components of a reagent sample are as follows: 240 ul of the first reagent, 60 ul of the second reagent and 30 ul of the calibrator.
If a Hitachi series full-automatic biochemical analyzer is adopted, the reagent sample comprises the following components: reagent one 128 μ l: reagent two 32. mu.l: the calibrator was 16. mu.l.
The detection method of the N-terminal pro-brain natriuretic peptide detection kit is characterized by comprising the following steps of:
the method comprises the following steps: setting parameters on an instrument parameter interface according to a corresponding parameter table of the full-automatic biochemical analyzer;
step two: collecting blood of a patient, centrifuging within 30 minutes, taking supernatant as a detection sample, setting sample grade according to parameters, and putting the sample into a sample tray;
step three: setting reagent positions according to the parameters, respectively and correspondingly placing a first reagent and a second reagent in a reagent tray, and simultaneously placing a calibration product in a sample tray corresponding to the sample positions;
a first reagent:
phosphate buffer (pH 6.5) 100-150mmol/L,
0.005 to 0.06 percent of preservative,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
phosphate buffer (pH 8.0) 100-150mmol/L,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody;
calibration products:
phosphate buffer (pH 8.0) 100-150mmol/L,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
1-5ml/L of N-terminal-pre B-type natriuretic peptide antigen;
step four: starting a calibration test, and passing calibration;
step five: starting sample detection, respectively sucking a reagent I and a sample through a reagent needle and a sample needle, putting the reagent I and the sample into a reaction cup, stirring the reagent I and the sample, and reacting for 5 minutes;
step six: sucking a reagent II and a sample respectively through a reagent needle and a sample needle, putting the reagent II and the sample into a reaction cup, stirring and incubating for 5 minutes to ensure that NT-proBNP (antigen) in the sample meets specific anti-NT-proBNP (antibody) in a liquid phase to form an insoluble antigen-antibody complex and generate turbidity;
step seven: measuring the absorbance change value of the NT-proBNP in the sample according to the specific absorbance change of the NT-proBNP at 570nm, and calculating the content of the NT-proBNP in the sample through the following formula by comparing the absorbance change value with the concentration of a calibrator in the kit and the absorbance change value of the calibrator;
the positive defining values for NT-proBNP were: patients <50 years of age and serum < 450 ng/L; patients aged 50-75 years and having serum < 900 ng/L; patients > 75 years of age and serum < 1800 ng/L.
The invention has the advantages that:
the detection kit of N-terminal brain natriuretic peptide precursor (NT-proBNP) adopts a microparticle enhanced immunoassay method, wherein NT-proBNP (antigen) in a sample meets specific anti-NT-proBNP (antibody) in a liquid phase to immediately form an insoluble antigen-antibody complex, and certain turbidity is generated. The turbidity is in direct proportion to the content of NT-proBNP in the sample, and the content of NT-proBNP in the sample can be calculated by comparing the turbidity with a calibrator treated in the same way;
the kit adopts 15-60nm latex, combines sheep polyclonal antibody to form sensitized latex particles, and enables the detection sensitivity to reach 1 ng/L;
the kit adopts the Proclin300 biological preservative with the concentration range of 0.02-0.05 to improve the stability of the kit;
the invention can simultaneously and quantitatively detect a large number of samples by matching different full-automatic biochemical analyzers with different reagent sample ratios;
the kit adopted by the invention contains a calibrator to achieve the purpose of quantitative detection.
Drawings
FIG. 1 is a scatter plot of the results of the testing of serum from a sample patient of the present invention;
FIG. 2 is a scattergram of the results of the measurement of serum from a sample patient according to the present invention arranged in descending order;
FIG. 3 is a graph showing the resulting distribution of N-terminal-pre-B natriuretic peptide from serum of a patient sample according to the present invention.
Detailed Description
The invention is described in detail below with reference to the figures and the embodiments.
An N-terminal pro-brain natriuretic peptide detection kit comprising:
a first reagent:
phosphate buffer (pH 6.5) 100-150mmol/L,
0.005 to 0.06 percent of preservative,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
phosphate buffer (pH 8.0) 100-150mmol/L,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody;
calibration products:
phosphate buffer (pH 8.0) 100-150mmol/L,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
n-terminal-pre B-type natriuretic peptide antigen 1-5 ml/L.
It should be noted that: the raw materials adopted by the components of the invention are obtained by external purchase, and the preparation is not needed, and the components are specifically shown in the table I:
watch 1
A detection method of an N-terminal pro-brain natriuretic peptide detection kit comprises the following steps:
the method comprises the following steps: and setting parameters on an instrument parameter interface according to the corresponding parameter table of the full-automatic biochemical analyzer.
Step two: collecting blood of a patient, centrifuging within 30 minutes, taking supernatant as a detection sample, setting the sample grade according to parameters, and placing the sample into a sample tray.
Step three: setting reagent positions according to the parameters, correspondingly placing a reagent I and a reagent II in the reagent tray respectively, and placing a calibration product in the sample tray correspondingly to the sample position.
Step four: and starting a calibration test to pass calibration.
Step five: starting sample detection, respectively sucking a reagent I and a sample through a reagent needle and a sample needle, putting the reagent I and the sample into a reaction cup, stirring the reagent I and the sample, and reacting for 5 minutes; it is noted that such manipulation can reduce the effect of interfering substances in the sample on the detection of NT-proBNP.
Step six: sucking a reagent II and a sample respectively through a reagent needle and a sample needle, putting the reagent II and the sample into a reaction cup, stirring and incubating for 5 minutes to ensure that NT-proBNP (antigen) in the sample meets specific anti-NT-proBNP (antibody) in a liquid phase to form an insoluble antigen-antibody complex and generate turbidity; it should be noted that: the turbidity is proportional to the amount of NT-BNP in the sample.
Step seven: measuring the absorbance change value of the NT-proBNP in the sample according to the specific absorbance change of the NT-proBNP at 570nm, and calculating the content of the NT-proBNP in the sample through the following formula by comparing the absorbance change value with the concentration of a calibrator in the kit and the absorbance change value of the calibrator;
the content of N-terminal pro-brain natriuretic peptide (NT-proBNP) was correlated with the age of the patients, and was confirmed by a sample of 200 healthy individuals,
the verification process is as follows:
first, selection sample requirement and experimental method
Since the content of N-terminal brain natriuretic peptide (NT-PROBNP) is related to the age of the patient, this experiment was performed with a sample group collection test. Three groups were divided according to age: serum from <50 year old patients; serum for patients 50-75 years old; serum for > 75 year old patients.
The human serum samples are all collected from physical examination normal human samples of the clinical laboratory of hospitals, and the samples are required to be fresh without hemolysis, jaundice and lipid turbidity.
Number of samples: 200 specimens were tested for males and females <50 years old; 200 samples are tested by male and female patients of 50-75 years old; 200 specimens were tested for males and females > 75 years old.
An experimental instrument: hitachi 7060, which ensures the normal state of the instrument before testing.
Experimenters require: laboratory staff can be skilled in operating the inspection instrument, familiar with the experimental contents, and can analyze and count the experimental data.
Setting the on-machine determination parameters according to the kit use instruction, determining the collected serum sample, and then performing statistical analysis on the determination result.
Second, data requirement and analysis
1. In order to ensure the reliability of the reference value data, 200 sample data are selected.
2. And judging the suspected outliers in the data, and dividing the difference D between the suspected outliers and the adjacent points thereof by the data overall distance R, wherein if the D/R is less than or equal to 1/3, the suspected outliers are considered as non-outliers. If there are two or more outliers, the smallest suspected outliers can be treated as above, if all > 1/3, all points must be rejected; if both are ≦ 1/3, all data is retained.
3. If the outliers are eliminated, other data are supplemented.
4. And drawing a distribution map to know the distribution characteristics of the data. If the data is normally distributed or the data is converted to be normally distributed, the data can be distributed according to
Representing 95% of the data distribution range. If the data are not normally distributed, non-parametric methods can be adopted for processing, and 2.5% and 97.5% digit reference limits are determined by a percentile method, so that a 95% reference interval is determined.
Third, experimental data and statistical process
1. The sample test data table is as follows: (Unit ng/L)
Serum of <50 year old patient:
50-75 years old
Patient older than 75 years
2. The sample scattergram is shown in FIG. 1;
and (3) judging outliers:
2.1 arranging all data from small to big, making a scatter diagram as shown in FIG. 2: 2.2 visual inspection, the data points clearly resemble outliers.
3. Sample data distribution map
3-1N-terminal-pro B-type natriuretic peptide frequency distribution table
Serum of <50 year old patient:
the resulting profile of 3-2N-terminal-pro B-type natriuretic peptide is shown in FIG. 3 (a);
according to the normal distribution rule, 95% of the observed values are
Within the range. I.e. 34.08-479.37 ng/L. Obviously, the distribution of the whole data of the experiment belongs to a skewed distribution, and the 95% distribution range cannot be obtained
And (4) calculating and determining. The percentile method is required. The reference limits for the 2.5% and 97.5% digits were determined, from which the 95% reference interval was determined.
In the formula:
pr: the r percentile;
l: the lower limit of the group where the r percentile is located;
w: the width of the group where the r percentile is located;
f: frequency of the group in which the r percentile is located
n: the total frequency;
c: the cumulative frequency of the group before the group where the r percentile is located.
And (3) calculating:
1. the 95 th percentile is counted to be 200, the 95 th percentile thereof should be the 200 × 95 th percentile to be 190, and the percentile is known to be in the 7 th group from the frequency distribution table, so as to determine each value in the formula: the calculation result shows that P95 is 433.33ng/L
2. The 2.5 th percentile is counted as 200, the 2.5 th percentile is the 200 x 2.5 th percentile which is 5, the percentile is known to be in the 1 st group from the frequency distribution table, and the values in the formula are determined, and the P2.5 is calculated as 74.74ng/L
3. The 97.5 th percentile, which is statistically 200, should have the 97.5 th percentile of 200 × 97.5% — 195, and this percentile is found in group 7 from the frequency distribution table, thereby determining the respective values in the formula:
the calculation result shows that P97.5 is 442.67ng/L
Serum for 50-75 years old patient
3-2N-terminal-pro B-type natriuretic peptide results profile; as shown in FIG. 3(b)
According to the normal distribution rule, 95% of the observed values are
Within the range. Namely 158.57-952.55 ng/L. Obviously, the distribution of the whole data of the experiment belongs to a skewed distribution, and the 95% distribution range cannot be obtained
And (4) calculating and determining. The percentile method is required. The reference limits for the 2.5% and 97.5% digits were determined, from which the 95% reference interval was determined.
In the formula:
pr: the r percentile;
l: the lower limit of the group where the r percentile is located;
w: the width of the group where the r percentile is located;
f: frequency of the group in which the r percentile is located
n: the total frequency;
c: the cumulative frequency of the group before the group where the r percentile is located.
And (3) calculating:
1. the 95 th percentile is counted to be 200, the 95 th percentile thereof should be the 200 × 95 th percentile to be 190, and the percentile is known to be in the 7 th group from the frequency distribution table, so as to determine each value in the formula: the calculation result shows that P95 is 864.29ng/L
2. The 2.5 th percentile is counted as 200, the 2.5 th percentile is the 200 x 2.5 th percentile which is 5, the percentile is known to be in the 1 st group from the frequency distribution table, and the values in the formula are determined, and the P2.5 is calculated as 217.24ng/L
3. The 97.5 th percentile, which is statistically 200, should have the 97.5 th percentile of 200 × 97.5% — 195, and this percentile is found in group 7 from the frequency distribution table, thereby determining the respective values in the formula:
the calculation result shows that P97.5 is 882.14ng/L
Patient older than 75 years
The resulting profile of 3-2N-terminal-pro B-type natriuretic peptide is shown in FIG. 3 (c);
according to the normal distribution rule, 95% of the observed values are
Within the range. Namely 82.20-1890.55 ng/L. Obviously, the distribution of the whole data of the experiment belongs to a skewed distribution, and the 95% distribution range cannot be obtained
And (4) calculating and determining. The percentile method is required. The reference limits for the 2.5% and 97.5% digits were determined, from which the 95% reference interval was determined.
In the formula:
pr: the r percentile;
l: the lower limit of the group where the r percentile is located;
w: the width of the group where the r percentile is located;
f: frequency of the group in which the r percentile is located
n: the total frequency;
c: the cumulative frequency of the group before the group where the r percentile is located.
And (3) calculating:
1. the 95 th percentile is counted to be 200, the 95 th percentile thereof should be the 200 × 95 th percentile to be 190, and the percentile is known to be in the 7 th group from the frequency distribution table, so as to determine each value in the formula: the calculation result shows that P95 is 1695.65ng/L
2. The 2.5 th percentile is counted as 200, the 2.5 th percentile is the 200 x 2.5 th percentile which is 5, the percentile is known to be in the 1 st group from the frequency distribution table, and the values in the formula are determined, and the P2.5 is calculated as 164.44ng/L
3. The 97.5 th percentile, which is statistically 200, should have the 97.5 th percentile of 200 × 97.5% — 195, and this percentile is found in group 7 from the frequency distribution table, thereby determining the respective values in the formula:
the calculation result shows that P97.5 is 1747.83ng/L
The experiment finally obtains a normal healthy human serum sample of a patient of <50 years old; patients 50-75 years old; test data of 200 parts of each patient aged > 75 years have no outliers. The sample distribution is skewed, and the distribution range of 2.5-97.5% is determined by an objective and simple percentile method as follows: 74.74-442.67 ng/L; 217.24-882.14 ng/L; 164.44-1747.83 ng/L.
The positive definite value of NT-proBNP after being adjusted according to age by combining the literature reference data at home and abroad is as follows: patients <50 years of age and serum < 450 ng/L; patients aged 50-75 years and having serum < 900 ng/L; patients > 75 years of age and serum < 1800 ng/L.
In order to verify the respective preferences and effects of the present invention, the following experiments were carried out:
experiment one, selecting and testing the diameter of latex;
preferably, the diameter of the sensitized latex particles of the goat anti-human N-terminal pre-B type natriuretic peptide antibody is in the range of 20 to 60 nm.
Experiment 1.1: the sensitized latex particles of the goat anti-human N-terminal-pre-B type natriuretic peptide antibody formed by combining the same antibody with latex particles with different diameters are purchased, the same N-terminal-pre-B type natriuretic peptide antigen is added, the mixture is uniformly mixed for 1 to 2 minutes, the agglutination result is observed within 5 minutes, and the test result is shown in the second table.
Watch two
As is clear from the Table II, the latex particles having a diameter of 20,40, and 60nm bound to the sensitized latex particles of the goat anti-human N-terminal-pre-B natriuretic peptide antibody formed from the goat anti-human N-terminal-pre-B natriuretic peptide antibody, and the results were as good as possible.
Experiment 2.2 included the following steps:
step one, respectively detecting the absorbance of sensitized latex particles of a goat anti-human N-terminal-pre-B type natriuretic peptide antibody formed by combining latex with a goat anti-human N-terminal-pre-B type natriuretic peptide antibody, wherein the latex has the diameter of 20,40,60, 80, 100 and 200nm, and the target required absorbance A1 is more than or equal to 1.2.
And step two, after the step one is finished, adding the same antigen, and measuring the absorbance A2 again. The requirement that A2-A1 is more than or equal to 0.05; see table three.
Watch III
The experiments can show that when the diameter of the latex is within the range of 20-60nm, the agglutination reaction is sufficiently and uniformly, the absorbance test change is obvious, and the sensitivity is high; considering the research and development cost problem, meeting the performance index requirement, and carrying out subsequent experiments by selecting sensitized latex particles with the diameter of 40 nm.
Experiment two: selecting the ratio of reagents;
preferably, when the metering ratio of the first reagent to the second reagent is 4:1, the residual quantity of the reagents is the least, and the utilization rate of the reagents is the highest.
Dividing the prepared reagent I (R1) and reagent II (R2) into 3 groups according to the proportion of 5:1, 4:1 and 3: 1; the reaction amount of the first reagent and the reaction amount of the second reagent are respectively set as follows according to the reagent proportion: 250, 50; 240, 60; 225, a step of mixing; 75, the total amount of the reagent is consistent. In the same full-automatic biochemical analysis (Hitachi 7080), samples with the same sample amount are detected at the same time until the reagent needle alarms that the reagent amount cannot be sucked, and the residual amounts of the first reagent, the second reagent and the number of the detected samples are observed. And calculating the utilization rate of the reagent according to the result. The results are shown in Table four and Table five.
Watch four
Watch five
As can be seen from tables IV and V, the reagent ratios of 4:1 and 3:1 are not different from the actual number of samples to be detected, the residual amount of the reagent is observed, the ratio of 4:1 is the minimum, the ratio of 4:1 is the highest, and therefore, the reagent ratio of 4:1 is selected for subsequent experiments.
Experiment three: selection of a reagent sample calibrator dose;
preferably, the ratio of the reagent to the calibrator is as follows: 10, reagent: 1, calibrating a product;
the amount of reagent determines the concentration of effective antibody in the reaction volume, and the ratio of the sample of reagent affects the linear range of the reagent. According to the reference value situation and the detection linearity requirement of a clinical reagent sample, the linearity range of the detection kit for the N-terminal brain natriuretic peptide precursor (NT-proBNP) is set to be 50-35000ng/L, and the following requirements are met:
a) the linear correlation coefficient r is more than or equal to 0.990;
b) linear deviation: the absolute deviation in the range of [ 100-; the relative deviation in the range of [5000 + 32000] ng/L should not exceed +/-10%.
Fixing the amounts of the R1 reagent and the R2 reagent, and respectively sucking different sample amounts for 7 point samples of the linear segmentation, wherein the sample adding amount is set as shown in the sixth table; calculating deviation and correlation coefficient by using regression equation, wherein the linear detection experiment result is shown in table seven;
watch six
Watch seven
From the above 5 groups of test results, it can be seen that under the condition of fixed reagent amount, the sample amount is 30 μ l, the reagent linearity effect is the best, and the correlation coefficient and deviation can both meet the test requirements. There were no false positives (excess antibody, detection result out of reagent linearity) when the sample size was insufficient, and no false negatives (excess antigen, incomplete reaction of reagent, no detection of actual content in sample) when the sample size was excessive
The comprehensive experiments II and III show that: the optimal metering ratio of the first reagent, the second reagent and the calibrator is 8:2: 1.
Experiment four: selecting and using amount experiment of the stabilizer;
preferably, the preservative is Proclin300, and the optimal dosage is 0.02-0.05%.
Experiment 4.1: selecting the dosage of the stabilizer;
dividing the same batch of N-terminal pro-brain natriuretic peptide (NT-proBNP) detection kit (latex immunoturbidimetry) into a plurality of equal parts, adding different amounts of Proclin300 into each part, and respectively detecting each performance index of each part of kit on the premise that the effective components and the performance indexes of the kit are not influenced by the amount of the stabilizer, wherein the results are shown in the table eight:
table eight
The experimental data show that the dosage of the Proclin300 is within the range of 0.005-0.06%, all technical indexes of the NT-proBNP measuring kit are within acceptable ranges, but the measuring result of all indexes within the range of 0.02-0.05% is the most ideal state.
Experiment 4.2: experiment on stabilizing effect of stabilizer
Experiment 4.1 shows that the test results of each index of the kit are in the most ideal state when the dosage of the Proclin300 is within the range of 0.02-0.05 percent, so that the preparation respectively contains 0.20 per thousand, 0.25 per thousand, 0.30 per thousand, 0.35 per thousand, 0.40 per thousand, 0.45 per thousand and 0.50 per thousand; and (3) carrying out a stability effect experiment on the NT-proBNP assay kit of the Proclin300, and simultaneously preparing the NT-proBNP assay kit containing 1g/L of sodium azide to compare the stability effects of the NT-proBNP assay kit and the NT-proBNP assay kit at the end of the stability effect period. The in-vitro diagnostic kit has the effective period of 12 months, so that the performance of the detection reagent is detected and contrasted and observed in the prepared kit respectively in 11 months, 12 months and 13 months; the results of the Proclin300 stabilization effect experiments are shown in table nine:
watch nine
The results of comparison of the stabilization effect of Proclin300 with that of 1g/L sodium azide at 13 months are shown in Table ten:
watch ten
From 13 months later, the result of adding different stabilizers to the same kit shows that 0.02-0.05% of proclin300 stabilizer has better effect than the common sodium azide stabilizer.
According to the results obtained from the above different experiments, the best choice is: preparing a 4:1 reagent by selecting a sensitizing latex particle raw material with 40nm latex combined with an NT-proBNT antibody and selecting 0.03% proclin300 dosage stabilizer; in the parameter setting, the ratio of the reagent metering to the calibrator is 10: 1. The total dosage can be properly adjusted according to the total sample adding amount of different instruments, but the ratio of the reagents and the ratio of the reagent samples are not changed.
The specific case is as follows:
if the delta adopts a Beckman DXC800 full-automatic biochemical analyzer, the proportion of reagent samples is as follows: 160, reagent one: 40, reagent II: 20, a calibrator.
The reagent sample ratio meets the requirement of detecting 350 persons at one time by adopting 160/40/20 according to the test result and the proportion of the reagent sample ratio when the reagent sample ratio is matched with a Beckmann DXC800 series full-automatic biochemical analyzer for use.
If a full-automatic biochemical analyzer of Kehua excellent 330 series is adopted, the proportion of the reagent samples is as follows: 240, reagent one: 60, reagent II: 30, a calibrator.
The reagent sample ratio meets the requirement of detecting 230 persons at one time by adopting 240/60/30 according to the test result by matching with a Kehuazhuo 330 full-automatic biochemical analyzer.
If a Hitachi 7060 full-automatic biochemical analyzer is adopted, the proportion of the reagent samples is as follows: 128, reagent one: 32, reagent II: 16 calibration article.
The reagent sample ratio meets the requirement of detecting 450 persons at a time by adopting 128/32/16 according to the test result by matching with Hitachi 7060 series full-automatic biochemical analyzers.
The invention provides an N-terminal brain natriuretic peptide precursor detection kit and a detection method thereof; the kit adopts a microparticle enhanced immunoassay method, and NT-proBNP (antigen) in a sample meets specific anti-NT-proBNP (antibody) in a liquid phase to immediately form an insoluble antigen-antibody complex to generate certain turbidity; the turbidity is in direct proportion to the content of NT-proBNP in the sample, and the content of NT-proBNP in the sample can be calculated by comparing the turbidity with a calibrator treated in the same way; the kit adopts 15-60nm latex, combines sheep polyclonal antibody to form sensitized latex particles, and enables the detection sensitivity to reach 1 ng/L; the kit adopts the Proclin300 biological preservative with the concentration range of 0.02-0.05 to improve the stability of the kit; the invention can simultaneously and quantitatively detect a large number of samples by matching different full-automatic biochemical analyzers with different reagent sample ratios; the kit adopted by the invention contains a calibrator, so that the aim of quantitative detection is fulfilled.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It should be understood by those skilled in the art that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the scope of the present invention.
Claims (3)
1. An N-terminal pro-brain natriuretic peptide detection kit, comprising:
a first reagent:
100-150mmol/L of phosphate buffer solution,
0.005 to 0.06 percent of preservative,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
100-150mmol/L of phosphate buffer solution,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody; the diameter of the sensitized latex particles of the sheep antihuman N-terminal-pre-B type natriuretic peptide antibody is 40 nm;
calibration products:
100-150mmol/L of phosphate buffer solution,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
0.005 to 0.06 percent of preservative,
1-5ml/L of N-terminal-pre B-type natriuretic peptide antigen;
the metering ratio of the first reagent, the second reagent and the calibrator is 8:2: 1;
if a Beckmann series full-automatic biochemical analyzer is adopted, the components of a reagent sample are as follows: 160 mul of reagent I, 40 mul of reagent II and 20 mul of calibrator;
if a Koehua excellent series full-automatic biochemical analyzer is adopted, the components of a reagent sample are as follows: 240 mul of reagent I, 60 mul of reagent II and 30 mul of calibrator;
if a Koehua excellent series full-automatic biochemical analyzer is adopted, the components of a reagent sample are as follows: 240 mul of reagent I, 60 mul of reagent II and 30 mul of calibrator;
if a Hitachi series full-automatic biochemical analyzer is adopted, the components of a reagent sample are as follows: reagent one 128 μ l: reagent two 32. mu.l: the calibrator was 16. mu.l.
2. The kit for detecting N-terminal pro-brain natriuretic peptide according to claim 1,
a first reagent:
phosphate buffer PH = 6.5100-150 mmol/L,
3000.005-0.06 percent of biological preservative Proclin,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
phosphate buffer PH = 8.0100-150 mmol/L,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
3000.005-0.06 percent of biological preservative Proclin,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody;
calibration products:
phosphate buffer PH = 8.0100-150 mmol/L,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
3000.005-0.06 percent of biological preservative Proclin,
n-terminal-pre B-type natriuretic peptide antigen 1-5 ml/L.
3. The kit for detecting N-terminal pro-brain natriuretic peptide according to claim 2,
a first reagent:
phosphate buffer PH = 6.5100-150 mmol/L,
3000.02% -0.05% of biological preservative Proclin,
tween 200.5-2.0 mL/L,
polyethylene glycol 600025-60 g/L;
and a second reagent:
phosphate buffer PH = 8.0100-150 mmol/L,
20-60g/L of disodium hydrogen phosphate,
2-5g/L of sodium dihydrogen phosphate,
10-50g/L of sodium chloride,
3000.02% -0.05% of biological preservative Proclin,
1-5ml/L of sensitized latex particles of sheep anti-human N-terminal-pre-B type natriuretic peptide antibody;
calibration products:
phosphate buffer PH = 8.0100-150 mmol/L,
10-50g/L of bovine serum albumin,
10-50g/L of sodium chloride,
3000.02% -0.05% of biological preservative Proclin,
n-terminal-pre B-type natriuretic peptide antigen 1-5 ml/L.
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| CN111505302A (en) * | 2019-01-31 | 2020-08-07 | 博阳生物科技（上海）有限公司 | Homogeneous immunoassay kit for rapidly detecting N-terminal B-type brain natriuretic peptide, preparation method, detection method and device |
| CN114207444A (en) | 2019-08-13 | 2022-03-18 | 挪威金田有限公司 | High sensitivity particle-enhanced assay for the quantification of NT-proBNP |
| CN110596367B (en) * | 2019-09-06 | 2023-03-28 | 广州科方生物技术股份有限公司 | Stabilizer capable of prolonging effective period of brain natriuretic peptide calibrator and preparation method thereof |
| CN110940815B (en) * | 2019-12-16 | 2023-11-17 | 成都普利泰生物科技有限公司 | Emulsion immunonephelometry detection kit for quantitatively determining cat serum amyloid A |
| WO2023190560A1 (en) * | 2022-03-31 | 2023-10-05 | デンカ株式会社 | Method, composition, and kit for detecting human b-type natriuretic peptide, or precursor or degradation product of same |
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