CN103575892A - Kit for quantitatively detecting CEA (Carcino-Embryonic Antigen) as well as preparation method and application method of kit - Google Patents
Kit for quantitatively detecting CEA (Carcino-Embryonic Antigen) as well as preparation method and application method of kit Download PDFInfo
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- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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Abstract
The invention discloses a kit for quantitatively detecting a CEA (Carcino-Embryonic Antigen). The kit comprises following reagents: a biological functional magnetic nano composite particle coupled with a CEA antibody, an enzyme-labeled CEA antibody and a chemiluminiscence substrate, wherein the biological functional magnetic nano composite particle coupled with the CEA antibody is formed by compounding a nano-grade magnetic nucleus with a high molecular material containing various functional groups. According to the kit for quantitatively detecting the CEA, a nano magnetic bead separation technology, a chemiluminiscence technology and an enzyme immunoassay technology are combined together; the kit has the advantages of high sensitivity, wide linearity range, high specificity, rapidness in reaction and the like.
Description
Technical field
The invention belongs to external clinical examination and field of nanometer technology, relate to particularly a kind of for quantitatively detecting kit, its preparation method of carcinomebryonic antigen (CEA) and using this kit to detect the method for CEA.
Background technology
Carcinomebryonic antigen (Cacinoembryonic antigen, CEA) is the cell surface glycoprotein that a kind of molecular weight of high glycosylation is 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.Detecting the content of body fluid (serum, pleural effusion, sputum, saliva, seminal fluid, urine, ascites etc.) CEA observes and has important clinical meaning for diagnosis, prognosis, the postoperative curative effect of tumour.Normal serum CEA reference value is 5ng/mL.
1969, radio immunoassay was the earliest for the detection of CEA.Although radio immunoassay is to be most commonly used to a kind of method that antigen detects, for the radioelement of mark, operator's health is had to very large injury.Therefore, the method such as enzyme-linked immunosorbent assay (ELISA), piezoelectric immuno analytic approach, chemiluminometry, colourimetry, fluoroimmunoassay and liposome immunoassay is developed successively.Yet, 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 highly operating personnel of specialty, etc.
Summary of the invention
(1) technical matters that will solve
Technical matters to be solved by this invention is to propose a kind of new kit quantitatively detecting for CEA and preparation thereof, using method, cannot be simple, quick, sensitive to solve prior art, at low cost CEA is detected.
(2) technical scheme
For solving the problems of the technologies described above, the kit quantitatively detecting for CEA of the present invention comprises following reagent: coupling has the biological functional magnetic nano-complex particle of CEA antibody, CEA antibody and the chemical luminous substrate of enzyme labeling.
According to a kind of embodiment of the present invention, described coupling has the biological functional magnetic nano-complex particle of CEA antibody to be composited by nano level magnetic core and the macromolecular material that comprises various functional groups.
According to a kind of embodiment of the present invention, described macromolecular material is one or more in polyethyleneimine, polyvinyl alcohol (PVA), polystyrene, Polyvinylchloride, silicon dioxide, polysaccharide and bovine serum albumin(BSA).
According to a kind of embodiment of the present invention, the powder that described magnetic core is iron, cobalt, nickel or oxide or its alloy.
According to a kind of embodiment of the present invention, described chemical luminous substrate comprises luminol and superoxide.
According to a kind of embodiment of the present invention, monoclonal antibody or polyclonal antibody that the CEA antibody of described enzyme labeling is CEA.
According to a kind of embodiment of the present invention, the concentration of the CEA antibody of described enzyme labeling is 300～2000ng/mL.
The present invention also proposes a kind of corresponding manufacture for the method for the quantitative kit detecting of CEA,
The method comprises manufactures the step that coupling has the biological functional magnetic nano-complex particle of CEA antibody,
And this step comprises as follows step by step: the first step, biotinylated CEA antibody is added in magnetic bead,
And shake reaction a period of time, make the two carry out coupling and form biological functional magnetic nano-complex particle; Second step, BSA is added and in magnetic bead, again shakes reaction with unreacted site on sealing magnetic bead; The 3rd step, in magnetic bead, add PBS solution preparation to become biological functional magnetic nano-complex particle.
According to a kind of embodiment of the present invention, before the first step, also comprise the step of the magnetic bead of modifying with cleansing solution washing Avidin.
The present invention also proposes a kind of method of using kit to detect CEA, the method utilizes a calibration object to calibrate, obtain the calibration curve of CEA concentration, and according to this calibration curve, detecting the CEA concentration of sample, described calibration steps and detecting step include following steps: the first step, utilize biological functional magnetic nano-complex particle to catch the CEA in calibration object or sample; Second step, the CEA antibody that utilizes enzyme labeling and the CEA catching in the described first step and biological functional magnetic nano-complex particle form double-antibody sandwich structure; The 3rd step, in described double-antibody sandwich structure, add described chemical luminous substrate, then put in chemiluminescence detector, read light intensity maximal value.。
(3) beneficial effect
The present invention combines nanometer magnetic bead isolation technics, chemiluminescence and Enzyme Immunoassay, has the advantages such as highly sensitive, the range of linearity is wide, specificity is high, be swift in response.By the CEA detection kit of method of the present invention exploitation, can detect sensitive, quick, reliably the content of CEA, thus for the diagnosis of tumour clinically, by stages, observe postoperative curative effect and prognosis provides a reference value accurately.
Accompanying drawing explanation
Fig. 1 is the graph of relation (take 30ng/mL CEA be example) of chemiluminescence intensity and time;
Fig. 2 is the calibration curve being recorded by calibration solution.
Embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in further detail.
The present invention includes two aspects, the one, biological functional magnetic nano-complex particle and preparation method thereof, the 2nd, utilize the detection method of this biological functional magnetic nano-complex particle to CEA.
According to a first aspect of the invention, the method for preparing the magnetic nano-composite particle of biological functional comprises: the coated magnetic nano-complex particle of Avidin and the antibody of biotin modification are shaken 30 minutes in the shaking table of 37 ℃, and seal with 2%BSA.The present invention has replaced the solid supported of traditional euzymelinked immunosorbent assay (ELISA) (ELISA) with the magnetic nano-composite particle of biological functional as the moving load of antibody,
Advantage is to have accelerated immune response, has shortened incubation time.The one-time detection cycle of conventional ELISA is 3～4 hours, and sense cycle of the present invention is 25～30 minutes.
According to a further aspect in the invention: utilize this biological functional magnetic nano-complex particle to comprise the steps: the first step to the detection method of CEA, in sample, add biological functional magnetic nano-complex particle to catch the CEA in sample; Second step, then add the CEA antibody of enzyme labeling to form double-antibody sandwich structure; The 3rd step, adds chemical luminous substrate, produces light intensity and detects, and the size of light intensity represents the height of CEA concentration.
According to the step described in detection method, detect, finally draw the graph of relation of light intensity and time as shown in Figure 1.As shown in Figure 1, through maximal value and the CEA concentration linear dependence of experimental verification light intensity, so the maximal value of getting light intensity is as signal.
Embodiment 1: kit
This embodiment is a kind of CEA immue quantitative detection reagent box, and this kit is based on biological functional magnetic nano-complex particle technology, and has high sensitivity and response fast.So-called kit is a kind of complete product that comprises plurality of reagents, and the various reagent in kit are jointly for completing specific function.
According to the present invention, kit at least comprises following reagent: coupling has the biological functional magnetic nano-complex particle of CEA antibody, CEA antibody and the Chemoluminescent substrate of enzyme labeling.The biological functional magnetic nano-complex particle that wherein coupling has CEA antibody is for catching CEA antigen and externally CEA is separated with other media under the effect in magnetic field; The CEA antibody of enzyme labeling for form sandwich structure with CEA and biological functional magnetic nano-complex particle and cataluminescence substrate luminous, Chemoluminescent substrate is for generation of optical signalling and this signal intensity and the proportional relation of CEA.
In this embodiment, coupling has the biological functional magnetic nano-complex particle of CEA antibody to be composited by nano level magnetic core and the macromolecular material that comprises various functional groups, and wherein macromolecular material is one or more in polyethyleneimine, polyvinyl alcohol (PVA), polystyrene, Polyvinylchloride, silicon dioxide, polysaccharide and bovine serum albumin(BSA).Magnetic core is powder or oxide or its alloy of iron, cobalt, nickel.Biological functional magnetic nano-complex particle is that the antibody coupling of the magnetic bead modified by Avidin and biotin modification or amido modified magnetic bead and magnetic bead and the antibody coupling of antibody coupling or carboxyl modified are made.In the present embodiment, magnetic bead and antibody mass ratio are 250: 1, can be also the some values between 150: 1～300: 1.
In this embodiment, described chemical luminous substrate comprises luminol (luminol) and superoxide,
Superoxide is for example hydrogen peroxide, and chemical luminous substrate is for generation of chemiluminescence signal.
In this embodiment, the CEA antibody of described enzyme labeling is CEA monoclonal antibody or polyclonal antibody.In the present embodiment, the concentration of enzyme labelled antibody is 2 μ g/mL, can be also certain value between 300～2000ng/mL.
According to the present invention, described kit also can comprise concentrated cleaning solution and calibration object.
In this embodiment, described concentrated cleaning solution is PBST (adds 5% Tween-20 formulated in phosphate buffer, refer to embodiment 2), and still, it can be also 0.1molL
-1tris-HCl damping fluid, or other can be used for washing the damping fluid of magnetic bead.
In this embodiment, described calibration object is that concentration is 0,5,10,20,40,80, seven points of 200ng/mL, and still, it can be also within the scope of 0～200ng/mL, to have the CEA of several concentration point of reasonable gradient.
Embodiment 2: the preparation method of kit
The preparation method of kit comprises the step of each reagent in this kit of preparation, and in the present invention, it is mainly the preparation method of biological functional magnetic nano-complex particle, and it is also innovation of the present invention and key point.Biological functional magnetic nano-complex particle is a kind of microballoon being formed by Nanocomposites with superparamagnetism, and on this microballoon, coupling biological antibody forms biological functional magnetic nano-complex particle.
The first step, biotinylated CEA antibody is added in magnetic bead, and shakes reaction a period of time,
Make the two carry out coupling and form biological functional magnetic nano-complex particle; Second step, BSA (bovine serum albumin) is added and in magnetic bead, again shakes reaction with unreacted site on sealing magnetic bead; The 3rd step, in magnetic bead, add PBS (phosphate buffered saline) solution preparation to become biological functional magnetic nano-complex particle.
In this embodiment, the magnetic bead that the Avidin of take is modified is example, getting the magnetic bead of the Avidin modification that 100 μ L concentration are 10mg/mL washs 3～5 times with the PBST (concentrated cleaning solution obtains according to dilution in 1: 100) of 250 μ L, add the biotinylated CEA antibody of 40 μ L, be placed in 120 turn, the shaking table of 37 ℃ takes out after 10～30 minutes, with 200 μ LPBST washing 4 times.Then add the BSA of 1mL 2% to carry out capping 20min, this reaction is carried out in shaking table.Then taking out with 200 μ LPBST washings adds for 4 times 500 μ L phosphate buffer PBS to be made into the immunomagnetic beads of 1mg/mL.Being placed in 4 ℃ keeps in Dark Place.
The preparation of CEA calibration object, similar with the preparation process of general kit alignment product.In this embodiment, by CEA solution (the Biodesign company of 100 μ g/mL imports, people source, calibration level) with antigenic dilution (cow's serum, antiseptic etc.), carry out gradient dilution, 0ng/mL, 5ng/mL, 10ng/mL, 20ng/mL, 40ng/mL, 80ng/mL, 200ng/mL, carry out packing.
The Chemoluminescent substrate of this embodiment is purchased from Beijing Ke Yuezhong pattern Bioisystech Co., Ltd.
This reality is from the preparation of routine concentrated cleaning solution: concentrated cleaning solution is by being that 1mol/L, pH add 5% Tween-20 formulated in 7.4 phosphate buffer in concentration.
In the performance history of kit, inventor explores and optimizes the long-pending optimization of optimization, the luminous substrate liquid of the optimization of incubation time in the stability of the coupling efficiency of biological functional magnetic nano-complex particle, biological functional magnetic nano-complex particle, reaction, interference experiment, HR mark CEA antibody concentration, the change procedure of light intensity time etc.The coupling efficiency of CEA antibody and magnetic particle can reach 75%; Magnetic bead by same batch of preparation that 4 ℃ are kept in Dark Place, detects the CEA of 10ng/mL for every 3 days, measures 10 times in 30 days, and relative standard deviation is 4.02%, shows that this biological functional magnetic nano-complex particle had good stability in 30 days; In reaction, the time-optimized of water-bath is 10 minutes, makes to be controlled at total detection time in 25 minutes; In interference experiment, in CEA antigenic solution, add AFP and gC A125, along with the light intensity signal that increases of AFP and CA125 concentration does not have significant change, than light intensity signal value, increase by 2.1% with pure CEA solution phase, can ignore, show that this kit has good anti-interference.
Embodiment 3: use kit to detect the method for CEA
1, for the pre-service of kit and sample.
In this embodiment, kit is placed in to room temperature (18-26 ℃) balance 10-15 minute; Getting concentrated cleaning solution 100 μ L uses deionized water according to 1: 100 diluted for use; Sample to be measured (blood, pleural effusion, sputum, saliva, seminal fluid, urine, ascites) etc. is carried out to pre-service, get supernatant, be placed in equilibrium at room temperature 10-15 minute.
2, utilize described kit sample to be carried out to the detection of CEA.
Step 1: the testing process that this step is calibration object, utilize calibration object to calibrate.This step comprises following three steps:
The first step, this step utilizes biological functional magnetic nano-complex particle to catch the CEA in solution.In this embodiment, 50 μ L biological functional magnetic nano-complex particles are mixed with 50 μ L calibration objects, 37 ℃ of water-baths were taken out after 5 minutes, and incubation time can also be 5～15 minutes.
Second step, this step utilizes CEA and the biological functional magnetic nano-complex particle in enzyme labelled antibody and previous step to form double-antibody sandwich structure.In this embodiment, the CEA antibody that adds 50 μ L horseradish peroxidase-labeled, 37 ℃ of water-baths 10 minutes, according to the present invention, Horseradish Peroxidase Conjugates volume and biological functional magnetic nano-complex particle volume can also be certain volume between 20～100 μ L; Incubation time can also be 5～15 minutes.
This step also comprises washing process, and sample is washed in magnetic field, washes away unnecessary enzymic-labelled antibody.In this embodiment, with 200 μ L cleansing solutions, repeatedly clean 5 times, the volume of cleansing solution can also be certain value between 200～500 μ L.
The 3rd step, this step is chemiluminescence detection process.In this embodiment, get Chemoluminescent substrate 50 μ L and be added in the double-antibody sandwich structure of above-mentioned steps formation, then put in chemiluminescence detector, read light intensity maximal value.The volume of chemical luminous substrate can also be certain value between 30～80 μ L.Seven calibration points detect according to aforesaid operations step successively, draw light intensity value separately.Preferably, each some Parallel testing three times, using light intensity value as the longitudinal axis, CEA concentration, as transverse axis, is made the calibration graph of light intensity and concentration and is drawn calibration equation.Testing result can be with reference to accompanying drawing 2.
The testing process that step 2, this step are actual sample.
In this embodiment, 50 μ L biological functional magnetic nano-complex particles are mixed with 50 μ L samples, according to four steps described above, operate equally.According to the calibration equation of calibration curve gained, calculate the concentration of CEA in sample.Being preferably each sample surveys three to five times.
Above-described specific embodiment; object of the present invention, technical scheme and beneficial effect are further described; be understood that; the foregoing is only specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any modification of making, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (11)
1. the kit quantitatively detecting for CEA, is characterized in that, comprises following reagent: coupling has the biological functional magnetic nano-complex particle of CEA antibody, CEA antibody and the chemical luminous substrate of enzyme labeling.
2. the kit quantitatively detecting for CEA as claimed in claim 1, is characterized in that, described coupling has the biological functional magnetic nano-complex particle of CEA antibody to be composited by nano level magnetic core and the macromolecular material that comprises various functional groups.
3. the kit quantitatively detecting for CEA as claimed in claim 2, is characterized in that, described macromolecular material is one or more in polyethyleneimine, polyvinyl alcohol (PVA), polystyrene, Polyvinylchloride, silicon dioxide, polysaccharide and bovine serum albumin(BSA).
4. the kit quantitatively detecting for CEA as claimed in claim 2, is characterized in that the powder that described magnetic core is iron, cobalt, nickel or oxide or its alloy.
5. the kit quantitatively detecting for CEA as claimed in claim 1, is characterized in that, described chemical luminous substrate comprises luminol and superoxide.
6. the kit quantitatively detecting for CEA as claimed in claim 1, is characterized in that monoclonal antibody or polyclonal antibody that the CEA antibody of described enzyme labeling is CEA.
7. the kit quantitatively detecting for CEA as claimed in claim 6, is characterized in that, the concentration of the CEA antibody of described enzyme labeling is 300～2000ng/mL.
8. a method for the kit that manufacture quantitatively detects for CEA, is characterized in that, the method comprises manufactures the step that coupling has the biological functional magnetic nano-complex particle of CEA antibody, and this step comprises as follows step by step:
The first step, biotinylated CEA antibody is added in magnetic bead, and shake reaction a period of time, make the two carry out coupling and form biological functional magnetic nano-complex particle;
Second step, BSA is added and in magnetic bead, again shakes reaction with unreacted site on sealing magnetic bead;
The 3rd step, in magnetic bead, add PBS solution preparation to become biological functional magnetic nano-complex particle.
9. the method for the kit that manufacture as claimed in claim 8 quantitatively detects for CEA, is characterized in that, before the first step, also comprises the step of the magnetic bead of modifying with cleansing solution washing Avidin.
10. a method of using the concentration that detects the CEA in sample, the method utilizes a calibration object to calibrate, and obtains the calibration curve of CEA concentration, and according to this calibration curve, detects the CEA concentration of sample, it is characterized in that, described calibration steps and detecting step include following steps:
The first step, utilize biological functional magnetic nano-complex particle to catch the CEA in calibration object or sample;
Second step, the CEA antibody that utilizes enzyme labeling and the CEA catching in the described first step and biological functional magnetic nano-complex particle form double-antibody sandwich structure;
The 3rd step, in described double-antibody sandwich structure, add described chemical luminous substrate, then put in chemiluminescence detector, read light intensity maximal value.
11. uses as claimed in claim 10 detect the method for the concentration of the CEA in sample, it is characterized in that, also comprise washing step, for washing away unnecessary enzymic-labelled antibody in described second step.
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| CN105693862A (en) * | 2016-03-07 | 2016-06-22 | 中国人民解放军第二军医大学 | Monoclonal antibody 4E8 resistant to CEA576-669HSP70L1 and application of monoclonal antibody 4E8 |
| CN105693863A (en) * | 2016-03-07 | 2016-06-22 | 中国人民解放军第二军医大学 | Monoclonal antibody 3C1 resistant to fusion protein CEA576-669HSP70L1 and application of monoclonal antibody 3C1 |
| CN110196324A (en) * | 2019-05-31 | 2019-09-03 | 扬州大学 | Magnetic mesoporous Nano particles of silicon dioxide probe and its preparation method and application |
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| CN110196324A (en) * | 2019-05-31 | 2019-09-03 | 扬州大学 | Magnetic mesoporous Nano particles of silicon dioxide probe and its preparation method and application |
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Application publication date: 20140212 |