CN106093416B - A kind of kit and preparation method thereof of one-step method detection Procalcitonin - Google Patents
A kind of kit and preparation method thereof of one-step method detection Procalcitonin Download PDFInfo
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- CN106093416B CN106093416B CN201610325807.1A CN201610325807A CN106093416B CN 106093416 B CN106093416 B CN 106093416B CN 201610325807 A CN201610325807 A CN 201610325807A CN 106093416 B CN106093416 B CN 106093416B
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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
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
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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
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- G01N21/763—Bioluminescence
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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Abstract
The present invention provides the kit and preparation method thereof of Procalcitonin content in a kind of one-step method detection human serum.This detection kit uses a step sandwich method reaction principle, and the kit includes：Calibration object, enzyme conjugates, coating plate, luminous substrate liquid, concentrated cleaning solution, wherein the enzyme conjugates is the anti-PCT monoclonal antibodies of horseradish peroxidase-labeled, and the coating plate is that PCT monoclonal antibodies will be resisted to be coated on surface of solid phase carriers.The present invention provide a kind of fast and easy, high sensitivity, high specificity, reproducible, quantitative true scope is wide and can be obtained in 90 minutes testing result and it is at low cost the advantages that.PCT detection kits provided by the invention, its instrument compatibility is strong, and testing cost is low, compensates for the demand that product is clinically detected to PCT.
Description
Technical field
The present invention relates to external diagnosis reagent fields, in particular it relates to which a kind of detecting people's blood with chemoluminescence method
The kit and preparation method thereof of Procalcitonin content in clear.
Background technology
Procalcitonin (PCT) is made of 116 amino acid, and molecular weight is about 12.7KD.PCT is by neuroendocrine cell
(the C cells for including thyroid gland, lung and pancreatic tissue) is expressed, and (prematurity) calcitonin, c-terminal peptides and ammonia are decomposed into through digestion
Cardinal extremity peptide.Only contain a small amount of PCT in Healthy People blood.PCT can be significantly raised after bacterium infection.
When animal model test shows that septicopyemia occurs for body, multigroup knit can express PCT.In patients with sepsis body
PCT contain only 114 amino acid, lack the Ala-Pro of amino terminal.
The raising of PCT levels sees bacillary septicopyemia, especially severe septicopyemia and infectious shock.PCT can make
For the prognostic indicator and acute critical pancreatitis of septicopyemia and its reliability index of major complications.
For Community Acquired Respiratory Tract Infection and air-conditioning inductivity patients with pneumonia, PCT can be used as antibiotic selection and
The index that curative effect judges.
The blood plasma PCT mass concentrations of Healthy People are less than 0.05ng/ml.The elderly, patients with chronic diseases and less than 10%
Human normal plasma PCT mass concentrations be higher than 0.05ng/ml, reach as high as 0.1ng/ml, but be usually no more than 0.3ng/ml.Purulence
The diagnosis dividing value of toxication patient PCT is more than 0.5ng/ml, severe sepsis and the fluctuation of patients with septic shock PCT mass concentrations
Between 5~500ng/ml.Only a few severe infections patients blood plasma PCT levels are more than 1000ng/ml.
Human calcitonin original (PCT) is the precursor of human calcitonin, no hormonal activity, the horizontal pole in Healthy Human Serum
Low, but content increases rapidly in systemic bacterial infections patients serum, infecting latter two hour can detect, and when continuing
Between it is long, level and the severity of infectious diseases in serum are proportionate, after effective antibiotics are treated, PCT water
It is flat to decline rapidly, and viral infection, tumour, autoimmune disease, wound (more wounds or operation wound), clinical application,
Chronic inflammation and local infection person, PCT levels are maintained within normal range or have slight raising.Therefore, PCT is bacterium sense
The good index of severe systemic inflammatory reaction caused by dye, in systemic bacterial infections and the antidiastole of pyemia auxiliary, prognosis
Judgement, observation of curative effect etc. have very high clinical value.
Invention content
The object of the present invention is to provide a kind of kits of detection Procalcitonin (PCT).Detection examination provided by the present invention
Agent box uses a step sandwich method reaction principle, and combines Chemiluminescence Immunoassay.Kit include calibration object, enzyme conjugates,
It is coated with plate, luminous substrate liquid, concentrated cleaning solution.
Wherein, the enzyme conjugates is the anti-PCT monoclonal antibodies of horseradish peroxidase-labeled.
The coating plate is that PCT monoclonal antibodies will be resisted to be coated on surface of solid phase carriers.
The technical scheme is that using 2 plants of anti-PCT monoclonal antibodies, one plant of PCT monoclonal antibody is coated with first
In surface of solid phase carriers, after being separately added into calibration object or blood serum sample in each hole of microwell plate, adds enzyme and mark another plant of PCT
Monoclonal antibody forms solid matrix antibody-antigen-hrp-antibody complex after incubation.Chemiluminescence bottom fully is added after washing
Thing liquid, luminous intensity are directly proportional to PCT concentration.When measurement, the liquid in coating plate is first washed away, luminous substrate liquid is added,
Its luminous intensity is directly proportional to PCT concentration.It measures per hole luminous value (RLU), (is needed with the logarithm of each calibration object luminous value (RLU)
Subtract S0Luminous value) be ordinate (Y-axis), the logarithm of each calibration object concentration is that abscissa (X-axis) is mapped, and it is bent to obtain standard
Line.PCT concentration can be found from standard curve in sample.
It is a further object to provide a kind of preparation methods such as above-mentioned detection kit, including following step
Suddenly：
1. calibration object is prepared：It is divided into the phosphate buffer that 0.01~0.05M pH are 7.0~9.6, volume ratio 2 with group
PCT antigen bulks are diluted to 0.2 by~20% newborn bovine serum and 0.1%~1%Proclin-300 standard dilutions~
50ng/ml, 4 DEG C save backup, and are calibration object.
2. the preparation of enzyme conjugates：Improvement sodium periodate oxidizing process is selected in the connection of HRP and anti-PCT monoclonal antibodies.This
Method is that the hydroxyl on HRP molecules is directly oxidized to aldehyde radical with sodium metaperiodate and forms hydroformylation enzyme, is added under conditions of pH4~6
Enter ethylene glycol to neutralize NaIO4.The amino of this aldehyde radical and antibody molecule forms Schiff, adds NaBH4Reduction is formed steady
Fixed enzyme-antibody conjugates.Then use 0.01~0.05M pH for 7.0~9.6 phosphate buffer, volume ratio be 2~
20% newborn bovine serum, 0.1%~1%Proclin-300 and 0.1%~0.5% Food Red buffer solution are by enzyme-antibody conjugates
It is diluted according to 1: 1000~1: 10000 volume ratio, 4 DEG C save backup, and are enzyme conjugates.
3. being coated with the preparation of plate：It is that 7.0~9.6 phosphate makees coating buffer solution to select 0.01~0.05M pH, will be resisted
PCT monoclonal antibodies are added in microwell plate, 100 holes the μ l/ of μ l~200 by 2~10 μ g/ml dilutions, are got rid of after 2~8 DEG C of overnight coatings
Coating buffer is gone, 100 holes the μ l/ of μ l~200 of confining liquid are added, 2~8 DEG C are closed overnight, with the blank site on closed plate hole wall, so
After get rid of deblocking liquid, button is dry, room temperature drying, to be coated with plate.
4. the preparation of luminous substrate liquid：For 2~10mmol/L hydrogen peroxide, 2~10mmol/L Luminol, 2~
The Tris-HCL buffer solutions of 10mmol/L p-iodopHenol, 10~100mmol/L pH 7.0~9.6.
5. the preparation of concentrated cleaning solution：The phosphate for being 7.0~9.6 for 0.01~0.05M pH, mass ratio be 10~
20% NaCl and the buffer solution for containing 0.2~2%Tween20.
The kit of the present invention has easy to operate, it is only necessary to a step incubation reaction, high sensitivity, high specificity, again
Renaturation is good, it is quantitative it is accurate, range is wide, testing result can be obtained in 90 minutes and it is at low cost the advantages that.Its application value is：
(1) septicemia is early diagnosed；(2) early stage is done to systematic severe bacterial infections (peritonitis or soft tissue infection etc.) to examine
It is disconnected；(3) it makes a differential diagnosis to septicemia and SIRS；(4) bacterium infection and non-bacteria inflammation react (autoimmune disease
Deng) antidiastole；(5) antidiastole (encephalomyelitis etc.) of bacterium infection and virus infection；(6) the postoperative discriminating of organ transplant
Diagnosis (bacterium infection, virus infection, absorption heat, rejection, fungal infection etc.)；(7) unknown cause fever (UFO) is examined
It is disconnected and special infection high-risk patient (intensive care unit, organ transplant postoperative, immunosupress phase) is monitored；(8) septic shock
With the antidiastole of non-septic shock.
The present invention provides a kind of fast and easy, high sensitivity, high specificity, reproducible, quantitative true scope wide PCT
Detection kit, the kit instrument compatibility is strong, and testing cost is low, compensates for the demand clinically to PCT products.
Description of the drawings
.A batches of kit log-log linear graphs of Fig. 1, through regression analysis：Y=0.937x+4.572；R2=0.997.
.B batches of kit log-log linear graphs of Fig. 2, through regression analysis：Y=0.946x+4.571；R2=0.998.
.C batches of kit log-log linear graphs of Fig. 3, through regression analysis：Y=0.946x+4.571；R2=0.998.
Specific implementation mode
Below in conjunction with the accompanying drawings, the implementation that the present invention will be described in detail operation, but the claimed technical solution of the present invention not by
It is limited to following embodiments, in the case where not changing its main points, various changes can be done and implemented.
The reagent wherein used in embodiment in the case where meeting requirement of experiment, is purchased from if not otherwise specified
Reagent Company.
1 kit preparation process of embodiment
Kit preparation process includes the following steps：
(1) preparation of calibration object
1. formulation components are the phosphate of a concentration of pH7.0~9.6 0.01~0.05M, volume ratio is 2~20% newborn
The calibration object dilution of cow's serum and 0.1%~1%Proclin-300.
It is calibration object 2. PCT antigen bulks are diluted to each concentration point within the scope of 0.2~50ng/ml.
3. being dispensed according to 0.5ml/ bottles, 2~8 DEG C save backup.
(2) preparation of enzyme conjugates；
The label of 1.PCT monoclonal antibodies：Improvement sodium periodate oxidation is selected in the connection of HRP and anti-PCT monoclonal antibodies
Method forms stable enzyme-antibody conjugates.
2. by the enzyme-antibody conjugates marked by 1: 1000~1: 10000 volume ratio be added to formula for 0.01~
The phosphate buffer that 0.05M pH are 7.0~9.6, volume ratio are 2~20% newborn bovine serum, 0.1%~1%Proclin-
300 and 0.1%~0.5% Food Red enzyme dilution in.
3. being dispensed according to 6ml/ bottles, 2~8 DEG C save backup.
(3) it is coated with the preparation of plate
1. PCT monoclonal antibodies are diluted to 2~10 μ with 0.01~0.05M pH phosphate buffers for being 7.0~9.6
G/ml is added by 100 holes the μ l/ of μ l~200 in microwell plate, and 2~8 DEG C are coated with (16~24 hours) overnight.
2. getting rid of coating buffer, buckles and do on blotting paper, general 100 holes the μ l/ of μ l~200 of BSA confining liquids of addition per hole, 2~8
DEG C overnight (16~24 hours) close.
3. getting rid of deblocking liquid, dry, (15~28 DEG C) dryings of room temperature are buckled on blotting paper, by dry pre-coated plate loading aluminium
It is encapsulated in foil bag, 2~8 DEG C save backup.
(4) preparation of luminous substrate liquid
1. formulation components are 2~10mmol/L hydrogen peroxide, 2~10mmol/L Luminol, 2~10mmol/L p-
The luminous substrate liquid of the Tris-HCL of pH7.0~9.6 iodopHenol, 10~100mmol/L.
2. being dispensed according to 7ml/ bottles, 2~8 DEG C save backup.
(5) preparation of concentrated cleaning solution
1. the phosphate buffer that it is 7.0~9.6 that formulation components, which are 0.01~0.05M pH, mass ratio are 10~20%
NaCl and the concentrated cleaning solution for containing 0.2~2%Tween20.
2. being dispensed according to 20ml/ bottles, 2~8 DEG C save backup.
2 kit of embodiment detects operating process
1. taking out kit and blood serum sample, equilibrium at room temperature 15~30 minutes；Concentrated cleaning solution is diluted with distilled water 1: 20
For use.
2. taking out coating plate number, 50 μ l standard items (S are added per hole0~S5), quality controlled serum and sample.
3. being separately added into 50 μ l of enzyme conjugates per hole, notice that being loaded pipette tips keeps off the liquid being coated in wooden partition or hole.
So that it is uniformly mixed 4. micro oscillator vibrates, covered with adhesive sticker sealer, sets 37 DEG C and incubate 1 hour.
5. drying mixture in hole, each hole is filled with cleaning solution, stands 20 seconds or so, liquid in hole is dried, is repeated 5 times,
Finally patted dry on blotting paper.
6. luminous substrate liquid 2 is added per hole drips (or 100 μ l).
7. micro oscillator vibrates 30 seconds, room temperature (14~28 DEG C) is protected from light, and in 5~10 minutes, uses light-emitting appearance
Measure each hole luminous value (RLU).
8. data processing
1) online process：It is automatically processed and is obtained a result by computer.It is recommended that user uses log-log data processing modes.
2) manual plotting (deducts S with the counting of each standard point0Counting afterwards) it is ordinate, each a concentration of horizontal seat of standard point
Mark, draws standard curve in semilog or log-log paper, sample can be checked on standard curve according to the counting of sample
This concentration.
3) calculator is handled：It is counted with standard point and makees log-log linear regressions with concentration value, obtain standard curve.Use sample
Counting acquire concentration of specimens.Concrete operation method please refers to the operation instructions of the calculator.
3 result judgement of embodiment
Suggestion of the table 1 for PCT result interpretations
4 kit performance evaluation of embodiment
1. sensitivity for analysis
Make Log-Log fittings according to each calibration point luminous value and respective concentration.Calculate 10 hole, zero calibration object S0Luminous value
(RLU) average value (Mean) and standard deviation (SD) are calculated dense when luminous value (RLU) is Mean+2 × SD by fit equation
Angle value is that minimum detectability is sensitivity for analysis,
We use the kit measurement sensitivity for analysis of three batches, respectively less than 0.01ng/ml, therefore can be by this kit
Sensitivity for analysis is set to not higher than 0.05ng/ml, and the results are shown in Table 2.
The measurement result of 2 sensitivity for analysis of table
2. analysis specificity assessment
Use the kit measurement 1mg/ml calcitonins of three batches, 1mg/ml c reactive proteins (CRP), 10mg/ml people
Seralbumin (ALB), 1mg/ml hemoglobins, the results are shown in Table 3.
3 kit specific outcome of table
3. measurement range
By PCT sterlings according to mark concentration dilution at 0.05ng/ml, 0.2ng/ml, 0.5ng/ml, 5ng/ml, 50ng/
ml、100ng/ml、500ng/ml、1000ng/ml.The luminous value (RLU) of sample should be with concentration within the scope of kit measurement
In good linear relationship, dose-response curve related coefficient should reach 0.9900 or more.Related coefficient reaches as seen from Table 4
For 0.9900 or more maximum linearity range between 0.05~100ng/ml, luminous value (RLU) is shown in Table 5.Consider normal human serum
Concentration and the concentration range of related disorder patients consider in conjunction with the limitation of reagent range of linearity itself, this kit is surveyed
Determine range and is set to 0.05~50ng/ml.
4 each range section related coefficient result of table
5 three batches of kit luminous value (RLU) experimental results of table
4. measuring accuracy
The recycling sample of 5,15ng/ml is prepared according to PCT enterprises calibration object calibration value.
PCT recycles sample A1：5ng/ml
PCT recycles sample A2：15ng/ml
With normal human serum sample B according to 1: 9 dilution proportion：C1：0.5ng/ml, C2：1.5ng/ml.Toward coated antibody plate
50 μ l calibration objects of middle addition, above-mentioned PCT recycling samples (B, C1：0.5ng/ml、C2：1.5ng/ml), the combination of 50 μ l enzymes is added
Object, micro oscillator, which vibrates, makes it be uniformly mixed for 30 seconds, is covered with adhesive sticker sealer, and 37 DEG C incubate 1 hour, wash 5 times and clap
It is dry, each 1 drops of luminous substrate liquid A and luminous substrate liquid B are added per hole, micro oscillator vibrates 30 seconds, and room temperature (14~28 DEG C) is kept away
Light reaction measures each hole contrast and experiment after five minutes, with luminometer.
Make Log-Log fittings according to each calibration point luminous value and respective concentration.The concentration of each sample is calculated by fit equation,
Calculate the rate of recovery.The rate of recovery=recycling sample measured concentration/recycling sample theory concentration × 100%.Detailed data is shown in Table 6.
6 kit accuracy result table of table
Be measured with kit calibration object, be fitted with Log-Log patterns, the rate of recovery 96.02%~
Between 102.60%, average recovery rate is in 90.0~110% allowable ranges.
The log-log linear graphs of three batches of kits are the results show that the PCT kits of gained have pole by the method for the invention
High stability, good repeatability, correlation and linear.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Claims (1)
1. a kind of one-step method detects Procalcitonin（PCT）Kit, this detection kit use a step sandwich method reaction principle,
And Chemiluminescence Immunoassay is combined, the kit includes：Calibration object, enzyme conjugates, coating plate, luminous substrate liquid, concentration
Cleaning solution, wherein the enzyme conjugates is the anti-PCT monoclonal antibodies of horseradish peroxidase-labeled, and the coating plate is will
Anti- PCT monoclonal antibodies are coated on surface of solid phase carriers, which is characterized in that it is described coating plate preparation method be：Select 0.01
The phosphate that~0.05M pH are 7.0~9.6 makees coating buffer solution, by anti-PCT monoclonal antibodies by 2~10 μ g/ml dilutions, adds
Enter in microwell plate, 100 holes the μ l/ of μ l~200, gets rid of coating buffer after 2~8 DEG C of overnight coatings, 100 μ l of general BSA confining liquids are added
~200 holes μ l/, 2~8 DEG C, overnight with the blank site on closed plate hole wall, are then got rid of deblocking liquid, button is dry, room temperature dries up, institute
It is the phosphate buffer for being divided into 0.01~0.05M pH 7.0~9.6 using group to state calibration object, and volume ratio is 2~20% newborn
PCT antigen diluents to 0.2~50ng/ml are prepared by the calibration object dilution of cow's serum and 0.1%~1%proclin-300
Arrive, the enzyme conjugates is to use formula for the phosphate buffer of pH7.0~9.6 0.01~0.05M, volume ratio for 2~
The enzyme dilution of 20% newborn bovine serum, 0.1%~1%Proclin-300 and 0.1%~0.5% Food Red is by enzyme-antibody knot
It closes object to be prepared according to 1: 1000~1: 10000 volume ratio dilution, the luminous substrate liquid group is divided into 2~10mmol/L mistakes
Hydrogen oxide, 2~10mmol/L luminols, 2~10mmol/L to iodophenol, 10~100mmol/L pH's 7.0~9.6
Tris-HCL buffer solutions, the concentrated cleaning solution be include phosphate buffer that 0.01~0.05M pH are 7.0~9.6, matter
Amount is than the NaCl for 10~20% and containing the buffer solution of 0.2~2%Tween 20.
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| CN201610325807.1A CN106093416B (en) | 2016-05-18 | 2016-05-18 | A kind of kit and preparation method thereof of one-step method detection Procalcitonin |
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| WO2021123249A1 (en) * | 2019-12-20 | 2021-06-24 | Gerencia Regional de Salud de Castilla y León | In vitro method for the differential diagnosis between septic shock and non-septic shock patients |
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| CN107688096A (en) * | 2017-08-19 | 2018-02-13 | 杭州飞悦生物技术有限公司 | Detect the enzyme linked immunological kit and preparation method and detection method of Procalcitonin |
| CN108387734A (en) * | 2017-11-27 | 2018-08-10 | 南京天纵易康生物科技股份有限公司 | A kind of PCT detection kits, method of preparation and use based on bimolecular fluorescence complementary technology |
| CN108445230B (en) * | 2018-03-21 | 2020-08-07 | 北京科卫临床诊断试剂有限公司 | Procalcitonin chemiluminescence detection reagent based on nano antibody and detection method |
| CN109061163B (en) * | 2018-05-24 | 2021-01-15 | 上海良润生物医药科技有限公司 | CST1 chemiluminescence detection kit and detection method thereof |
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