CN104345155A - Detection method for alpha fetoprotein content - Google Patents
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Abstract
The invention discloses a detection method for alpha fetoprotein content. The detection method comprises the following steps: mixing a sample to be detected, an anti-alpha fetoprotein antibody solution marked by semiconductor quantum dots and an anti-alpha fetoprotein antibody solution marked by carboxylation standard magnetic beads, and incubating for 20 min at 37 DEG C after adding a buffer solution; using a washing liquid to wash a reaction system after incubation is completed for impurity removal, then adding the buffer solution, and using fluorospectro photometers with the excitation wavelength of 300 nm and the emission wavelength of 605 nm to measure a fluorescence value; obtaining the alpha fetoprotein content of the sample to be detected according to the detected fluorescence value. The anti-alpha fetoprotein antibody marked by the semiconductor quantum dots and the anti-alpha fetoprotein antibody marked by carboxylation standard magnetic beads are combined with alpha fetoproteins, a sandwich immune response detected and monitored by the fluorospectro photometers is formed, and the detection method for alpha fetoprotein content is higher in sensitivity and accuracy.
Description
Technical field
The present invention relates to immunofluorescence field, particularly relate to a kind of detection method of α-Fetoprotein.
Background technology
1956, Bergstrandh and Czar found alpha-fetoprotein (AFP).AFP is a kind of single poly-peptide glycoprotein, and its mean molecular weight is about 68000Da.As a kind of Special Proteins in fetal serum, the liver cell of adult normal loses the ability of synthesis AFP.In health adult's body, the concentration ratio of AFP is lower than the mean value of 3.4ng/mL.
Clinical research shows, the susceptibility of Serum AFP is 41-65%, specificity 80-94% (critical value is ng/mL).AFP is general only for the supervision of liver cancer, and diagnosis and monitoring are as serologic marker thing.If Level of Alpha Fetoprotein does not reduce in operation, the possibility of recurrence is larger.Patient with high-level AFP is lower than the survival rate of common horizontal patient after the treatment.
Enzyme linked immunosorbent assay (ELISA) (ELISA) and chemiluminescent enzyme immunoassay (CLEIA) are often used to the level detecting AFP in serum.But enzyme linked immunosorbent assay has as repeatability is poor, muting sensitivity and the narrow restriction of the range of linearity.CLEIA is considered to accurately with reliably, but its shortcoming is of short duration lighting hours.
Semiconductor-quantum-point (QDs) has the optical property of the uniquenesses such as the duration length of high brightness and fluorescence, so its appearance causes sizable concern.At present, QDs be developed to be high sensitivity that a class is new and high luminous intensity probe and there is no the inherent limitation of organic dyestuff and fluorescin.Compared with other luminous markers, QDs has unique optics and characteristic electron, such as high brightness and narrow emission band and other be better than traditional organic fluorescence group.QDs in the animal lived and cell imaging for as the fluorescence labeling to tumor imaging.
Summary of the invention
Based on this, be necessary the detection method of the α-Fetoprotein that a kind of based semiconductor quantum dot is provided.
A detection method for α-Fetoprotein, is characterized in that, comprises the steps:
By the anti-alpha-fetoprotein antibody solution of testing sample, semiconductor-quantum-point mark and the anti-alpha-fetoprotein antibody solution mixing of carboxylated standard marked by magnetic bead, to add after damping fluid incubation 20min at 37 DEG C, wherein, the anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is different with anti-alpha-fetoprotein antibody respective in the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead;
Reaction system cleansing solution washing impurity-removing after incubation completes, then adds described damping fluid, and with the fluorescent spectrophotometer assay fluorescent value of the excitation wavelength of 300nm and the emission wavelength of 605nm; And
The α-Fetoprotein of testing sample is obtained according to the fluorescent value recorded.
In one embodiment, carry out in the reaction system of incubation, the volume ratio of the addition of the anti-alpha-fetoprotein antibody solution of described testing sample, described semiconductor-quantum-point mark and the anti-alpha-fetoprotein antibody solution of described carboxylated standard marked by magnetic bead is 2:3:3, the concentration of the anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is 16.5 μ g/mL ~ 18.3 μ g/mL, and the concentration of the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead is 20.4 μ g/mL ~ 23.8 μ g/mL.
In one embodiment, the anti-alpha-fetoprotein antibody solution of described semiconductor-quantum-point mark is prepared as follows:
Semiconductor quantum dot solution is mixed with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride solution, then anti-alpha-fetoprotein antibody and described damping fluid is added, 37 DEG C of lower incubations of shake carry out coupling reaction for 3 hours, then add pH be 7.4 TIRS-HCl coupling reaction is stopped, finally by reaction system with the centrifugation 20 minutes of 3000rpm/min, supernatant discarded also adds described damping fluid, obtains the anti-alpha-fetoprotein antibody solution of described semiconductor-quantum-point mark.
In one embodiment, described semiconductor quantum dot solution is CdSe semiconductor quantum dot solution.
In one embodiment, the anti-alpha-fetoprotein antibody solution of described carboxylated standard marked by magnetic bead is prepared by following operation:
By carboxylated standard magnetic bead activating solution washing at least three times, then 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride solution mixing, 37 DEG C of shake incubation 10min, then anti-alpha-fetoprotein antibody 37 DEG C of lower incubation 2h of shake are simultaneously added, finally add described damping fluid, obtain the anti-alpha-fetoprotein antibody solution of described carboxylated standard marked by magnetic bead.
In one embodiment, described activating solution is the PBS damping fluid of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride containing 4mg/mL.
In one embodiment, described damping fluid is PBS damping fluid, (methylol) aminoethane (Tris) solution of described cleansing solution to be the concentration being the polysorbas20 of 0.05% containing mass percent be 0.05mol/L.
In one embodiment, being operating as of α-Fetoprotein in testing sample is obtained: the process identical with described testing sample is carried out to the alpha-fetoprotein solution of the known content of some groups of variable concentrations according to the fluorescent value recorded, according to the fluorescent value of the alpha-fetoprotein solution recorded, obtain linear fit curve, bring the fluorescent value of the described testing sample obtained into described linear fit curve, obtain the α-Fetoprotein of described testing sample.
In one embodiment, the concentration of the alpha-fetoprotein in the alpha-fetoprotein solution of described some groups of variable concentrations is respectively: 0ng/mL, 2ng/mL, 10ng/mL, 5ng/mL, 100ng/mL and 400ng/mL.
In one embodiment, reaction system incubation complete after to complete fluorescent value measure the time interval be no more than 3h.
The detection method of this α-Fetoprotein, goes the monoclonal antibody of mark two kinds of different anti-alpha-fetoproteins by semiconductor-quantum-point and carboxylated standard magnetic bead.Semiconductor-quantum-point mark anti-alpha-fetoprotein antibody and carboxylated standard marked by magnetic bead anti-alpha-fetoprotein antibody in conjunction with alpha-fetoprotein define detected by fluorospectrophotometer and monitor sandwich immunoassay reaction.The detection method of this α-Fetoprotein is used for α-Fetoprotein and has higher sensitivity and degree of accuracy, demonstrates the great potential that it applies in clinical labororatory.
Accompanying drawing explanation
Fig. 1 is the process flow diagram of the preparation method of the organic polymer-based heat conduction microballoon of an embodiment;
Fig. 2 is the spectrogram that the spectrum of QDs antibody coupling matter by using three different excitation wave scannings reaches optimum signal and obtains;
Fig. 3 is the curved line relation of the sensitivity of incubative time and immunoassays.
Embodiment
For enabling above-mentioned purpose of the present invention, feature and advantage become apparent more, are described in detail the specific embodiment of the present invention below in conjunction with accompanying drawing.Set forth a lot of detail in the following description so that fully understand the present invention.But the present invention can be much different from alternate manner described here to implement, those skilled in the art can when without prejudice to doing similar improvement when intension of the present invention, therefore the present invention is by the restriction of following public concrete enforcement.
The Some Drugs used in the present invention and instrument as follows: 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is purchased from Sigma-Aldrich company (Missouri State, the U.S.).Bovine serum albumin(BSA) (BSA) is bought from Beijing JingKeHongDa Bioisystech Co., Ltd (Beijing, China).CdSeQDs605 buys from Life Technologies (AB & Invitrogen company) (Carlsbad, California, the U.S.).Carboxylated standard magnetic bead (pearl) is bought from Ademtech company limited (Pei Sake, France).Two kinds of different mouse-anti human a-fetoproteins monoclonal antibody (AFP-3, AFP-28) obtain from company of Fapon biotech inc (Shenzhen, China).Breeding and the program of rocking are carried out in thermostatic container (FYL-YS, China) and vibratory screening apparatus (ZXWL-100, China).Hitachi F-4600 fluorospectrophotometer (Tokyo) is used to carry out immune programme for children.
Composition graphs 1, the detection method of the α-Fetoprotein of an embodiment, comprises the steps:
S10, by the anti-alpha-fetoprotein antibody solution of testing sample, semiconductor-quantum-point mark and the anti-alpha-fetoprotein antibody solution mixing of carboxylated standard marked by magnetic bead, to add after damping fluid incubation 20min at 37 DEG C.
Testing sample can be serum.
In the anti-alpha-fetoprotein antibody solution of the anti-alpha-fetoprotein antibody solution that semiconductor-quantum-point marks and carboxylated standard marked by magnetic bead, the anti-alpha-fetoprotein antibody marked by semiconductor-quantum-point is different from by the anti-alpha-fetoprotein antibody of carboxylated standard marked by magnetic bead.In the present embodiment, two kinds of anti-alpha-fetoprotein antibodies are respectively AFP-3, AFP-28 (obtaining from company of Fapon biotech inc, Shenzhen).
Carry out in the reaction system of incubation, the volume ratio of the addition of the anti-alpha-fetoprotein antibody solution of testing sample, semiconductor-quantum-point mark and the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead is 2:3:3, the concentration of the anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is 16.5 μ g/mL ~ 18.3 μ g/mL, and the concentration of the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead is 20.4 μ g/mL ~ 23.8 μ g/mL.
The pH that damping fluid can be 0.1mol/L for concentration is the PBS damping fluid of 7.4.
In the present embodiment, the volume of testing sample is 20 μ L, the volume of the anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is 30 μ L, the volume of the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead is 30 μ L, the volume of damping fluid is 1mL.
The anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is prepared as follows: semiconductor quantum dot solution mixed with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride solution, then anti-alpha-fetoprotein antibody and described damping fluid is added, 37 DEG C of lower incubations of shake carry out coupling reaction for 3 hours, then add pH be 7.4 TIRS-HCl coupling reaction is stopped, finally by reaction system with the centrifugation 20 minutes of 3000rpm/min, supernatant discarded also adds described damping fluid, obtain the anti-alpha-fetoprotein antibody solution of described semiconductor-quantum-point mark.
Specifically, in the present embodiment, the anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is prepared as follows: 1-ethyl-3 (3-dimethylaminopropyl) the carbodiimide hydrochloride solution be semiconductor quantum dot solution and the 7 μ L concentration of 8 μm of ol/L by 10 μ L concentration being 1.3 μm of ol/L mixes, then to add 500 μ L concentration be the AntiAFP antibody of 0.2mg/mL and the pH of 100 μ L is the PBS of 6.0, 37 DEG C of lower incubations of shake carry out coupling reaction for 3 hours, then to add 100 μ L concentration be the pH of 50mmol/L be 7.4 TIRS-HCl coupling reaction is stopped, finally by reaction system with the centrifugation 20 minutes of 3000rpm/min, supernatant discarded to add 1mL concentration be the pH of 0.1mol/L is the PBS of 7.4, obtain the anti-alpha-fetoprotein antibody solution of described semiconductor-quantum-point mark.
In the present embodiment, semiconductor quantum dot solution is CdSe semiconductor quantum dot solution.In other examples, other semiconductor-quantum-point can also be adopted.The CdSe semiconductor-quantum-point (QDs) of the present embodiment is CdSeQDs605.
The anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead is prepared by following operation: by carboxylated standard magnetic bead activating solution washing at least three times, then 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride solution mixing, 37 DEG C of shake incubation 10min, then anti-alpha-fetoprotein antibody 37 DEG C of lower incubation 2h of shake are simultaneously added, finally add described damping fluid, obtain the anti-alpha-fetoprotein antibody solution of described carboxylated standard marked by magnetic bead.
Specifically, in the present embodiment, the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead is prepared by following operation: first, is that 3% carboxylated standard magnetic bead puts into required test tube then with activating solution washing bead three times by 810 μ L massfractions.Secondly, add every milligram of EDC solution (4 mg/ml) of the carboxylated standard magnetic bead of 80 μ L, 37 DEG C are shaken incubation 10 minutes.Then, joined by 25 μ L (2 mg/ml) AntiAFP antibody in the carboxylated standard magnetic bead activated, then 37 DEG C are shaken lower incubation 2 hours.Finally, add 2mL damping fluid, obtain the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead.
Activating solution is the PBS damping fluid of the EDC containing 4mg/mL.
Reaction system cleansing solution washing impurity-removing after S20, the incubation obtained by S10 complete, then adds above-mentioned damping fluid, and with the fluorescent spectrophotometer assay fluorescent value of the excitation wavelength of 300nm and the emission wavelength of 605nm.
In the present embodiment, the addition of damping fluid is 1mL.
By using three different excitation wave (300nm, 370nm, the spectrum of QDs antibody coupling matter 400nm) scanned reaches optimum signal, the monoclonal antibody obtaining coupling quantum dot 300nm, 370nm and 400nm excitation wavelength emission spectrum as shown in Figure 2.
As seen from Figure 2, transmission peak wavelength (605nm) is independently excitation wavelength.No matter be under 300nm, 370nm or 400nm, the form trait of transmission is constant, and the intensity of 300nm is the strongest.Therefore, best the exciting of setting is respectively 300nm and 605nm with transmitted wave.
(methylol) aminoethane (Tris) solution of cleansing solution to be the concentration being the polysorbas20 of 0.05% containing mass percent be 0.05mol/L.
Especially, reaction system incubation complete after to complete fluorescent value measure the time interval be no more than 3h.
In order to test the duration of QDs antibody coupling matter, select the fluorescence intensity of same sample respectively at 1 hour, 2 hours, 4 hours, 12 hours, 1 day and 2 days.Found that, the fluorescence intensity of immune complex diminished after 3 hours, lost 1/3rd after 12 hours, disappeared after 1 day.
Therefore, set after reaction system incubation completes and be no more than 3h to completing the time interval that fluorescent value measures.
S30, the fluorescent value recorded according to S20 obtain the α-Fetoprotein of testing sample.
Being operating as of α-Fetoprotein in testing sample is obtained: the process identical with described testing sample is carried out to the alpha-fetoprotein solution of the known content of some groups of variable concentrations according to the fluorescent value recorded, according to the fluorescent value of the alpha-fetoprotein solution recorded, obtain linear fit curve, bring the fluorescent value of the testing sample obtained into linear fit curve, obtain the α-Fetoprotein of testing sample.
Obtaining linear fit curve can be: in dual wells, measure calibrating device and sample, average fluorescent strength value; A linear fit standard curve of four parameters is obtained by drafting AU (Y) and analyte concentration (X).
Specifically, in the present embodiment, the concentration of the alpha-fetoprotein in the alpha-fetoprotein solution of some groups of variable concentrations is respectively: 0ng/mL, 2ng/mL, 10ng/mL, 5ng/mL, 100ng/mL and 400ng/mL, and is designated as S respectively
0, S
1, S
2, S
3, S
4and S
5.Alpha-fetoprotein solution BSA/PBS solution (sodium hydrogen phosphate of 0.01mol/L, the KH of 0.003mol/L
2pO
4, the BSA of the NaCl of 0.15mol/L, 10g/L, pH are 7.4) and preparation.
May measure the sensitivity of immunoassays incubative time, AFP standard concentration is 2ng/ml, the AFP antibody dilution ratio of quantum point coupling is 1:80, the AFP antibody dilution ratio of carboxylated standard magnetic bead coupling is 1:50, obtains immune response incubative time effect of optimization figure as shown in Figure 3.
As shown in Figure 3, because the amount of immune complex increases, A.U. (flat fluorescent intensity level) value is increased to 20 minutes with the increase in reaction time.After 20 minutes, A.U. value slowly declines, and this shows that reaction reaches balance between antigen and antibody.
Therefore, setting incubative time is 20 minutes.
Immunoassay reagent is optimized.
Immune response reagent is the key parameter affecting immunoassays sensitivity.In an experiment, the dilution rate of the AntiAFP antibody of QDs mark and the AntiAFP antibody stoste of carboxylated standard marked by magnetic bead is studied.The dilution ratio of the dilution buffer of the AntiAFP antibody stoste dilution of QDs mark is 1:10 to 1:100.The optimization of the immunoassay reagent studied with calibration curve.As Table I display, the dilution ratio of 1:80 is selected as best ratio, because its correspondence the widest range of linearity (the highest AUS5/AUS0) and maximum sensitivity (the highest AUS1/AUS0).
Table I: the coupling AFP MAb concentration dilution effect of quantum dot.
The concentration of the anti-AFP of carboxylated standard marked by magnetic bead is also affect one of the sensitivity of immunoassays and the key factor of reach.It is 1:10 to 1:50 that the AntiAFP antibody stoste dilution buffer of carboxylated standard marked by magnetic bead is diluted to dilution ratio.Similarly, according to the value of AUS1/AUS0 and AUS5/AUS0, the dilution ratio of 1:30 is selected for further research (Table II).
Table II: the coupling AFP monoclonal antibody dilution effect of carboxylated standard magnetic bead
Summary for this experiment:
Sensitivity: the average A .U. signal being obtained 10 S0 repetitions by first time has calculated detection limit, then adds 2 SDs mean values.This result of calculation is inferred and is obtained from typical curve, and represents the sensitivity of this mensuration.The value of AFP detection limit is 1.2 ngs/ml.
Degree of accuracy: two different concentration detect the degree of accuracy obtaining intra test for 10 times in single test.Similarly, use identical code (each 2 repetitions) to analyze these samples different number of days (5 days) is inner, obtain the rate of change of inter test.The incomplete precise results [mean (CV)] of intra test is 5.0ng/mL (5.81%) and 10.0ng/mL (5.53%); The incomplete precise results of inter test is [5.0ng/mL (6.26%)], [10.0ng/mL (6.15%)].
The immunofluorescence of high sensitivity based on QDs that have of the AFP horizontal analysis of human serum is established.This test comprises AntiAFP antibody pearl, the AntiAFP antibody of QDs mark and IF detection system.In immunology " sandwich " reaction, the concentration of immunoreagent and immune response time are the key parameters of the sensitivity affecting immunoassays.As in Table Ⅰ and Table Ⅱ display, when the AntiAFP antibody of QDs mark and the AntiAFP antibody dilution rate of carboxylated standard marked by magnetic bead raise, the A.U. of S1 and S5 is on a declining curve.Consider the range of linearity (AUS5/AUS1 value) and sensitivity (A.U.S1/A.U.S0 value), we select the ratio of 1:80 and 1:50.Immunoreactive incubative time is one of key parameter of the sensitivity affecting immunoassays.As Fig. 2 display, A.U., along with the prolongation of incubation time, reaches peak value after 20 minutes.We set 20 minutes as incubation time, are because non-specific adsorption can extend the immunoreactive time.
The parameter of the method, as sensitivity and degree of accuracy are all verified in advance.This result shows, the method has pointed out the possibility of clinical analysis.
The major advantage of this test is its stability and powerful fluorescence intensity.Result shows that the fluorescence intensity of immune complex reduces after 3 hours, loses 1/3rd after 12 hours.Other luminous markers are as ABEI and acridinium ester, and the duration that they transmit is short, and this signal is normally less than 1 second.Therefore, the bond of QDs may be used for a valuable especially instrument of the luminesceence analysis as clinical labororatory's application.In addition, the high sensitivity of the method that proposes display 1.2ng/ milliliter, and it for ELISA method be 2.0ng/mL, CLEIA method is 1.29ng/mL.Clearly remolding sensitivity ELISA and CLEIA of the method is better.
In sum, employ QDs as a kind of new immunofluorescence technique of fluorescent marker exploitation to detect the level of AFP in serum, the applicability of QDs even can expand to clinical analysis field, is the new test that AFP provides.
The above embodiment only have expressed several embodiment of the present invention, and it describes comparatively concrete and detailed, but therefore can not be interpreted as the restriction to the scope of the claims of the present invention.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement, these all belong to protection scope of the present invention.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. a detection method for α-Fetoprotein, is characterized in that, comprises the steps:
By the anti-alpha-fetoprotein antibody solution of testing sample, semiconductor-quantum-point mark and the anti-alpha-fetoprotein antibody solution mixing of carboxylated standard marked by magnetic bead, to add after damping fluid incubation 20min at 37 DEG C, wherein, the anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is different with anti-alpha-fetoprotein antibody respective in the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead;
Reaction system cleansing solution washing impurity-removing after incubation completes, then adds described damping fluid, and with the fluorescent spectrophotometer assay fluorescent value of the excitation wavelength of 300nm and the emission wavelength of 605nm; And
The α-Fetoprotein of testing sample is obtained according to the fluorescent value recorded.
2. the detection method of α-Fetoprotein according to claim 1, it is characterized in that, carry out in the reaction system of incubation, the volume ratio of the addition of the anti-alpha-fetoprotein antibody solution of described testing sample, described semiconductor-quantum-point mark and the anti-alpha-fetoprotein antibody solution of described carboxylated standard marked by magnetic bead is 2:3:3, the concentration of the anti-alpha-fetoprotein antibody solution of semiconductor-quantum-point mark is 16.5 μ g/mL ~ 18.3 μ g/mL, and the concentration of the anti-alpha-fetoprotein antibody solution of carboxylated standard marked by magnetic bead is 20.4 μ g/mL ~ 23.8 μ g/mL.
3. the detection method of α-Fetoprotein according to claim 1, is characterized in that, the anti-alpha-fetoprotein antibody solution of described semiconductor-quantum-point mark is prepared as follows:
Semiconductor quantum dot solution is mixed with 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride solution, then anti-alpha-fetoprotein antibody and described damping fluid is added, 37 DEG C of lower incubations of shake carry out coupling reaction for 3 hours, then add pH be 7.4 TIRS-HCl coupling reaction is stopped, finally by reaction system with the centrifugation 20 minutes of 3000rpm/min, supernatant discarded also adds described damping fluid, obtains the anti-alpha-fetoprotein antibody solution of described semiconductor-quantum-point mark.
4. the detection method of α-Fetoprotein according to claim 3, is characterized in that, described semiconductor quantum dot solution is CdSe semiconductor quantum dot solution.
5. the detection method of α-Fetoprotein according to claim 1, is characterized in that, the anti-alpha-fetoprotein antibody solution of described carboxylated standard marked by magnetic bead is prepared by following operation:
By carboxylated standard magnetic bead activating solution washing at least three times, then 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride solution mixing, 37 DEG C of shake incubation 10min, then anti-alpha-fetoprotein antibody 37 DEG C of lower incubation 2h of shake are simultaneously added, finally add described damping fluid, obtain the anti-alpha-fetoprotein antibody solution of described carboxylated standard marked by magnetic bead.
6. the detection method of α-Fetoprotein according to claim 5, is characterized in that, described activating solution is the PBS damping fluid of 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide hydrochloride containing 4mg/mL.
7. the detection method of α-Fetoprotein according to claim 1, it is characterized in that, described damping fluid is PBS damping fluid, (methylol) aminoethane (Tris) solution of described cleansing solution to be the concentration being the polysorbas20 of 0.05% containing mass percent be 0.05mol/L.
8. the detection method of α-Fetoprotein according to claim 1, it is characterized in that, being operating as of α-Fetoprotein in testing sample is obtained: the process identical with described testing sample is carried out to the alpha-fetoprotein solution of the known content of some groups of variable concentrations according to the fluorescent value recorded, according to the fluorescent value of the alpha-fetoprotein solution recorded, obtain linear fit curve, bring the fluorescent value of the described testing sample obtained into described linear fit curve, obtain the α-Fetoprotein of described testing sample.
9. the detection method of α-Fetoprotein according to claim 8, it is characterized in that, the concentration of the alpha-fetoprotein in the alpha-fetoprotein solution of described some groups of variable concentrations is respectively: 0ng/mL, 2ng/mL, 10ng/mL, 5ng/mL, 100ng/mL and 400ng/mL.
10. the detection method of α-Fetoprotein according to claim 8, is characterized in that, after reaction system incubation completes to complete fluorescent value measure the time interval be no more than 3h.
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| CN108469445A (en) * | 2018-03-29 | 2018-08-31 | 福州大学 | For the kit of the lanthanide fluoro nano-particle based on excitation of X-rays of cancer antigen content and preparation in no background detection blood |
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| CN108469445A (en) * | 2018-03-29 | 2018-08-31 | 福州大学 | For the kit of the lanthanide fluoro nano-particle based on excitation of X-rays of cancer antigen content and preparation in no background detection blood |
| CN108469445B (en) * | 2018-03-29 | 2020-09-01 | 福州大学 | Kit for detecting content of cancer antigen in blood without background based on lanthanide fluorescent nanoparticles excited by X-rays and preparation method thereof |
| CN114034865A (en) * | 2021-11-09 | 2022-02-11 | 石家庄斯巴克生物科技有限公司 | Method for coating magnetic beads with alpha-fetoprotein antibodies, coupled magnetic beads and kit |
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