CN105548549B - Carcinoembryonic antigen (CEA) immue quantitative detection reagent box and preparation method thereof - Google Patents
Carcinoembryonic antigen (CEA) immue quantitative detection reagent box and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to field of biological detection, be specifically related to a kind of carcinoembryonic antigen quantification kit and preparation method thereof.Its reagent comprised has Magneto separate reagent, the CEA antibody-solutions of enzyme labelling, standard substance, cleaning mixture and chemical luminous substrate solution.
Description
Technical field
The invention belongs to field of biological detection, be specifically related to a kind of carcinoembryonic antigen quantification kit and preparation method thereof.
Background technology
Carcinoembryonic antigen (carcinoembryonic antigen, CEA): for being present in a kind of glycoprotein of colon cancer and embryo's Colon mucosa cell.By Gastrointestinal Tract of Fetus epithelial tissue, pancreas regulating liver-QI cell synthesized by, generally in first 6 months of gestation, CEA content increases, and after birth, in serum, content is the lowest, 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 make change of serum C EA raise, when CEA is higher than 20 μ g/L, then mean there is digestive tract tumor.CEA raises and is common in colorectal cancer, cancer of pancreas, gastric cancer, small cell lung cancer, breast carcinoma, medullary thyroid carcinoma etc..But the disease such as smoking, trimester of pregnancy and cardiovascular disease, diabetes, nonspecific colonitis, the patients serum CEA of 15%～53% also can raise, so CEA is not the specificity marker of malignant tumor, only assists value in diagnosis.Additionally, CEA level and the pathological changes having definite relation, more late period of colorectal cancer by stages, CEA concentration is the highest.Carcinoembryonic antigen (Cacinoembryonic antigen, CEA) is the cell surface glycoprotein that molecular weight is 180-220kDa of a kind of high glycosylation.CEA is the tumor markers of a broad spectrum activity, and the disease such as colorectal carcinoma, cancer of pancreas, gastric cancer, pulmonary carcinoma, breast carcinoma all can high-caliber be expressed.The content of detection body fluid (serum, hydrothorax, sputum, saliva, seminal fluid, urine, ascites etc.) CEA for the diagnosis of tumor, 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 most commonly used for a kind of method of Detection of antigen, but the radioelement being used for labelling has the biggest injury to operator's health.Therefore, the method such as enzyme-linked immunosorbent assay (ELISA), piezoelectric immuno analytic process, chemiluminometry, colorimetry, fluoroimmunoassay and liposome immunization analytic process is developed successively.But, the most of complex steps in these methods, or the most oversize, or need the instrument and equipment of complex and expensive, or need height professional operator, etc..
Magnetic particle separation enzyme-linked immunoassay technology is a kind of with magnetic particle for solid phase carrier of separating, immunity 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, antigen, the association reaction of antibody are carried out on solid phase (elisa plate reacting hole) surface, and magnetic particle separation enzyme-linked immunoassay, antigen, the association reaction of antibody are also carried out under conditions of approximation liquid phase, thus reaction is quickly, thoroughly.Have highly sensitive compared with traditional E LISA, the advantage that the detection used time is few.
Summary of the invention
It is an object of the present invention to provide the quantitative determination reagent kit of a kind of carcinoembryonic antigen (CEA), its reagent comprised has Magneto separate reagent, the CEA antibody-solutions of enzyme labelling, standard substance, cleaning mixture and chemical luminous substrate solution..
Described 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 of the 70mmol/L of the PEG200 of 0.3mg/ml, and pH value is 8.5.
The CEA antibody-solutions of described enzyme labelling is the anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase labelling and the PBS of the caseic 20mmol/L of 0.5mg/ml, and pH value is 8.5.
Described standard substance are the CEA standard solution of variable concentrations, and it contains the 20mmol/l PBS of CEA standard substance of 0,5,10,20,40,80 and 200ng/ml, pH7.4 respectively.
Cleaning mixture by concentration be 20mol/L, pH be 7.4 phosphate buffer in add 1% Tween-20 formulated.
Described chemical luminous substrate solution is divided into luminous substrate solution A and luminous substrate B solution；The Tris-HCl buffer that luminous substrate A is 0.1M, pH value is 8.5, and this buffer contains the luminol of final concentration of 5.0mg/mL, described luminous substrate B be pH value be the 0.1M citrate buffer solution of 4.5, and containing the hydrogen peroxide of final concentration of 100mg/mL and 15mg/mL horseradish peroxidase in this buffer.
It is a further object to provide the preparation method of described test kit, specifically comprise the following steps that
1) preparation of CEA monoclonal antibody: hybridoma (preserving number the is CGMCC NO:2504) In vitro culture of CEA monoclonal antibody will be expressed；Inoculating cell number 2 × 105/ml；Culture fluid consists of 97%DMEM/F12:RPMI1640=1:1；3%FBS；Rolling speed 0.5rpm；Changed liquid once every 96 hours totally, carry out twice changing liquid altogether, gather in the crops three times；And when cell is cultivated, add 10IU/10ml LIF (leukocyte inhibitory factor), 2IU/10ml HCG, 2IU/10ml bovine insulin in the medium；Culture supernatant is clarified through filtering, and purification is collected antibody and get final product；
2) preparation of CEA Magneto separate reagent: use chemical crosslink technique by CEA monoclonal antibody and magnetic microsphere coupling, magnetic microsphere diameter is about 0.9 μm, flush three times with PBS after preparation, then with the PBS Eddy diffusion of 70mmol/L, add gelatin and the PEG200 of 0.3mg/ml of final concentration 0.1mg/ml, adjust pH value to 8.5 and to get final product；
3) preparation of the CEA antibody-solutions of enzyme labelling: use NaIO4Method labelling CEA monoclonal antibody, purification of dialysing after reaction, then dissolves with the PBS of 20mmol/L, adds final concentration 0.5mg/ml casein, adjust pH value to 8.5 and to get final product；
4) conventional method configuration standard product, cleaning mixture and chemical substrate solution.
Detailed description of the invention
The present invention is illustrated below in conjunction with accompanying drawing and further detailed description in detail.It is pointed out that following description is only the illustration to claimed technical scheme, the not any restriction 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 test kit
1) preparation of CEA monoclonal antibody: hybridoma (preserving number the is CGMCC NO:2504) In vitro culture of CEA monoclonal antibody will be expressed；Inoculating cell number 2 × 105/ml；Culture fluid consists of 97%DMEM/F12:RPMI1640=1:1；3%FBS；Rolling speed 0.5rpm；Changed liquid once every 96 hours totally, carry out twice changing liquid altogether, gather in the crops three times；And when cell is cultivated, add 10IU/10ml LIF (leukocyte inhibitory factor), 2IU/10ml HCG, 2IU/10ml bovine insulin in the medium；Culture supernatant is clarified through filtering, and purification is collected antibody and get final product；
2) preparation of CEA Magneto separate reagent: use disuccinimidyl suberate method by CEA monoclonal antibody and magnetic microsphere coupling (reaction method sees CN201110235584), magnetic microsphere diameter is about 0.9 μm, flush three times with PBS after preparation, then with the PBS Eddy diffusion of 70mmol/L, add gelatin and the PEG200 of 0.3mg/ml of final concentration 0.1mg/ml, adjust pH value to 8.5 and to get final product；
3) preparation of the CEA antibody-solutions of enzyme labelling: use NaIO4Method labelling CEA monoclonal antibody, purification of dialysing after reaction, then dissolves with the PBS of 20mmol/L, adds final concentration 0.5mg/ml casein, adjust pH value to 8.5 and to get final product；
4) conventional method configuration standard product, cleaning mixture and chemical substrate solution.
After preparation, test kit composition is 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 of the 70mmol/L of the PEG200 of 0.3mg/ml, and pH value is 8.5.
The CEA antibody-solutions of enzyme labelling is the anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase labelling and the PBS of the caseic 20mmol/L of 0.5mg/ml, and pH value is 8.5.
Standard substance are the CEA standard solution of variable concentrations, and it contains the 20mmol/l PBS of CEA standard substance of 0,5,10,20,40,80 and 200ng/ml, pH7.4 respectively.
Cleaning mixture by concentration be 20mol/L, pH be 7.4 phosphate buffer in add 1% Tween-20 formulated.
Chemical luminous substrate solution is divided into luminous substrate solution A and luminous substrate B solution；The Tris-HCl buffer that luminous substrate A is 0.1M, pH value is 8.5, and this buffer contains the luminol of final concentration of 5.0mg/mL, described luminous substrate B be pH value be the 0.1M citrate buffer solution of 4.5, and containing the hydrogen peroxide of final concentration of 100mg/mL and 15mg/mL horseradish peroxidase in this buffer.
Embodiment 2 kit test method
A. it is separately added into serum sample and CEA standard substance, every hole 20 μ l to microwell plate；
Add CEA antibody-solutions and each 50 μ l of CEA Magneto separate reagent of enzyme labelling the most successively to every hole, after vibration 30s, put 37 DEG C of water-baths 30 minutes.
C. use magnetic separator, precipitate 2 minutes.Supernatant poured out by reversing separator slowly, and the microwell plate of reversing is placed on filter paper together with separator, firmly bounces all drops that separator bottom is bonded on tube wall with removing.
D. adding 200 μ l cleaning mixture in every hole, vibration mixing 30s, Magneto separate removes cleaning mixture, in triplicate.
E. add luminous substrate A, each 50 μ l in the every hole of B, fully mix, be added immediately detected signal value in Chemiluminescence Apparatus.
The study on the stability of embodiment 3 CEA Magneto separate reagent
Use the Magneto separate reagent of different solution compositions, place one month with 60 revs/min of shakes at 37 DEG C, the method using embodiment 2 measures CEA standard substance (concentration 5ng/ml), other detectable, with embodiment 1, calculate the percentage ratio of chemical signal value and the chemical signal value measured when 0 day measured different standing time.Each group Magneto separate reagent composition is 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 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 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 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 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 labelling
The method of the employing embodiment 3 CEA antibody-solutions study on the stability to enzyme labelling, difference is not shake, and the CEA antibody-solutions component of concrete each group enzyme labelling is as follows:
Group 6: with embodiment 1
Group 7: the anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase labelling and the PBS of the 20mmol/L of 0.5mg/ml BSA, pH value is 8.5
Group 8: the anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase labelling and the PBS 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 test kit performance indications
Sensitivity for analysis is defined as: the mensuration to 20 zero standard product, takes its average deviation of 2 times, and its concentration corresponding on standard curve is sensitivity for analysis；
Sensitivity for analysis: 0.50ng/ml
Elaboration: 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: measure the AFP of 1000 μ g/L, degree of disturbance ＜ 0.05%；Measure the PSA of 100 μ g/L, degree of disturbance ＜ 1%.
Present invention merely illustrates some claimed specific embodiments; technical characteristic described in one of them or more technical scheme can be combined with arbitrary one or more technical schemes; the technical scheme that these are combined and obtain is also in the application protection domain, technical scheme that is combined just as these and that obtain specifically is recorded in the disclosure of invention.
Claims (2)
1. a quantitative determination reagent kit for carcinoembryonic antigen (CEA), its reagent comprised has Magneto separate reagent, the CEA antibody-solutions of enzyme labelling, standard substance, cleaning mixture and chemical luminous substrate solution；
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 of the 70mmol/L of the PEG200 of 0.3mg/ml, and pH value is 8.5；
The CEA antibody-solutions of described enzyme labelling is the anti-CEA monoclonal antibody containing 1mg/ml horseradish peroxidase labelling and the PBS of the caseic 20mmol/L of 0.5mg/ml, and pH value is 8.5；
Described standard substance are the CA125 standard solution of variable concentrations, and it contains the 20mmol/l PBS of CA125 standard substance of 0,5,10,20,40,80 and 200ng/ml, pH7.4 respectively；
Cleaning mixture by concentration be 20mol/L, pH be 7.4 phosphate buffer in add 1% Tween-20 formulated；
Described chemical luminous substrate solution is divided into luminous substrate solution A and luminous substrate B solution；The Tris-HCl buffer that luminous substrate A is 0.1M, pH value is 8.5, and this buffer contains the luminol of final concentration of 5.0mg/mL, described luminous substrate B be pH value be the 0.1M citrate buffer solution of 4.5, and containing the hydrogen peroxide of final concentration of 100 mg/mL and 15mg/mL horseradish peroxidase in this buffer.
2. a preparation method for test kit described in claim 1, specifically comprises the following steps that
1) preparation of CEA monoclonal antibody: the hybridoma In vitro culture of CEA monoclonal antibody will be expressed；Described hybridoma preserving number is CGMCC NO:2504, inoculating cell number 2 × 105/ml；Culture fluid consists of 97%DMEM/F12:RPMI1640=1:1；3%FBS；Rolling speed 0.5rpm；Changed liquid once every 96 hours totally, carry out twice changing liquid altogether, gather in the crops three times；And when cell is cultivated, add 10 IU/10ml LIF(leukocyte inhibitory factors in the medium), 2 IU/10ml HCG, 2 IU/10ml bovine insulins；Culture supernatant is clarified through filtering, and purification is collected antibody and get final product；
2) preparation of CEA Magneto separate reagent: use chemical crosslink technique by CEA monoclonal antibody and magnetic microsphere coupling, magnetic microsphere diameter 0.9 μm, flush three times with PBS after preparation, then with the PBS Eddy diffusion of 70mmol/L, add gelatin and the PEG200 of 0.3mg/ml of final concentration 0.1mg/ml, adjust pH value to 8.5 and to get final product；
3) preparation of the CEA antibody-solutions of enzyme labelling: use NaIO4Method labelling CEA monoclonal antibody, purification of dialysing after reaction, then dissolves with the PBS of 20mmol/L, adds final concentration 0.5mg/ml casein, adjust pH value to 8.5 and to get final product；
4) conventional method configuration standard product, cleaning mixture 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 |
| CN101377490B (en) * | 2007-08-30 | 2012-07-04 | 北京科美生物技术有限公司 | Magnetic microparticle separating chemiluminescence immune analysis determination reagent kit for detecting related sign object and preparing method thereof |
| CN101607985B (en) * | 2008-12-24 | 2013-03-27 | 中国科学院生物物理研究所 | Monoclonal antibody for anti-human CEA, a composition containing same and application thereof |
| CN101762700A (en) * | 2009-06-24 | 2010-06-30 | 北京科美东雅生物技术有限公司 | Magnetic immuno-chromatographic test paper strip for quantitatively detecting carcinoembryonic antigen in blood and preparation method thereof |
| CN102426257A (en) * | 2011-08-16 | 2012-04-25 | 内蒙古科慧生物科技有限责任公司 | Carcinoma embryonic antigen (CEA) quantitative determination kit and detection method thereof |
| CN203164191U (en) * | 2013-04-19 | 2013-08-28 | 苏州长光华医生物试剂有限公司 | Rapid carcino embryonie antigen (CEA) detection kit |
| CN103364558A (en) * | 2013-07-17 | 2013-10-23 | 江阴泽成生物技术有限公司 | Human tumor marker carcinoembryonic antigen (CEA) magnetic particle chemiluminiscence immunoassay kit and detection method |
| CN103472229B (en) * | 2013-09-17 | 2016-01-13 | 武汉生之源生物科技有限公司 | A kind of carcinomebryonic antigen magnetic microparticle chemiluminescence immune assay detection kit |
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