CN105548549A - Kit for quantitative detection of carcinoembryonic antigen (CEA) and preparation method of kit - Google Patents
Kit for quantitative detection of carcinoembryonic antigen (CEA) and preparation method of kit Download PDFInfo
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- 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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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G01N33/553—Metal or metal coated
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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- 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
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Abstract
The invention belongs to the field of biological detection, and specifically relates to a kit for quantitative detection of carcinoembryonic antigen, and a preparation method of the kit. The kit comprises reagents including a magnetic separation reagent, an enzyme-labeled CEA antibody solution, a standard substance, a washing liquid, and a chemiluminescence substrate solution.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of carcinomebryonic antigen quantification kit and preparation method thereof.
Background technology
Carcinomebryonic antigen (carcinoembryonicantigen, CEA): for being present in a kind of glycoprotein of colon cancer and embryo's Colon mucosa cell.Synthesized by the cell of Gastrointestinal Tract of Fetus epithelial tissue, pancreas and liver, in first 6 months of gestation, CEA content increases usually, and after birth, in serum, content is very low, and in healthy adult human serum, CEA concentration is less than 2.5 μ g/L.Because of loss of polarity during gastroenteric tumor, CEA reflux enter lymph or blood and and change of serum C EA is raised, when CEA is higher than 20 μ g/L, then mean there is tumor in digestive tract.CEA raises and is common in colorectal cancer, cancer of pancreas, cancer of the stomach, small-cell carcinoma of the lung, breast cancer, medullary carcinoma of thyroid gland etc.But the diseases such as smoking, the gestational period and angiocardiopathy, diabetes, nonspecific colonitis, the patients serum CEA of 15% ~ 53% also can raise, so CEA is not the specificity marker of malignant tumour, diagnosis only has auxiliary value.In addition, CEA level and colorectal cancer have definite relation by stages, the more pathology in late period, CEA concentration is higher.Carcinomebryonic antigen (Cacinoembryonicantigen, CEA) is a kind of molecular weight of high glycosylation is the cell surface glycoprotein of 180-220kDa.CEA is the tumor markers of a broad spectrum activity, and the diseases such as colorectal cancer, cancer of pancreas, cancer of the stomach, lung cancer, breast cancer all can high-caliberly be expressed.The content detecting body fluid (serum, pleural effusion, sputum, saliva, seminal fluid, urine, ascites etc.) CEA for the diagnosis of tumour, prognosis, postoperative curative effect is observed has important clinical meaning.Normal serum CEA reference value is 5ng/mL.
1969, radio immunoassay was the earliest for the detection of CEA.Although radio immunoassay is a kind of method being most commonly used to antigen detection, there is very large injury for the radioelement marked to operator's health.Therefore, the method such as enzyme-linked immunosorbent assay (ELISA), piezoelectric immuno analytic approach, chemiluminometry, colourimetry, fluoroimmunoassay and liposome immunization analytic approach is developed successively.But, the most of complex steps in these methods, or consuming time oversize, or need the instrument and equipment of complex and expensive, or need the operating personnel of highly specialty, etc.
Magnetic particle separation enzyme-linked immunoassay technology is a kind of is solid phase carrier of separating with magnetic particle, immune magnetic particle isolation technics is combined with enzyme linked immunosorbent detection technology and a kind of Novel immune detection method of setting up.Conventional ELISA method, the association reaction of antigen, antibody carries out on solid phase (elisa plate reacting hole) surface, and magnetic particle separation enzyme-linked immunoassay, the association reaction of antigen, antibody also carries out under the condition of approximate liquid phase, and thus reaction is fast, thoroughly.Have highly sensitive compared with traditional E LISA, the advantage that the detection used time is few.
Summary of the invention
An object of the present invention is to provide the quantitative determination reagent kit of a kind of carcinomebryonic antigen (CEA), its reagent comprised has Magneto separate reagent, the CEA antibody-solutions of enzyme labeling, standard items, cleansing solution and chemical luminous substrate solution..
Described CEA Magneto separate reagent is the magnetic microsphere that coupling containing 1mg/ml has anti-CEA monoclonal antibody, and the gelatin of 0.1mg/ml, the PBS damping fluid of the 70mmol/L of the PEG200 of 0.3mg/ml, pH value is 8.5.
The CEA antibody-solutions of described enzyme labeling is the PBS damping fluid of anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase mark and the caseic 20mmol/L of 0.5mg/ml, and pH value is 8.5.
Described standard items are the CEA standard solution of variable concentrations, and it is respectively containing 0,5,10,20,40,80 and the 20mmol/lPBS damping fluid of CEA standard items of 200ng/ml, pH7.4.
Cleansing solution add in the phosphate buffer of 7.4 by be 20mol/L, pH in concentration be 1% Tween-20 formulated.
Described chemical luminous substrate solution is divided into luminous substrate solution A and luminous substrate B solution; The Tris-HCl damping fluid that luminous substrate A is 0.1M, pH value is 8.5, and in this damping fluid containing final concentration be the luminol of 5.0mg/mL, described luminous substrate B is to be pH value be 4.5 0.1M citrate buffer solution, and be hydrogen peroxide and the 15mg/mL horseradish peroxidase of 100mg/mL containing final concentration in this damping fluid.
Another object of the present invention is to provide the preparation method of described kit, and concrete steps are as follows:
1) preparation of CEA monoclonal antibody: will hybridoma (preserving number the is CGMCCNO:2504) in vitro culture of CEA monoclonal antibody be expressed; Inoculating cell number 2 × 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1; 3%FBS; Roll fast 0.5rpm; Changed liquid once every 96 hours totally, carry out altogether changing liquid twice, gather in the crops three times; And during cell chulture, add 10IU/10mlLIF (leukocyte inhibitory factor) in the medium, 2IU/10mlHCG, 2IU/10ml bovine insulin; Culture supernatant, through filtering clarification, purifying, is collected antibody and get final product;
2) preparation of CEA Magneto separate reagent: adopt chemical crosslink technique by CEA monoclonal antibody and magnetic microsphere coupling, magnetic microsphere diameter is at about 0.9 μm, PBS wash buffer three times are used after preparation, then the PBS damping fluid Eddy diffusion of 70mmol/L is used, add the gelatin of final concentration 0.1mg/ml and the PEG200 of 0.3mg/ml, adjust pH to 8.5 and get final product;
3) preparation of the CEA antibody-solutions of enzyme labeling: adopt NaIO
4method mark CEA monoclonal antibody, purifying of dialysing after reaction, then uses the PBS buffer solution of 20mmol/L, adds final concentration 0.5mg/ml casein, adjust pH to 8.5 and get final product;
4) conventional method configuration standard product, cleansing solution and chemical substrate solution.
Embodiment
The present invention is illustrated below in conjunction with accompanying drawing and further detailed description in detail.It is pointed out that following explanation is only illustrating the technical scheme that application claims is protected, any restriction not to these technical schemes.The content that protection scope of the present invention is recorded with appended claims is as the criterion.
Prepared by embodiment 1 kit
1) preparation of CEA monoclonal antibody: will hybridoma (preserving number the is CGMCCNO:2504) in vitro culture of CEA monoclonal antibody be expressed; Inoculating cell number 2 × 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1; 3%FBS; Roll fast 0.5rpm; Changed liquid once every 96 hours totally, carry out altogether changing liquid twice, gather in the crops three times; And during cell chulture, add 10IU/10mlLIF (leukocyte inhibitory factor) in the medium, 2IU/10mlHCG, 2IU/10ml bovine insulin; Culture supernatant, through filtering clarification, purifying, is collected antibody and get final product;
2) preparation of CEA Magneto separate reagent: adopt disuccinimidyl suberate method by CEA monoclonal antibody and magnetic microsphere coupling (reaction method is see CN201110235584), magnetic microsphere diameter is at about 0.9 μm, PBS wash buffer three times are used after preparation, then the PBS damping fluid Eddy diffusion of 70mmol/L is used, add the gelatin of final concentration 0.1mg/ml and the PEG200 of 0.3mg/ml, adjust pH to 8.5 and get final product;
3) preparation of the CEA antibody-solutions of enzyme labeling: adopt NaIO
4method mark CEA monoclonal antibody, purifying of dialysing after reaction, then uses the PBS buffer solution of 20mmol/L, adds final concentration 0.5mg/ml casein, adjust pH to 8.5 and get final product;
4) conventional method configuration standard product, cleansing solution and chemical substrate solution.
After preparation, kit is composed as follows:
CEA Magneto separate reagent is the magnetic microsphere that the coupling containing 1mg/ml has anti-CEA monoclonal antibody, the gelatin of 0.1mg/ml, the PBS damping fluid of the 70mmol/L of the PEG200 of 0.3mg/ml, and pH value is 8.5.
The CEA antibody-solutions of enzyme labeling is the PBS damping fluid of anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase mark and the caseic 20mmol/L of 0.5mg/ml, and pH value is 8.5.
Standard items are the CEA standard solution of variable concentrations, and it is respectively containing 0,5,10,20,40,80 and the 20mmol/lPBS damping fluid of CEA standard items of 200ng/ml, pH7.4.
Cleansing solution add in the phosphate buffer of 7.4 by be 20mol/L, pH in concentration be 1% Tween-20 formulated.
Chemical luminous substrate solution is divided into luminous substrate solution A and luminous substrate B solution; The Tris-HCl damping fluid that luminous substrate A is 0.1M, pH value is 8.5, and in this damping fluid containing final concentration be the luminol of 5.0mg/mL, described luminous substrate B is to be pH value be 4.5 0.1M citrate buffer solution, and be hydrogen peroxide and the 15mg/mL horseradish peroxidase of 100mg/mL containing final concentration in this damping fluid.
Embodiment 2 kit test method
A. serum sample and CEA standard items are added respectively to microwell plate, every hole 20 μ l;
B. add CEA antibody-solutions and each 50 μ l of CEA Magneto separate reagent of enzyme labeling successively to every hole, after vibration 30s, put 37 DEG C of water-baths 30 minutes.
C. adopt magnetic separator, precipitate 2 minutes.Supernatant poured out by the separation vessel that reverses slowly, and the microwell plate of reversing is placed on filter paper together with separation vessel, firmly bounces separator bottom to remove all drops be bonded on tube wall.
D. add in 200 μ l cleansing solutions to every hole, vibration mixing 30s, Magneto separate removes cleansing solution, in triplicate.
E. add each 50 μ l in the every hole of luminous substrate A, B, fully mix, add detected signal value in Chemiluminescence Apparatus at once.
The study on the stability of embodiment 3CEA Magneto separate reagent
Adopt the Magneto separate reagent of different solution compositions, with 60 revs/min of shake placements one month at 37 DEG C, the method of embodiment 2 is adopted to measure CEA standard items (concentration 5ng/ml), other detect reagent with embodiment 1, the number percent of the chemical signal value measured when calculating the chemical signal value and 0 day that measure different standing time.Each group of Magneto separate reagent is composed as follows:
Group 1: with embodiment 1
The coupling of group 2:1mg/ml has the magnetic microsphere of anti-CEA monoclonal antibody, and the gelatin of 0.1mg/ml, the PBS damping fluid of the 70mmol/L of the PEG200 of 0.3mg/ml, pH value is 8.0.
The coupling of group 3:1mg/ml has the magnetic microsphere of anti-CEA monoclonal antibody, and the gelatin of 0.1mg/ml, the PBS damping fluid of the 70mmol/L of the PEG200 of 0.3mg/ml, pH value is 9.0.
The coupling of group 4:1mg/ml has the magnetic microsphere of anti-CEA monoclonal antibody, and the PBS damping fluid of the 70mmol/L of the PEG200 of 0.4mg/ml, pH value is 8.5.
Group 5: the coupling containing 1mg/ml has the magnetic microsphere of anti-CEA monoclonal antibody, the gelatin of 0.4mg/ml, the PBS damping fluid of 70mmol/L, and pH value is 8.5.
Concrete outcome is as follows:
| N＝5 | Group 1 | Group 2 | Group 3 | Group 4 | Group 5 |
| 5 days | 99.1％ | 91.4％ | 93.5％ | 89.3％ | 87.9％ |
| 15 days | 98.3％ | 81.6％ | 85.8％ | 79.4％ | 76.5％ |
| 30 days | 96.4％ | 74.7％ | 77.2％ | 72.6％ | 70.3％ |
The study on the stability of the CEA antibody-solutions of embodiment 4 enzyme labeling
Adopt the method for embodiment 3 to the CEA antibody-solutions study on the stability of enzyme labeling, difference is not shake, and the CEA antibody-solutions component of concrete each group enzyme labeling is as follows:
Group 6: with embodiment 1
Group 7: the PBS damping fluid of the anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase mark and the 20mmol/L of 0.5mg/mlBSA, pH value is 8.5
Group 8: the anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase mark and the PBS damping fluid of the caseic 20mmol/L of 1.0mg/ml, pH value is 8.5
Concrete outcome is as follows:
| N＝5 | Group 6 | Group 7 | Group 8 |
| 5 days | 99.7％ | 88.1％ | 94.5％ |
| 15 days | 97.4％ | 81.8％ | 87.8％ |
| 30 days | 95.8％ | 75.4％ | 81.3％ |
Embodiment 5 embodiment of the present invention 1 kit performance index
Sensitivity for analysis is defined as: to the mensuration of 20 zero standard product, gets its mean deviation of 2 times, and its concentration corresponding on typical curve is sensitivity for analysis;
Sensitivity for analysis: 0.50ng/ml
Accuracy: variation within batch CV%≤5.0%; Batch variation CV%≤10.0%
Linear coefficient: r >=0.9900
The range of linearity: 0.50-160ng/ml
Specificity: the AFP measuring 1000 μ g/L, degree of disturbance < 0.05%; Measure the PSA of 100 μ g/L, degree of disturbance < 1%.
Content of the present invention merely illustrates some claimed specific embodiments; one of them or more described technical characteristic can be combined with arbitrary one or more technical scheme in technical scheme; these technical schemes obtained through combination also in the application's protection domain, just as these technical schemes obtained through combination in the disclosure of invention concrete record.
Claims (7)
1. a quantitative determination reagent kit for carcinomebryonic antigen (CEA), its reagent comprised has Magneto separate reagent, the CEA antibody-solutions of enzyme labeling, standard items, cleansing solution and chemical luminous substrate solution.
2. quantitative determination reagent kit according to claim 1, it is characterized in that, described Magneto separate reagent is the magnetic microsphere that the coupling containing 1mg/ml has anti-CEA monoclonal antibody, the gelatin of 0.1mg/ml, the PBS damping fluid of the 70mmol/L of the PEG200 of 0.3mg/ml, pH value is 8.5.
3. quantitative determination reagent kit according to claim 1, it is characterized in that, the CEA antibody-solutions of described enzyme labeling is the PBS damping fluid of anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase mark and the caseic 20mmol/L of 0.5mg/ml, and pH value is 8.5.
4. quantitative determination reagent kit according to claim 1, it is characterized in that, described standard items are the CEA standard solution of variable concentrations, and it is respectively containing 0,5,10,20,40,80 and the 20mmol/lPBS damping fluid of CEA standard items of 200ng/ml, pH7.4.
5. quantitative determination reagent kit according to claim 1, is characterized in that, cleansing solution add in the phosphate buffer of 7.4 by be 20mol/L, pH in concentration be 1% Tween-20 formulated.
6. quantitative determination reagent kit according to claim 1, is characterized in that, described chemical luminous substrate solution is divided into luminous substrate solution A and luminous substrate B solution; The Tris-HCl damping fluid that luminous substrate A is 0.1M, pH value is 8.5, and in this damping fluid containing final concentration be the luminol of 5.0mg/mL, described luminous substrate B is to be pH value be 4.5 0.1M citrate buffer solution, and be hydrogen peroxide and the 15mg/mL horseradish peroxidase of 100mg/mL containing final concentration in this damping fluid.
7. a preparation method for kit described in claim 1, concrete steps are as follows:
1) preparation of CEA monoclonal antibody: will hybridoma (preserving number the is CGMCCNO:2504) in vitro culture of CEA monoclonal antibody be expressed; Inoculating cell number 2 × 10
5/ ml; Nutrient solution consists of 97%DMEM/F12:RPMI1640=1:1; 3%FBS; Roll fast 0.5rpm; Changed liquid once every 96 hours totally, carry out altogether changing liquid twice, gather in the crops three times; And during cell chulture, add 10IU/10mlLIF (leukocyte inhibitory factor) in the medium, 2IU/10mlHCG, 2IU/10ml bovine insulin; Culture supernatant, through filtering clarification, purifying, is collected antibody and get final product;
2) preparation of CEA Magneto separate reagent: adopt chemical crosslink technique by CEA monoclonal antibody and magnetic microsphere coupling, magnetic microsphere diameter is at about 0.9 μm, PBS wash buffer three times are used after preparation, then the PBS damping fluid Eddy diffusion of 70mmol/L is used, add the gelatin of final concentration 0.1mg/ml and the PEG200 of 0.3mg/ml, adjust pH to 8.5 and get final product;
3) preparation of the CEA antibody-solutions of enzyme labeling: adopt NaIO
4method mark CEA monoclonal antibody, purifying of dialysing after reaction, then uses the PBS buffer solution of 20mmol/L, adds final concentration 0.5mg/ml casein, adjust pH to 8.5 and get final product;
4) conventional method configuration standard product, cleansing solution and chemical substrate solution.
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| JP2006162467A (en) * | 2004-12-08 | 2006-06-22 | Kyowa Medex Co Ltd | Immunity measurement method using light transmissive magnetic particle |
| CN101377490A (en) * | 2007-08-30 | 2009-03-04 | 北京科美东雅生物技术有限公司 | Magnetic microparticle separating chemiluminescence immune analysis determination reagent kit for detecting related sign object and preparing method thereof |
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