CN106093416A - A kind of test kit of one-step method detection Procalcitonin. and preparation method thereof - Google Patents
A kind of test kit of one-step method detection Procalcitonin. and preparation method thereof Download PDFInfo
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- G—PHYSICS
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- 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
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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 test kit of Procalcitonin. content and preparation method thereof in a kind of one-step method detection human serum.This detection kit uses a step sandwich assay reaction principle, described test kit includes: calibration object, enzyme conjugates, be coated plate, luminous substrate liquid, concentrated cleaning solution, wherein, described enzyme conjugates is the anti-PCT monoclonal antibody of horseradish peroxidase-labeled, described in be coated plate for anti-PCT monoclonal antibody is coated on surface of solid phase carriers.The present invention provides a kind of fast and easy, highly sensitive, high specificity, true scope reproducible, quantitative wide and can obtain testing result and low cost and other advantages in 90 minutes.The PCT detection kit that the present invention provides, its instrument compatibility is strong, and testing cost is low, compensate for the demand clinically to PCT detection product.
Description
Technical field
The present invention relates to external diagnosis reagent field, in particular it relates to one chemoluminescence method detects human blood
Test kit of Procalcitonin. content and preparation method thereof in clear.
Background technology
Procalcitonin. (PCT) is made up of 116 aminoacid, and molecular weight is about 12.7KD.PCT is by neuroendocrine cell
(including the C cell of thyroid, lung and pancreatic tissue) expresses, and is decomposed into (immaturity) calcitonin, c-terminal peptides and ammonia through enzyme action
Cardinal extremity peptide.Healthy human blood contains only a small amount of PCT.After antibacterial infects, PCT can be significantly raised.
During animal model test display body generation pyemia, many tissues all can express PCT.In patients with sepsis body
PCT comprise only 114 aminoacid, lack aminoterminal Ala-Pro.
PCT level raises and sees bacillary pyemia, especially serious symptom pyemia and septic shock.PCT can make
For the prognostic indicator of pyemia, it also it is the reliability index of acute critical pancreatitis and major complications thereof.
For Community Acquired Respiratory Tract Infection and air-conditioning inductivity patients with pneumonia, PCT can select as antibiotic and
The index of Outcome measure.
The blood plasma PCT mass concentration of Healthy People is less than 0.05ng/ml.Old people, patients with chronic diseases and less than 10%
Human normal plasma PCT mass concentration higher than 0.05ng/ml, reach as high as 0.1ng/ml, but be usually no more than 0.3ng/ml.Pus
The diagnosis dividing value of toxication patient PCT is for more than 0.5ng/ml, severe sepsis and the fluctuation of patients with septic shock PCT mass concentration
Between 5～500ng/ml.Only a few severe infections patients blood plasma PCT horizontal exceeding 1000ng/ml.
Human calcitonin former (PCT) is the precursor of human calcitonin, and without hormonal activity, it is level pole in Healthy Human Serum
Low, but content raises rapidly in systemic bacterial infections patients serum, infecting latter two hour i.e. can detect that, and when continuing
Between long, its level in serum is proportionate with the order of severity of infectious disease, through effective antibiotics treat after, PCT water
Flat can decline rapidly, and virus infection, tumor, autoimmune disease, wound (many wounds or operation wound), clinical application,
Chronic inflammatory disease and local infection person, PCT level maintains in normal range or has slight rising.Therefore, PCT is antibacterial sense
The good index of the caused serious symptom systemic inflammatory reaction of dye, assists Differential Diagnosis, prognosis at systemic bacterial infections and sepsis
The aspects such as judgement, observation of curative effect have the highest clinical value.
Summary of the invention
It is an object of the invention to provide the test kit of a kind of detection Procalcitonin. (PCT).Detection examination provided by the present invention
Agent box uses a step sandwich assay reaction principle, and combines Chemiluminescence Immunoassay.Test kit include calibration object, enzyme conjugates,
It is coated plate, luminous substrate liquid, concentrated cleaning solution.
Wherein, described enzyme conjugates is the anti-PCT monoclonal antibody of horseradish peroxidase-labeled.
The described plate that is coated is for be coated on surface of solid phase carriers by anti-PCT monoclonal antibody.
The technical scheme is that employing 2 strain anti-PCT monoclonal antibody, first a strain PCT monoclonal antibody is coated
In surface of solid phase carriers, after being separately added into calibration object or blood serum sample in each hole of microwell plate, add enzyme labelling another strain PCT
Monoclonal antibody, i.e. forms solid matrix antibody-antigen-hrp-antibody complex after incubation.Fully add at the bottom of chemiluminescence after washing
Thing liquid, its luminous intensity is directly proportional to PCT concentration.During mensuration, first wash away the liquid being coated in plate, add luminous substrate liquid,
Its luminous intensity is directly proportional to PCT concentration.Measure every hole luminous value (RLU), (need with the logarithm of each calibration object luminous value (RLU)
Deduct S0Luminous value) be vertical coordinate (Y-axis), the logarithm of each calibration object concentration is abscissa (X-axis) mapping, obtains standard bent
Line.In sample, PCT concentration can be found from standard curve.
It is a further object to provide the preparation method of a kind of detection kit described above, including following step
Rapid:
1. calibration object preparation: with component be 0.01～0.05M pH be the phosphate buffer of 7.0～9.6, volume ratio is 2
～20% new-born calf serum and 0.1%～1%Proclin-300 standard dilution PCT antigen bulk is diluted to 0.2～
50ng/ml, 4 DEG C save backup, for calibration object.
2. the preparation of enzyme conjugates: HRP selects improvement sodium periodate oxidizing process with the connection of anti-PCT monoclonal antibody.This
Method is under conditions of pH4～6, directly the hydroxyl oxygen on HRP molecule is melted into aldehyde radical with sodium metaperiodate and forms hydroformylation enzyme, adds
Enter ethylene glycol to neutralize NaIO4.This aldehyde radical forms Schiff with the amino of antibody molecule, adds NaBH4Reduction, is formed steady
Fixed enzyme-antibody conjugates.Then using 0.01～0.05M pH is the phosphate buffer of 7.0～9.6, volume ratio be 2～
20% new-born calf serum, 0.1%～1%Proclin-300 and 0.1%～0.5% Food Red buffer are by enzyme-antibody conjugates
Volume ratio according to 1: 1000～1: 10000 dilutes, and 4 DEG C save backup, for enzyme conjugates.
3. it is coated the preparation of plate: the phosphate selecting 0.01～0.05M pH to be 7.0～9.6 is coated buffer, will be anti-
PCT monoclonal antibody, by 2～10 μ g/ml dilutions, adds in microwell plate, 100 μ l～200 μ l/ holes, 2～8 DEG C be overnight coated after get rid of
Remove to be coated liquid, add confining liquid 100 μ l～200 μ l/ holes, overnight close for 2～8 DEG C, to close the blank site on plate hole wall, so
After get rid of deblocking liquid, button is dry, room temperature dries up, for being coated plate.
4. the preparation of luminous substrate liquid: be 2～10mmol/L hydrogen peroxide, 2～10mmol/L Luminol, 2～
10mmol/L p-iodopHenol, the Tris-HCL buffer of 10～100mmol/L pH 7.0～9.6.
5. the preparation of concentrated cleaning solution: be 0.01～0.05M pH be the phosphate of 7.0～9.6, mass ratio be 10～
The NaCl of 20% and containing 0.2～2%Tween20 buffer.
The test kit of the present invention, has easy and simple to handle, it is only necessary to a step incubation reaction, highly sensitive, high specificity, weight
Renaturation is good, the most accurate, scope is wide, can obtain testing result and low cost and other advantages in 90 minutes.Its using value is:
(1) septicemia is done early diagnosis；(2) systematic severe bacterial infections (peritonitis or soft tissue infection etc.) does early stage examine
Disconnected；(3) septicemia and SIRS are made a differential diagnosis；(4) antibacterial infects and non-bacteria inflammation reacts (autoimmune disease
Deng) Differential Diagnosis；(5) Differential Diagnosis (meningitis etc.) that antibacterial infects and virus infects；(6) the postoperative discriminating of organ transplantation
Diagnosis (antibacterial infects, virus infects, absorb heat, rejection, fungal infection etc.)；(7) unknown cause fever (UFO) is examined
Disconnected and to special infection high-risk patient (intensive care unit, organ transplantation postoperative, immunosuppressant phase) monitoring；(8) septic shock
Differential Diagnosis with non-septic shock.
The PCT that the present invention provides a kind of fast and easy, highly sensitive, high specificity, true scope reproducible, quantitative are wide
Detection kit, this test kit instrument compatibility is strong, and testing cost is low, compensate for the demand clinically to PCT product.
Accompanying drawing explanation
Fig. 1 .A criticizes test kit log-log linear graph, through regression analysis: y=0.937x+4.572；R2=0.997.
Fig. 2 .B criticizes test kit log-log linear graph, through regression analysis: y=0.946x+4.571；R2=0.998.
Fig. 3 .C criticizes test kit log-log linear graph, through regression analysis: y=0.946x+4.571；R2=0.998.
Detailed description of the invention
Below in conjunction with the accompanying drawings, describe the enforcement operation of the present invention in detail, but the technical scheme that the present invention is claimed is not subject to
It is limited to following embodiment, in the case of not changing its main points, various change can be made and implement.
The reagent wherein used in embodiment, if not otherwise specified, in the case of meeting requirement of experiment, is purchased from
Reagent Company.
Embodiment 1 test kit preparation technology
Test kit preparation process comprises the following steps:
(1) preparation of calibration object
1. formulation components be concentration be 0.01～0.05M pH7.0～the phosphate of 9.6, volume ratio is 2～20% newborn
Ox blood serum and the calibration object diluent of 0.1%～1%Proclin-300.
2. PCT antigen bulk is diluted to each concentration point in the range of 0.2～50ng/ml, for calibration object.
3., according to 0.5ml/ bottle subpackage, 2～8 DEG C save backup.
(2) preparation of enzyme conjugates；
The labelling of 1.PCT monoclonal antibody: HRP selects improvement sodium periodate oxidation with the connection of anti-PCT monoclonal antibody
Method, forms stable enzyme-antibody conjugates.
2. by enzyme-antibody conjugates good for labelling by the volume ratio of 1: 1000～1: 10000 join formula be 0.01～
0.05M pH is the phosphate buffer of 7.0～9.6, and volume ratio is 2～20% new-born calf serum, 0.1%～1%Proclin-
300 and 0.1%～0.5% Food Red enzyme diluent in.
3., according to 6ml/ bottle subpackage, 2～8 DEG C save backup.
(3) preparation of plate it is coated
1. the PCT monoclonal antibody phosphate buffer that 0.01～0.05M pH is 7.0～9.6 is diluted to 2～10 μ
G/ml, is added in microwell plate by 100 μ l～200 μ l/ holes, and 2～8 DEG C are overnight coated (16～24 hours).
2. getting rid of and be coated liquid, in absorbent paper, button is dry, and every hole adds general BSA confining liquid 100 μ l～200 μ l/ holes, 2～8
DEG C overnight (16～24 hours) are closed.
3. getting rid of deblocking liquid, in absorbent paper, button is dry, and room temperature (15～28 DEG C) dries up, and dry pre-coated plate is loaded aluminum
Encapsulating in paper tinsel bag, 2～8 DEG C save backup.
(4) preparation of luminous substrate liquid
1. formulation components is 2～10mmol/L hydrogen peroxide, 2～10mmol/L Luminol, 2～10mmol/L p-
IodopHenol, the luminous substrate liquid of the Tris-HCL of 10～100mmol/L pH7.0～9.6.
2., according to 7ml/ bottle subpackage, 2～8 DEG C save backup.
(5) preparation of concentrated cleaning solution
1. formulation components be 0.01～0.05M pH be 7.0～9.6 phosphate buffer, mass ratio be 10～20%
NaCl and containing 0.2～2%Tween20 concentrated cleaning solution.
2., according to 20ml/ bottle subpackage, 2～8 DEG C save backup.
The detection operating process of embodiment 2 test kit
1. take out test kit and blood serum sample, equilibrium at room temperature 15～30 minutes；Concentrated cleaning solution distilled water 1: 20 dilutes
Stand-by.
2. taking out and be coated plate numbering, every hole adds 50 μ l standard substance (S0～S5), quality controlled serum and sample.
3. every hole is separately added into enzyme conjugates 50 μ l, notices that sample loading gun head does not encounter the liquid being coated in wooden partition or hole.
4. micro oscillator vibration makes its mix homogeneously in 30 seconds, covers by adhesive sticker sealer, puts 37 DEG C of incubations 1 hour.
5. dry mixture in hole, fill each hole with cleaning mixture, stand about 20 seconds, dry liquid in hole, be repeated 5 times,
Finally pat dry in absorbent paper.
6. every hole adds luminous substrate liquid 2 (or 100 μ l).
7. micro oscillator vibrates 30 seconds, and room temperature (14～28 DEG C) lucifuge is reacted, and in 5～10 minutes, uses light-emitting appearance
Measure each hole luminous value (RLU).
8. data process
1) on-line processing: automatically processed by computer and obtain a result.Suggestion user uses log-log data processing mode.
2) manual plotting is with counting (the deduction S of each standard point0After counting) be vertical coordinate, each standard point concentration is horizontal seat
Mark, draws standard curve in semilog or log-log paper, can check in sample according to the counting of sample on standard curve
This concentration.
3) computer processes: makees log-log rectilinear regression with standard point counting with concentration value, obtains standard curve.Use sample
Counting try to achieve concentration of specimens.Concrete operation method refer to the operation instructions of this computer.
Embodiment 3 result judges
Table 1 is for the suggestion of PCT result interpretation
Embodiment 4 test kit performance evaluation
1. sensitivity for analysis
Log-Log matching is made according to each calibration point luminous value and respective concentration.Calculate 10 hole zero calibration object S0Luminous value
(RLU) meansigma methods (Mean) and standard deviation (SD), by fit equation calculate when luminous value (RLU) is Mean+2 × SD dense
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 test kit
Sensitivity for analysis is set to not higher than 0.05ng/ml, and result is as shown in table 2.
The measurement result of table 2 sensitivity for analysis
2. analyze specificity assessment
Use the kit measurement 1mg/ml calcitonin of three batches, 1mg/ml c reactive protein (CRP), 10mg/ml people
Serum albumin (ALB), 1mg/ml hemoglobin, result is as shown in table 3.
Table 3 test kit specific outcome
3. measurement range
PCT sterling is become 0.05ng/ml, 0.2ng/ml, 0.5ng/ml, 5ng/ml, 50ng/ according to indicating concentration dilution
ml、100ng/ml、500ng/ml、1000ng/ml.Within the scope of kit measurement, the luminous value (RLU) of sample should be with concentration
In good linear relationship, dose-response curve correlation coefficient should reach more than 0.9900.Correlation coefficient reaches as seen from Table 4
More than 0.9900 maximum linearity range is between 0.05～100ng/ml, and luminous value (RLU) is shown in Table 5.Consider normal human serum
Concentration and the concentration range of related disorder patients, in conjunction with the restriction of the range of linearity of reagent own, consider, and this test kit is surveyed
Determine scope and be set to 0.05～50ng/ml.
Table 4 each range section correlation coefficient result
5 three batches of test kit luminous value (RLU) experimental results of table
4. measure accuracy
According to PCT enterprise calibration object calibration value preparation 5, the recovery sample of 15ng/ml.
PCT reclaims sample A1:5ng/ml
PCT reclaims sample A2:15ng/ml
By normal human serum sample B according to 1: 9 dilution proportion: C1:0.5ng/ml, C2:1.5ng/ml.Toward coated antibody plate
Middle addition 50 μ l calibration object, above-mentioned PCT reclaim sample (B, C1:0.5ng/ml, C2:1.5ng/ml), add 50 μ l enzymes and combine
Thing, micro oscillator vibration makes its mix homogeneously in 30 seconds, covers by adhesive sticker sealer, and 37 DEG C of incubations 1 hour wash 5 times and clap
Dry, every hole adds each 1 of luminous substrate liquid A and luminous substrate liquid B, and micro oscillator vibrates 30 seconds, and room temperature (14～28 DEG C) is kept away
After photoreaction 5 minutes, measure each hole contrast and experiment with luminometer.
Log-Log matching is made according to each calibration point luminous value and respective concentration.The concentration of each sample is calculated by fit equation,
Calculate the response rate.The response rate=recovery sample measured concentration/recovery sample theory concentration × 100%.Detailed data is shown in Table 6.
Table 6 test kit accuracy result table
Be measured with test kit calibration object, be fitted with Log-Log pattern, the response rate 96.02%～
Between 102.60%, average recovery rate is 90.0～110% in allowed band.
The log-log linear graph result of three batches of test kits shows have pole by the PCT test kit of the inventive method gained
High stability, good repeatability, dependency and linear.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvements and modifications, these improvements and modifications
Also should be regarded as protection scope of the present invention.
Claims (7)
1. the test kit of one-step method detection Procalcitonin. (PCT), it is characterised in that this detection kit uses a step sandwich
Method reaction principle, and combine Chemiluminescence Immunoassay, described test kit includes: calibration object, enzyme conjugates, be coated plate, luminescence
Substrate solution, concentrated cleaning solution, wherein, described enzyme conjugates is the anti-PCT monoclonal antibody of horseradish peroxidase-labeled, described
It is coated plate for anti-PCT monoclonal antibody is coated on surface of solid phase carriers.
Test kit the most according to claim 1, it is characterised in that described calibration object is that employing 0.01～0.05M pH is
The phosphate buffer of 7.0～9.6, volume ratio is 2～20% new-born calf serum and the standard of 0.1%～1%proclin-300
PCT antigen diluent to 0.2～50ng/ml are prepared by diluent.
Test kit the most according to claim 1, it is characterised in that described enzyme conjugates employing 0.01～0.05M pH is
The phosphate buffer of 7.0～9.6, volume ratio is 2～20% new-born calf serum, 0.1%～1%Proclin-300 and 0.1%
～0.5% Food Red buffer by enzyme-antibody conjugates according to 1: 1000～1: 10000 volume ratio dilute prepare.
Test kit the most according to claim 1, it is characterised in that described in be coated the preparation method of plate and be: select 0.01～
0.05M pH be 7.0～9.6 phosphate be coated buffer, by anti-PCT monoclonal antibody by 2～10 μ g/ml dilution, add
In microwell plate, 100 μ l～200 μ l/ holes, 2～8 DEG C be overnight coated after get rid of and be coated liquid, add confining liquid 100 μ l～200 μ l/
Hole, gets rid of deblocking liquid after 2～8 DEG C of overnight closings, to close the blank site on plate hole wall, then gets rid of deblocking liquid, and button is dry, room
Temperature dries up.
Test kit the most according to claim 1, it is characterised in that described luminescent solution is 2～10mmol/L hydrogen peroxide, 2
～the Tris-of 10mmol/L Luminol, 2～10mmol/L p-iodopHenol, 10～100mmol/L pH 7.0～9.6
HCL buffer.
Test kit the most according to claim 1, it is characterised in that described concentrated cleaning solution is for including 0.01～0.05M pH
Be the phosphate buffer of 7.0～9.6, mass ratio be 10～the NaCl of 20% and containing 0.2～2%Tween 20 buffer.
Test kit the most according to claim 1, it is characterised in that wherein involved solid phase carrier is selected from microwell plate, plastics
Pearl, plastic tube or magnetic-particle.
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| Publication number | Priority date | Publication date | Assignee | Title |
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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 |
| CN108445230A (en) * | 2018-03-21 | 2018-08-24 | 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 |
| CN109061163A (en) * | 2018-05-24 | 2018-12-21 | 上海良润生物医药科技有限公司 | A kind of CST1 chemiluminescence detection kit and its detection method |
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| CN109061163A (en) * | 2018-05-24 | 2018-12-21 | 上海良润生物医药科技有限公司 | A kind of CST1 chemiluminescence detection kit and its detection method |
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