CN110058022A - Carcinoembryonic Antigen CEA immue quantitative detection reagent box and its application - Google Patents
Carcinoembryonic Antigen CEA immue quantitative detection reagent box and its application Download PDFInfo
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- CN110058022A CN110058022A CN201910455098.2A CN201910455098A CN110058022A CN 110058022 A CN110058022 A CN 110058022A CN 201910455098 A CN201910455098 A CN 201910455098A CN 110058022 A CN110058022 A CN 110058022A
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- cea
- carcinoembryonic antigen
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57473—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving carcinoembryonic antigen, i.e. CEA
Abstract
The invention discloses a kind of Carcinoembryonic Antigen CEA immue quantitative detection reagent box and its applications, are related to field of biotechnology.The Carcinoembryonic Antigen CEA immue quantitative detection reagent box includes calibration object, quality-control product, anti-reagent, magnetic particle reagent and luminous substrate, wherein, the preparation step of the anti-reagent are as follows: be coupled Carcinoembryonic Antigen CEA antibody with fluorescein isothiocynate and alkaline phosphatase respectively, obtain the CEA coated antibody of marked by fluorescein isothiocyanate and the CEA coated antibody of alkali phosphatase enzyme mark, the CEA labelled antibody of the CEA coated antibody of marked by fluorescein isothiocyanate and alkali phosphatase enzyme mark is added in anti-reagent buffer, is sufficiently stirred；The preparation step of the magnetic particle reagent are as follows: fluorescein isothiocynate monoclonal antibody and carboxyl magnetic bead are coupled, clean and save by cleaning carboxyl magnetic bead.The present invention can improve detection sensitivity and reliability and reduce cost.
Description
Technical field
The present invention relates to field of biotechnology, and in particular to a kind of Carcinoembryonic Antigen CEA immue quantitative detection reagent box and its application.
Background technique
Carcinomebryonic antigen (carcinoembryonic antigen, CEA) is a kind of with the decision of human embryos antigen-specific
The carcinomebryonic antigen of cluster is important tumor associated antigen, is derived in early stage fetus by entoderm, by Gastrointestinal Tract of Fetus epithelium
Synthesized by tissue, pancreas and liver cell.Nineteen sixty-five, Gold and Freedman have found from fetus and colon cancer tissue first, therefore incite somebody to action
It is known as carcinomebryonic antigen.The encoding gene of CEA is located at No. 19 chromosomes, is that a kind of polysaccharide protein that molecular weight is 22KD is compound
Object, 45% is protein.
CEA belongs to non-organ specificity tumor associated antigen, is primarily present in Rectum and colon cancer tissue and fetus intestinal mucosa
Interior, the tumour for secreting CEA is located at hollow organ, such as gastrointestinal tract, respiratory tract, the urinary tract mostly.Under normal circumstances, CEA is through stomach
Gut metabolism, and entered in local humor and blood and Lymphatic Circulation by the CEA that tumor cell secretion generates, therefore in above-mentioned cancer
The serum and chest of disease, ascites may occur in which that CEA increases extremely in digestive juice.
CEA is a kind of broad-spectrum tumor marker, is often detected in conjunction with other tumor markers.Clinically, when CEA is greater than
When 60 μ g/L, it is seen that in colon and rectum carcinoma, gastric cancer and lung cancer.Primary colorectal carcinoma when not shifting in early days, CEA sun
Property rate be 45%~80% or so, the concentration and positive rate of patient its CEA shifted increases.The measurement of CEA can be made
For one of the foundation of observation curative effect.CEA value increases, and shows there is lesion remaining or progress.Such as lung cancer, breast cancer, bladder cancer and ovum
Nest cancer patient, change of serum C EA amount can be significantly raised, are shown as tumor-infiltrated mostly, wherein about 70% is metastatic carcinoma.
Currently, domestic Carcinoembryonic Antigen CEA clinical detection is mainly based on external import reagent and immunohistochemistry reagent, it is external
Import reagent price is very expensive, and very big financial burden is brought to patient, is unfavorable for universal in base.It is produced with independent intellectual
The domestic Carcinoembryonic Antigen CEA immunochemiluminescence detection kit of power is also less at present, and detection sensitivity is lower, linear narrow, special
It is anisotropic poor, also it is unfavorable for high-throughput full-automatic detection.
Summary of the invention
The present invention provide one kind can improve detection sensitivity and reliability and reduce cost Carcinoembryonic Antigen CEA it is fixed
Measure detection kit and application.
In order to solve the above technical problems, present invention offer technical solution is as follows:
A kind of Carcinoembryonic Antigen CEA immue quantitative detection reagent box, including calibration object, quality-control product, anti-reagent, magnetic particle reagent and hair
Light substrate, wherein
The preparation step of the anti-reagent are as follows: by Carcinoembryonic Antigen CEA antibody respectively with fluorescein isothiocynate and alkaline phosphorus
The coupling of acid esters enzyme, obtains the CEA coated antibody of marked by fluorescein isothiocyanate and the CEA labelled antibody of alkali phosphatase enzyme mark,
Anti- reagent buffering is added in the CEA labelled antibody of the CEA coated antibody of marked by fluorescein isothiocyanate and alkali phosphatase enzyme mark
In liquid, it is sufficiently stirred；
The preparation step of the magnetic particle reagent are as follows: cleaning carboxyl magnetic bead, by fluorescein isothiocynate monoclonal antibody with
The coupling of carboxyl magnetic bead, cleans and saves.
Further, the preparation step of the CEA coated antibody of the marked by fluorescein isothiocyanate is further are as follows:
Fluorescein isothiocynate is added in anti-reagent buffer, fluorescein isothiocynate solution is configured, makes isothiocyanic acid
The ultimate density of luciferin solution is 1.0~5.0mg/mL；
It is being mixed well 10~20 hours to fluorescein isothiocynate solution and Carcinoembryonic Antigen CEA for 1.1:1 by mass ratio,
Bicarbonate buffer is sufficiently added after reaction to be balanced, then gel chromatography isolates and purifies.
Further, the preparation step of the CEA labelled antibody of the alkali phosphatase enzyme mark is further are as follows:
Alkaline phosphatase is added in anti-reagent buffer, alkaline phosphatase enzyme solutions is configured, makes alkaline phosphatase enzyme solutions
Ultimate density is 1.0~3.0mg/mL；
Alkaline phosphatase enzyme solutions and Carcinoembryonic Antigen CEA that molar ratio is 1.5:1 are mixed well 14~24 hours, it is sufficiently anti-
The Tris salt buffer containing surfactant should be added afterwards to be balanced, then gel chromatography isolates and purifies；
The component and content of the Tris salt buffer are as follows: Tris buffer 12.12mg, sodium chloride 5.82mg.
Further, the preparation step of the magnetic particle reagent is further are as follows:
Step 1: magnetic field separation being carried out to carboxyl magnetic bead cleaning concentrate, abandons supernatant after the sedimentation of carboxyl magnetic bead；
Step 2: the magnetic particle buffer of 2~5 times of carboxyl magnetic bead volumes is added in the solution that Xiang Suoshu step 1 obtains,
Mix 20~30min；
Step 3: magnetic field separation is carried out to the solution that the step 2 obtains, abandons supernatant after the sedimentation of carboxyl magnetic bead, finally
To the carboxyl magnetic bead solution of 10~50mg/mL；
Step 4: carboxyl magnetic bead solution and fluorescein isothiocynate monoclonal antibody that mass ratio is 100:1 are mixed well
React 18h；
Step 5: magnetic field separation is carried out to the solution that the step 4 obtains, it is slow using magnetic particle after the sedimentation of carboxyl magnetic bead
Fliud flushing cleaning, finally obtains the magnetic particle reagent of 10mg/mL.
Further, the group of the luminous substrate is divided into APS-5 and luminous substrate buffer, and volume ratio is 1:4~10,
In,
The component and content of the luminous substrate buffer are as follows: Tris buffer 12.12g/L, sodium chloride 5.82g/L, light
Damp essence 0.03g/L.
Further, the component and content of the anti-reagent buffer are as follows:
Further, the preparation step of the calibration object and quality-control product are as follows: use calibration object buffer solution carcinomebryonic antigen
CEA, obtain 0ng/mL, 5ng/mL, 10ng/mL, 25ng/mL, 50ng/mL and 150ng/mL calibration object and 10ng/mL,
The quality-control product of 50ng/mL, wherein
The component and content of the calibration object buffer are as follows: serum 500g/L, 0.01~0.05g/L of tetracycline, sulfuric acid are new
0.1~0.5g/L of mycin.
Further, the detection kit further includes cleaning solution, the component and content of the cleaning solution are as follows:
A kind of application of any of the above-described kit as external diagnosis reagent.
Compared with prior art, the invention has the following advantages:
1, detection kit of the invention passes through chemical reaction using fluorescein isothiocynate and alkaline phosphatase as marker enzyme
Labelled antibody improves the sensitivity of reaction；
2, detection kit of the invention is micro- with fluorescein isothiocynate monoclonal antibody and the obtained magnetic of carboxyl magnetic bead coupling
Grain reagent makes immune response be easier to mix and separate, and substantially increases reaction speed；
3, detection kit of the invention has good stability, and validity period can be to 1 year or more；
4, detection kit of the invention in clinical studies with external import reagent meet correlation be up to 99% with
On, and expense be only its 1/5, greatly reduce cost.
Detailed description of the invention
Fig. 1 is the standard curve that Carcinoembryonic Antigen CEA is detected using the carcinomebryonic antigen detection kit of the embodiment of the present invention 1；
Fig. 2 is the standard curve that Carcinoembryonic Antigen CEA is detected using the carcinomebryonic antigen detection kit of the embodiment of the present invention 2；
Fig. 3 is the standard curve that Carcinoembryonic Antigen CEA is detected using the carcinomebryonic antigen detection kit of the embodiment of the present invention 3.
Specific embodiment
To keep the technical problem to be solved in the present invention, technical solution and advantage clearer, below in conjunction with specific implementation
Example and attached drawing are described in detail.
It should be noted that each reagent material used is commercially available in embodiment, each instrument and equipment used is also institute
The existing instrument and equipment in category field, test method without specific conditions are conventional method and conventional strip known to fields
Part, or according to condition proposed by manufacturer.
Embodiment 1
The preparation of the Carcinoembryonic Antigen CEA immue quantitative detection reagent box of the present embodiment:
1, the ingredient and content of each buffer
1) ingredient and content of calibration object buffer
Horse serum 500g/L
Tetracycline 0.01g/L
Neomycinsulphate 0.1g/L.
2) ingredient and content of anti-reagent buffer
3) ingredient and content of Tris salt buffer
Tris buffer 12.12mg/L
Sodium chloride 5.82mg/L
Final pH value is 8.0.
4) ingredient and content of magnetic particle buffer
Tris buffer 14.21mg/L
Sodium chloride 6.21mg/L
Methyl cellulose ether 60g/L.
5) ingredient and content of luminous substrate buffer
Tris buffer 12.12g/L
Sodium chloride 5.82g/L
Lucigenin 0.03g/L
Final ph is 9.5.
6) ingredient and content of cleaning solution
2, the preparation of calibration object and quality-control product
Using calibration object buffer solution Carcinoembryonic Antigen CEA, it is configured to the calibration object and matter of aimed concn as shown in Table 1
Control product, the buying of Carcinoembryonic Antigen CEA used in the present embodiment is from fitzgerald company, producer.
Table 1: the concentration of calibration object and quality-control product
3, the preparation of anti-reagent
1) preparation of the CEA coated antibody of marked by fluorescein isothiocyanate
It is 2.5mg/mL fluorescein isothiocynate solution that fluorescein isothiocynate, which is configured to concentration, with anti-reagent buffer,
It is 1:1.1 according to Carcinoembryonic Antigen CEA and the mass ratio of fluorescein isothiocynate solution, the two is transferred to brown glass simultaneously
It in bottle, stirs 10 hours at room temperature, is sufficiently balanced after reaction using the bicarbonate buffer of pH=8, is coagulated later
Glue-line analysis isolates and purifies, and finally obtains the CEA coated antibody of marked by fluorescein isothiocyanate；
2) preparation of the CEA labelled antibody of alkali phosphatase enzyme mark
Alkaline phosphatase is configured to the alkaline phosphatase enzyme solutions that concentration is 2.5mg/mL with anti-reagent buffer first, is pressed
The two is transferred in brown bottle according to the amount that the molar ratio of alkaline phosphatase and Carcinoembryonic Antigen CEA is 1:1.5, stirs 14 at room temperature
Hour, it is sufficiently balanced after reaction using the buffer of pH=7, carries out gel chromatography later and isolate and purify, obtain alkaline phosphatase
The CEA labelled antibody of label；
3) CEA of the CEA coated antibody of the marked by fluorescein isothiocyanate of acquisition and alkali phosphatase enzyme mark is marked anti-
Body is separately added into the Tris salt buffer containing 0.1%Tween20, and the anti-reagent is obtained after being sufficiently stirred.
4, the preparation of magnetic particle reagent
1) preparation of carboxyl magnetic bead solution: the carboxyl magnetic bead cleaning concentrate after taking 10mL to mix well is placed in magnetic field
20min sucks supernatant after the sedimentation of carboxyl magnetic bead；The magnetic particle buffer of 5 times of carboxyl magnetic bead volumes is added later, concussion is clear
Wash 20min；Solution after cleaning is placed in magnetic field 20min, sucks supernatant after the sedimentation of carboxyl magnetic bead；Repeated washing carboxyl magnetic bead
3 times, finally by carboxyl magnetic bead solution constant volume to 25mg/mL；
2) connection reaction: being 100:1 according to carboxyl magnetic bead solution and the mass ratio of fluorescein isothiocynate monoclonal antibody
Ratio, in carboxyl magnetic bead solution be added fluorescein isothiocynate monoclonal antibody, in 2~8 DEG C keep mixing state it is anti-
It answers 18 hours；
3) solution that step 2) obtains is placed in magnetic field 20min, uses magnetic particle buffer solution for cleaning after the sedimentation of carboxyl magnetic bead
Carboxyl magnetic bead 3 times, carboxyl magnetic bead is finally settled to 10mg/mL, stand-by magnetic particle reagent needed for being made.
5, the preparation of luminous substrate
APS-5 is sufficiently dissolved using the luminous substrate buffer of 10 times of APS-5 volumes, obtains luminous substrate.
6, above-mentioned calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate and cleaning solution are dispensed and seals guarantor
It deposits to get Carcinoembryonic Antigen CEA immue quantitative detection reagent box is arrived.
Embodiment 2:
1, the ingredient and content of each buffer
1) ingredient and content of calibration object buffer
Horse serum 500g/L
Tetracycline 0.01g/L
Neomycinsulphate 0.1g/L.
2) ingredient and content of anti-reagent buffer
The ingredient and content of Tris salt buffer, magnetic particle buffer, the ingredient of luminous substrate buffer and content and
The ingredient and content of cleaning solution are the same as embodiment 1.
2, the preparation of calibration object and quality-control product
With embodiment 1.
3, the preparation of anti-reagent
1) preparation of the CEA coated antibody of marked by fluorescein isothiocyanate
It is 1mg/mL fluorescein isothiocynate solution that fluorescein isothiocynate, which is configured to concentration, with anti-reagent buffer, is pressed
It is 1:1.1 according to Carcinoembryonic Antigen CEA and the mass ratio of fluorescein isothiocynate solution, the two is transferred to Brown Glass Brown glass bottles and jars only simultaneously
In, it stirs 10 hours at room temperature, is sufficiently balanced after reaction using the bicarbonate buffer of pH=9, carries out gel later
Chromatography purifying, finally obtains the CEA coated antibody of marked by fluorescein isothiocyanate；
2) preparation of the CEA labelled antibody of alkali phosphatase enzyme mark
Alkaline phosphatase is configured to the alkaline phosphatase enzyme solutions that concentration is 1mg/mL with anti-reagent buffer first, according to
The molar ratio of alkaline phosphatase and Carcinoembryonic Antigen CEA is that the two is transferred in brown bottle by the amount of 1:1.5, and it is small to stir 14 at room temperature
When, it is sufficiently balanced after reaction using the buffer of pH=8, carries out gel chromatography later and isolate and purify, obtain alkaline phosphatase mark
The CEA labelled antibody of note；
3) CEA of the CEA coated antibody of the marked by fluorescein isothiocyanate of acquisition and alkali phosphatase enzyme mark is marked anti-
Body is separately added into the Tris salt buffer containing 0.05%Tween20, and the anti-reagent is obtained after being sufficiently stirred.
4, the preparation of magnetic particle reagent
1) preparation of carboxyl magnetic bead solution: the carboxyl magnetic bead cleaning concentrate after taking 10mL to mix well is placed in magnetic field
20min sucks supernatant after the sedimentation of carboxyl magnetic bead；The magnetic particle buffer of 2 times of carboxyl magnetic bead volumes is added later, concussion is clear
Wash 20min；Solution after cleaning is placed in magnetic field 20min, sucks supernatant after the sedimentation of carboxyl magnetic bead；Repeated washing carboxyl magnetic bead
3 times, finally by carboxyl magnetic bead solution constant volume to 10mg/mL；
2) connection reaction: being 100:1 according to carboxyl magnetic bead solution and the mass ratio of fluorescein isothiocynate monoclonal antibody
Ratio, in carboxyl magnetic bead solution be added fluorescein isothiocynate monoclonal antibody, in 2~8 DEG C keep mixing state it is anti-
It answers 18 hours；
3) solution that step 2) obtains is placed on magnetic field 20min, it is clear with magnetic particle buffer after the sedimentation of carboxyl magnetic bead
It washes carboxyl magnetic bead 3 times, carboxyl magnetic bead is finally settled to 10mg/mL, stand-by magnetic particle reagent needed for being made.
5, the preparation of luminous substrate
APS-5 is sufficiently dissolved using the luminous substrate buffer of 4 times of APS-5 volumes, obtains luminous substrate.
6, above-mentioned calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate and cleaning solution are dispensed and seals guarantor
It deposits to get Carcinoembryonic Antigen CEA immue quantitative detection reagent box is arrived.
Embodiment 3
1, the ingredient of each buffer and content are the same as embodiment 1
2, the preparation of calibration object and quality-control product
With embodiment 1.
3, the preparation of anti-reagent
1) preparation of the CEA coated antibody of marked by fluorescein isothiocyanate
It is 5.0mg/mL fluorescein isothiocynate solution that fluorescein isothiocynate, which is configured to concentration, with anti-reagent buffer,
It is 1:1.1 according to Carcinoembryonic Antigen CEA and the mass ratio of fluorescein isothiocynate solution, the two is transferred to brown glass simultaneously
It in bottle, stirs 20 hours at room temperature, is sufficiently balanced after reaction using the bicarbonate buffer of pH=8, is coagulated later
Glue-line analysis isolates and purifies, and finally obtains the CEA coated antibody of marked by fluorescein isothiocyanate；
2) preparation of the CEA labelled antibody of alkali phosphatase enzyme mark
Alkaline phosphatase is configured to the alkaline phosphatase enzyme solutions that concentration is 5.0mg/mL with anti-reagent buffer, according to alkali
The molar ratio of acid phosphatase and Carcinoembryonic Antigen CEA is that the two is transferred in brown bottle by the amount of 1:1.5, and it is small to stir 24 at room temperature
When, it is sufficiently balanced after reaction using the buffer of pH=7, carries out gel chromatography later and isolate and purify, obtain alkaline phosphatase mark
The CEA labelled antibody of note；
3) CEA of the CEA coated antibody of the marked by fluorescein isothiocyanate of acquisition and alkali phosphatase enzyme mark is marked anti-
Body is separately added into the Tris salt buffer containing 0.3%Tween20, and the anti-reagent is obtained after being sufficiently stirred.
4, the preparation of magnetic particle reagent
1) preparation of carboxyl magnetic bead solution: the carboxyl magnetic bead cleaning concentrate after taking 10mL to mix well is placed in magnetic field
20min sucks supernatant after the sedimentation of carboxyl magnetic bead；The magnetic particle buffer of 5 times of carboxyl magnetic bead volumes is added later, concussion is clear
Wash 20min；Solution after cleaning is placed in magnetic field 20min, sucks supernatant after the sedimentation of carboxyl magnetic bead；Repeated washing carboxyl magnetic bead
3 times, finally by carboxyl magnetic bead solution constant volume to 50mg/mL；
2) connection reaction: being 100:1 according to carboxyl magnetic bead solution and the mass ratio of fluorescein isothiocynate monoclonal antibody
Ratio, in carboxyl magnetic bead solution be added fluorescein isothiocynate monoclonal antibody, in 2~8 DEG C keep mixing state it is anti-
It answers 18 hours；
3) solution that step 2) obtains is placed in magnetic field and is centrifuged 20min, it is slow with magnetic particle after the sedimentation of carboxyl magnetic bead
Fliud flushing is cleaned carboxyl magnetic bead 3 times, and carboxyl magnetic bead is finally settled to 10mg/mL, stand-by magnetic particle reagent needed for being made.
5, the preparation of luminous substrate
APS-5 is sufficiently dissolved using the luminous substrate buffer of 10 times of APS-5 volumes, obtains luminous substrate.
6, above-mentioned calibration object, quality-control product, anti-reagent, magnetic particle reagent, luminous substrate and cleaning solution are dispensed and seals guarantor
It deposits to get Carcinoembryonic Antigen CEA immue quantitative detection reagent box is arrived.
Clinical data:
1, the Data Detections such as Carcinoembryonic Antigen CEA immue quantitative detection reagent box stability of the invention
In order to evaluate embodiment 1-3 Carcinoembryonic Antigen CEA immue quantitative detection reagent box stability, accuracy and variability,
The kit of embodiment 1-3 is tested for the property at regular intervals, test result is as shown in table 1-3.
The Carcinoembryonic Antigen CEA immue quantitative detection reagent box test result of 1 embodiment 1 of table
The Carcinoembryonic Antigen CEA immue quantitative detection reagent box test result of 2 embodiment 2 of table
The Carcinoembryonic Antigen CEA immue quantitative detection reagent box test result of 3 embodiment 3 of table
By table 1-3 it can be seen that the Carcinoembryonic Antigen CEA detection kit of embodiment 1-3 and the linear dependence of luminous value
It is held in 0.99 or more within continuous 15 months, minimum detectability is respectively less than 0.15, and the coefficient of variation is respectively less than 8%, meets national mark
It is quasi-.
2, the Carcinoembryonic Antigen CEA immue quantitative detection reagent box of embodiment 1 and import Carcinoembryonic Antigen CEA detection kit (Roche
Company buys) detection effect comparison
300 parts of serum samples are chosen, after the basic, normal, high value separation in serum sample, basic, normal, high value sample is carried out
Individual linear regression analysis has obtained equation of linear regression y=0.9924X+0.3786, R2=0.9855.The regression curve
Equation is as shown in Figure 1, wherein horizontal axis represents the testing result of import Carcinoembryonic Antigen CEA detection kit, and the longitudinal axis represents the present invention
The testing result of Carcinoembryonic Antigen CEA immue quantitative detection reagent box.
3, the Carcinoembryonic Antigen CEA immue quantitative detection reagent box of embodiment 2 and import Carcinoembryonic Antigen CEA detection kit (Roche
Company buys) detection effect comparison
300 parts of serum samples are chosen, after the basic, normal, high value separation in serum sample, basic, normal, high value sample is carried out
Individual linear regression analysis has obtained equation of linear regression y=0.9937X+0.4744, R2=0.9858.The regression curve
Equation is as shown in Figure 2, wherein horizontal axis represents the testing result of import Carcinoembryonic Antigen CEA detection kit, and the longitudinal axis represents the present invention
The testing result of Carcinoembryonic Antigen CEA immue quantitative detection reagent box.
4, the Carcinoembryonic Antigen CEA immue quantitative detection reagent box of embodiment 3 and import Carcinoembryonic Antigen CEA detection kit (Roche
Company buys) detection effect comparison
300 parts of serum samples are chosen, after the basic, normal, high value separation in serum sample, basic, normal, high value sample is carried out
Individual linear regression analysis has obtained equation of linear regression y=0.9868X+0.9753, R2=0.9860.The regression curve
Equation is as shown in Figure 3, wherein horizontal axis represents the testing result of import Carcinoembryonic Antigen CEA detection kit, and the longitudinal axis represents the present invention
The testing result of Carcinoembryonic Antigen CEA immue quantitative detection reagent box.
Carcinoembryonic Antigen CEA immue quantitative detection reagent box of the invention has preferable stability and standard it can be seen from Fig. 1-3
True property.
5, the reference interval of Carcinoembryonic Antigen CEA detection kit of the invention is measured with ROC curve method, carcinomebryonic antigen
CEA detection kit concentration measurement is analyzed, and all possible point of contact is carried out susceptibility as threshold value and specificity is counted
It calculates, using susceptibility as ordinate, using 1- specificity as abscissa, makes ROC curve, the rigid beginning of curve rises comparatively fast, says
Bright kit resolution ratio is higher, and area under the curve is that 0.999 > 0.9 indicates that curve accuracy is higher, and there are statistical significances.
According to the susceptibility and specificity at possibility each in statistical result point of contact, Youden index is calculated, finds out susceptibility and specificity
Making point with maximum point is critical point.
Using the Carcinoembryonic Antigen CEA detection kit of embodiment 1-3 respectively to carcinomebryonic antigen in 549 parts of proper manners this serum
CEA tests, to testing result be modified reference interval be 0-11.
The Carcinoembryonic Antigen CEA immue quantitative detection reagent box ROC curve testing result table of the invention of table 4
| P value (consistency) | |
| Embodiment 1 | 10.671 |
| Embodiment 2 | 11.0019 |
| Embodiment 3 | 10.8076 |
6, the range of normal value of Carcinoembryonic Antigen CEA detection kit of the invention
It is anti-to cancer embryo in 550 parts of normal person's sample serum respectively using the Carcinoembryonic Antigen CEA detection kit of embodiment 1-3
Former CEA tests, wherein the content peak of CEA is 9.994ng/mL, as shown in table 5 in 95% sample serum.Cause
This, when being detected using Carcinoembryonic Antigen CEA detection kit of the invention, it is proposed that the normal reference value of change of serum C EA is set to 0~
10ng/mL。
5 normal human serum CEA testing result statistical analysis of table
In conclusion Carcinoembryonic Antigen CEA immue quantitative detection reagent box reliable performance of the invention, high sensitivity, the range of linearity
Width can cooperate full-automatic instrument to use.
The above is a preferred embodiment of the present invention, it is noted that for those skilled in the art
For, without departing from the principles of the present invention, it can also make several improvements and retouch, these improvements and modifications
It should be regarded as protection scope of the present invention.
Claims (9)
1. a kind of Carcinoembryonic Antigen CEA immue quantitative detection reagent box, which is characterized in that micro- including calibration object, quality-control product, anti-reagent, magnetic
Grain reagent and luminous substrate, wherein
The preparation step of the anti-reagent are as follows: by Carcinoembryonic Antigen CEA antibody respectively with fluorescein isothiocynate and alkaline phosphate ester
Enzyme coupling, obtains the CEA coated antibody of marked by fluorescein isothiocyanate and the CEA coated antibody of alkali phosphatase enzyme mark, will be different
The CEA labelled antibody of the fluorescein-labeled CEA coated antibody of thiocyanic acid and alkali phosphatase enzyme mark is added in anti-reagent buffer,
It is sufficiently stirred；
The preparation step of the magnetic particle reagent are as follows: cleaning carboxyl magnetic bead, by fluorescein isothiocynate monoclonal antibody and carboxyl
Magnetic bead coupling, cleans and saves.
2. Carcinoembryonic Antigen CEA immue quantitative detection reagent box according to claim 1, which is characterized in that the isothiocyanic acid is glimmering
The preparation step of the CEA coated antibody of light element label is further are as follows:
Fluorescein isothiocynate is added in anti-reagent buffer, fluorescein isothiocynate solution is configured, makes isosulfocyanic acid fluorescence
The ultimate density of plain solution is 1.0~5.0mg/mL；
It is that 1.1:1 to fluorescein isothiocynate solution and Carcinoembryonic Antigen CEA mixes well 10~20 hours by mass ratio, sufficiently
Bicarbonate buffer is added after reaction to be balanced, then gel chromatography isolates and purifies.
3. Carcinoembryonic Antigen CEA immue quantitative detection reagent box according to claim 1, which is characterized in that the alkaline phosphatase
The preparation step of the CEA labelled antibody of label is further are as follows:
Alkaline phosphatase is added in anti-reagent buffer, alkaline phosphatase enzyme solutions is configured, makes the final of alkaline phosphatase enzyme solutions
Concentration is 1.0~3.0mg/mL；
Alkaline phosphatase enzyme solutions and Carcinoembryonic Antigen CEA that molar ratio is 1.5:1 are mixed well 14~24 hours, sufficiently after reaction
The Tris salt buffer containing surfactant is added to be balanced, then gel chromatography isolates and purifies；
The component and content of the Tris salt buffer are as follows: Tris buffer 12.12mg, sodium chloride 5.82mg.
4. Carcinoembryonic Antigen CEA immue quantitative detection reagent box according to claim 1, which is characterized in that the magnetic particle reagent
Preparation step it is further are as follows:
Step 1: magnetic field separation being carried out to carboxyl magnetic bead cleaning concentrate, abandons supernatant after the sedimentation of carboxyl magnetic bead；
Step 2: the magnetic particle buffer of 2~5 times of carboxyl magnetic bead volumes being added in the solution that Xiang Suoshu step 1 obtains, mixes
20~30min；
Step 3: magnetic field separation being carried out to the solution that the step 2 obtains, supernatant is abandoned after the sedimentation of carboxyl magnetic bead, finally obtains 10
The carboxyl magnetic bead solution of~50mg/mL；
Step 4: carboxyl magnetic bead solution and fluorescein isothiocynate monoclonal antibody that mass ratio is 100:1 are mixed well into reaction
18h；
Step 5: magnetic field separation being carried out to the solution that the step 4 obtains, uses magnetic particle buffer after the sedimentation of carboxyl magnetic bead
Cleaning, finally obtains the magnetic particle reagent of 10mg/mL.
5. according to claim 1 to any Carcinoembryonic Antigen CEA immue quantitative detection reagent box in 4, which is characterized in that the hair
The group of light substrate is divided into APS-5 and luminous substrate buffer, and volume ratio is 1:4~10, wherein
The component and content of the luminous substrate buffer are as follows: Tris buffer 12.12g/L, sodium chloride 5.82g/L, lucigenin
0.03g/L。
6. according to claim 1 to any Carcinoembryonic Antigen CEA immue quantitative detection reagent box in 4, which is characterized in that described anti-
The component and content of reagent buffer are as follows:
7. according to claim 1 to any Carcinoembryonic Antigen CEA immue quantitative detection reagent box in 4, which is characterized in that the school
The preparation step of quasi- product and quality-control product are as follows: use calibration object buffer solution Carcinoembryonic Antigen CEA, obtain 0ng/mL, 5ng/mL,
The calibration object of 10ng/mL, 25ng/mL, 50ng/mL and 150ng/mL and the quality-control product of 10ng/mL, 50ng/mL, wherein
The component and content of the calibration object buffer are as follows: serum 500g/L, 0.01~0.05g/L of tetracycline, neomycinsulphate
0.1~0.5g/L.
8. Carcinoembryonic Antigen CEA immue quantitative detection reagent box according to any one of claims 1-4, which is characterized in that the inspection
Test agent box further includes cleaning solution, the component and content of the cleaning solution are as follows:
9. a kind of application of kit as described in any in claim 1 to 8 as external diagnosis reagent.
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| CN105092834A (en) * | 2014-05-05 | 2015-11-25 | 江苏泽成生物技术有限公司 | Carbohydrate antigen 19-9 (CA 19-9) quantitative assay kit, preparation method and detection method thereof |
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