CN108387736A - A kind of CEA detection kits, method of preparation and use based on bimolecular fluorescence complementary technology - Google Patents

A kind of CEA detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Download PDF

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Publication number
CN108387736A
CN108387736A CN201711272662.4A CN201711272662A CN108387736A CN 108387736 A CN108387736 A CN 108387736A CN 201711272662 A CN201711272662 A CN 201711272662A CN 108387736 A CN108387736 A CN 108387736A
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China
Prior art keywords
cea
fluorescin
terminal segment
antibodie
coupling
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徐林
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Nanjing Tianzong Yikang Biological Science & Technology Co Ltd
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Nanjing Tianzong Yikang Biological Science & Technology Co Ltd
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
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  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

The present invention provides a kind of CEA diagnostic kits based on bimolecular fluorescence complementary technology.The kit includes：The fluorescin N-terminal segment of CEA antibodie coupling, the fluorescin C-terminal segment of CEA antibodie coupling；The invention also discloses a kind of preparation method of the CEA diagnostic kits based on bimolecular fluorescence complementary technology, this method includes：The preparation of the fluorescin C-terminal segment of preparation, the CEA antibodie coupling of the fluorescin N-terminal segment of CEA antibodie coupling；Finally also disclose the application method of the kit；Kit of the present invention has many advantages, such as that easy to operate, free of cleaning, precision is good, accuracy is high, is used convenient for clinical detection, is applied to the monitoring of tumour, can improve the accuracy rate of diagnosing tumor, has great market value.

Description

A kind of CEA detection kits, preparation and use based on bimolecular fluorescence complementary technology Method
Technical field
A kind of content the present invention relates to bimolecular fluorescence complementary technology for CEA in ion vitro immunization diagnosis detection human body, Belong to medical diagnosis on disease detection field.
Background technology
Carcinomebryonic antigen (carcino-embryonic antigen, CEA) is that one kind being present in colon cancer, normal fetus intestines A kind of proteoglycan compound in road, pancreas and liver.It can be widely present in the Alimentary System of endoderm origin, existed in In the digestion tubing of normal fetus, can also there be micro presence in normal human serum.
CEA is originally found in colon cancer and fetal gut tissue, therefore named carcinomebryonic antigen.CEA raisings are common in colorectal cancer, pancreas Gland cancer, gastric cancer, Small Cell Lung Cancer, breast cancer, medullary carcinoma of thyroid gland etc..But smoking, the gestational period and angiocardiopathy, diabetes, The patients serum CEA of the diseases such as nonspecific colonitis, 15%-53% can also be increased, so CEA is not the special of malignant tumour Property mark, in diagnosis only auxiliary value.In addition, CEA level and colorectal cancer has a definite relation by stages, more late period Lesion, CEA concentration are higher.
97% health adult's change of serum C EA concentration is in 2.5ng/mL or less.Primary colon cancer patient CEA, which increases, accounts for 45- 80%.In addition to primary colon cancer, gland pancreas cancer, cholangiocarcinoma, gastric cancer.Cancer of the esophagus, gland cancer, lung cancer, breast cancer and urinary system Tumer positive rate it is also very high, generally in 50-70%.
Benign tumour, inflammation and degenerative disease, as polyp of colon, ulcerative colitis, pancreatitis and alcoholic liver are hard Becoming patient CEA also has part raising, but well below malignant tumour, generally less than 20ng/mL, when CEA is more than 20ng/mL often Prompt has tumor in digestive tract.It can be as benign and malignant tumour antidiastole foundation so measuring CEA.
Common CEA detection methods mainly have enzyme-linked immunosorbent assay, radioimmunoassays and magnetic particle chemistry hair Light method.The enzyme-linked immunosorbent assay operating time is long, and process needs to clean, and step is cumbersome.Radioimmunoassays are the disadvantage is that production Raw radioactive pollution, offal treatment are complicated.Magnetic microparticle chemiluminescence belongs to heterogeneous reaction, and operating process needs to clean, drop The low precision of reagent.
To solve the above problems, if can be developed using bimolecular fluorescence complementary technology as platform using the antibody of CEA A kind of CEA quick detection reagents for diagnosing tumor.Make it compared with existing detection reagent, has and operate quick, sensitivity The advantages that high, detection is quickly, precision is high；It is applied to the monitoring of tumour, the accuracy rate of diagnosing tumor can be improved, then just Great market value can be will be widely welcomed and had by market.
Invention content
For the technical problems in the prior art, the present invention provides a kind of detection examination can be used for quantitative detection CEA Agent box, its method of preparation and use.
The invention is realized by the following technical scheme：
A method of it prepares based on bimolecular fluorescence complementary technology CEA detection kits, includes the following steps：
1) CEA antibodie is coupled fluorescin N-terminal segment；
2) CEA antibodie is coupled fluorescin C-terminal segment；
In the above-mentioned technical solutions, the CEA antibodie is the monoclonal antibody or Anti-TNF-α for CEA different epitopes Body.
In the above scheme, in the step of CEA antibodie coupling fluorescin N-terminal segment, fluorescin N-terminal segment Mass ratio with CEA antibodie is 1: 1-10.
In the above scheme, in the step of CEA antibodie coupling fluorescin C-terminal segment, fluorescin C-terminal segment Mass ratio with CEA antibodie is 1: 1-10.
According to the CEA detection reagents based on bimolecular fluorescence complementary technology prepared described in any of the above technical solution Box.It is mainly formed：
1) the fluorescin N-terminal segment of CEA antibodie coupling；
2) the fluorescin C-terminal segment of CEA antibodie coupling.
The application method of kit described in any of the above technical solution, it is characterised in that：Include the following steps：
1) it is anti-that sample, the fluorescin N-terminal segment of CEA antibodie coupling and anti-CEA are added in the reacting hole of kit The fluorescin C-terminal segment of body coupling, hybrid reaction 5-60 minutes；
2) exciting light irradiates reacting hole, measures each reacting hole luminous quantity and obtains fluorescence signal value.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Description of the drawings
Fig. 1 is the CEA detection kit principles signal provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology Figure, wherein 1- CEA antibodies, 2- bridging agents, 3- fluorescin N-terminal segments, 4- determinands (CEA), 5- CEA antibodies, 6- Fluorescin C-terminal segment, 7- bridging agents.
Fig. 2 is that the CEA detection kits detection provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology is linear Areal map.
Fig. 3 is that the CEA detection kits result provided in an embodiment of the present invention based on bimolecular fluorescence complementary technology is related Property compares.
Specific implementation mode
Below with reference to attached drawing to the present invention the CEA detection kits based on bimolecular fluorescence complementary technology, prepare and its Application method is described in detail.
Embodiment 1
CEA antibodie is coupled fluorescin N-terminal segment, with the segment YFPN of yellow fluorescence protein (YFP) 1-154 amino acid For, specific implementation process is：
1) 0.1mg YFPN albumen is added in centrifuge tube, is matched with 0.05mol/L pH9.5 carbonate buffer solutions (CB) YFPN albumen is diluted to 1mg/mL by system.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) desalting column Sephadex G-25 or 0.05mol/L pH9.5 CB dialysis is used to remove excessive glutaraldehyde.
5) 0.1mg CEA antibodies are taken, 1mg/mL antibody are prepared with 0.05mol/L pH9.5 CB, by the YFPN eggs of activation The mixing of white and antibody.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ L 0.2mol/L lysine solutions are added, room temperature closes 2h, to close remaining aldehyde radical, terminates anti- It answers.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by Sephadex G-200 gel columns or with 0.45 μm of filter membrane, used 0.01 mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 2
CEA antibodie is coupled fluorescin C-terminal segment, with the segment of yellow fluorescence protein (YFP) 155-238 amino acid For YFPC, specific implementation process is：
1) 0.1mg YFPC albumen is added in centrifuge tube, is matched with 0.05mol/L pH9.5 carbonate buffer solutions (CB) YFPC albumen is diluted to 1mg/mL by system.
2) final concentration of 1.25% glutaraldehyde is added in draught cupboard.
3) 37 DEG C of water-bath 2h.
4) desalting column Sephadex G-25 or 0.05mol/L pH9.5CB dialysis is used to remove excessive glutaraldehyde.
5) 0.1mg CEA antibodies are taken, 1mg/mL antibody are prepared with 0.05mol/L pH9.5 CB, by the YFPC eggs of activation The mixing of white and antibody.
6) 4 DEG C, reaction is overnight.
7) it closes：50 μ L 0.2mol/L lysine solutions are added, room temperature closes 2h, to close remaining aldehyde radical, terminates anti- It answers.
8) 4 DEG C of placement 2h.
9) polymer insoluble matter is removed by reactant by Sephadex G-200 gel columns or with 0.45 μm of filter membrane, used 0.01 mol/L pH7.2 PBS dialysis purification conjugates, are diluted to required concentration before use.
Embodiment 3
Kit mainly forms：
1) the fluorescin N-terminal segment of CEA antibodie coupling；
2) the fluorescin C-terminal segment of CEA antibodie coupling.
Embodiment 4
Kit application method, includes the following steps：
1) it is anti-that sample, the fluorescin N-terminal segment of CEA antibodie coupling and anti-CEA are added in the reacting hole of kit The fluorescin C-terminal segment of body coupling, hybrid reaction 5-60 minutes；
2) exciting light irradiates reacting hole, measures each reacting hole luminous quantity and obtains fluorescence signal value.
The reacting hole of this kit can be microwell plate, micro-fluidic reagent disc, reaction cup, reaction tube etc..
Embodiment 5
Kit method evaluation of the present invention：
1. linear
Compound concentration is 0ng/mL, 10ng/mL, 50ng/mL, 250ng/mL, 500ng/mL, the CEA marks of 1000ng/mL Quasi- product solution.The fluorescin N dististyles for being separately added into 20 μ L standard items in reacting hole, the coupling of 50 μ L CEA antibodies being added The fluorescin C-terminal segment of 50 μ L CEA antibodies coupling is added in section, and 37 DEG C incubate 10 minutes.After incubation, exciting light irradiation is anti- Ying Kong measures each reacting hole luminous quantity and obtains fluorescence signal value.
Using fluorescence signal value as ordinate, standard concentration is abscissa, draws standard working curve (see attached drawing 2).
2. accuracy
Recovery test：It is added in the serum specimen of normal person with known quantity CEA standard items, measures concentration value after being added It is compared with the theoretical value of addition, calculates the rate of recovery of CEA.Testing result is as follows：
Sample number CEA concentration (ng/mL) is added Measure the concentration (ng/mL) of CEA The rate of recovery (%)
1 50 49.1 98.2
2 300 310.2 103.4
3 500 491.3 98.3
4 800 791.1 98.9
3. precision
Choose the sample of 3 parts of various concentrations, respectively duplicate measurements 20 times according to the method described in the present invention.According to 20 times Measurement result calculates average deviation CV values.
4. sensitivity for analysis
The definition of sensitivity for analysis is：It refer to the amount that can be distinguished statistically with zero-dose.It is repeated 20 times measurement Zero-dose point, calculates its average value (X) and standard deviation (SD), and the concentration value with the calculating of X+2SD is the analysis of the kit Sensitivity.The sensitivity for analysis of kit of the present invention is 0.2ng/mL.
5. anti-interference
The immunological assay reagents based on bimolecular fluorescence complementary technology of the present invention are detected in interference substance (haemolysis, height Blood fat, high bilirubin) in the presence of detect serum specimen accuracy.Hemoglobin solutions are taken respectively and are added in right amount In CEA positive serum samples, it is respectively 0.5mg/mL, 1.0mg/mL to make the content of hemoglobin in serum specimen.By glycerine three Ester solution takes respectively to be added in CEA positive serum samples in right amount, makes the content of triglycerides in serum specimen be respectively 0.5mg/mL、 1.0mg/mL.Bilirubin solution is taken respectively and is added in CEA positive serum samples in right amount, is made in serum specimen The content of bilirubin is respectively 25 μ g/mL, 50 μ g/mL.It is positive to the CEA for adding hemoglobin, triglycerides and bilirubin Serum specimen is measured.Using the ratio of theoretical concentration and measured concentration as the rate of recovery, the rate of recovery is in 95.4%-104.1% Between.Show the CEA reagents based on bimolecular fluorescence complementary technology when detecting serum sample not by hemoglobin, glycerine three The interference of ester, bilirubin.
6. correlation
As shown in figure 3, the correlation with Roche Holding Ag CEA kits is：Y=0.996x-0.022, R2=0.998.
The present invention is compared with existing method and product, and with detection sensitivity height, specificity is good, cost is relatively low, to detection The low advantage of instrument requirements.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any Those familiar with the art within the technical scope disclosed by the invention, the change or replacement that can be readily occurred in, all It is covered by the protection scope of the present invention.

Claims (7)

1. a kind of CEA detection kits, method of preparation and use based on bimolecular fluorescence complementary technology, it is characterised in that：
1) kit mainly forms：The fluorescin C of the fluorescin N-terminal segment and CEA antibodie coupling of CEA antibodie coupling End fragment；
2) application method：Sample, the fluorescin N-terminal segment that CEA antibodie is coupled are added in the reacting hole of kit and resists The fluorescin C-terminal segment of CEA antibody couplings, hybrid reaction 5-60 minutes；
3) detection method：Exciting light irradiates reacting hole, measures each reacting hole luminous quantity and obtains fluorescence signal value.
2. a kind of preparation method of the CEA detection kits based on bimolecular fluorescence complementary technology, which is characterized in that including as follows Step：
1) preparation of CEA antibodie coupling fluorescin N-terminal segment；
2) preparation of CEA antibodie coupling fluorescin C-terminal segment.
3. CEA antibodie according to claim 1 is the monoclonal antibody or polyclonal antibody for CEA different epitopes.
4. fluorescin according to claim 1, including but not limited to green fluorescent protein, blue fluorescent protein, cyan Fluorescin, yellow fluorescence protein, red fluorescent protein.
5. method prepared by a kind of CEA detection kits based on bimolecular fluorescence complementary technology according to claim 2, It is characterized in that, in the CEA antibodie coupling fluorescin N-terminal segment step, fluorescin N-terminal segment and CEA antibody Mass ratio be 1: 1-10.
6. method prepared by the CEA detection kits according to claim 2 based on bimolecular fluorescence complementary technology, special Sign is that the CEA antibodie is coupled in fluorescin C-terminal segment step, the matter of fluorescin C-terminal segment and CEA antibody Amount is than being 1: 1-10.
7. reacting hole according to claim 1, including but not limited to microwell plate, micro-fluidic reagent disc, reaction cup, reaction Pipe.
CN201711272662.4A 2017-11-27 2017-11-27 A kind of CEA detection kits, method of preparation and use based on bimolecular fluorescence complementary technology Pending CN108387736A (en)

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107271661A (en) * 2017-05-18 2017-10-20 中国科学院烟台海岸带研究所 A kind of preparation method of liver cancer early detection kit

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107271661A (en) * 2017-05-18 2017-10-20 中国科学院烟台海岸带研究所 A kind of preparation method of liver cancer early detection kit

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CLIFF I. STAINS, ET AL.: "A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins Utilizing Split-Luciferase Reassembly", 《ACS CHEMICAL BIOLOGY》 *
黄欣媛等: "蛋白片段互补分析技术研究进展", 《中国生物工程杂志》 *

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Application publication date: 20180810