CN106198998A - Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit and preparation method thereof - Google Patents
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a kind of human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit and preparation method thereof, human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit includes: the coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody and the chemiluminescent labels of inhibin labeling of monoclonal antibody.This human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit can be with Full-automatic chemiluminescence immunoassay analysis meter for detection instrument, complete detection this human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit of human a-fetoprotein heteroplasmon 3, through experiment, its detection sensitivity reaches 1 μ g/L, at least improve 10 times relative to the detection method sensitivity of traditional human a-fetoprotein heteroplasmon 3, the accuracy of detection of this human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit is higher.
Description
Technical field
The present invention relates to vitro detection field, particularly relate to a kind of human a-fetoprotein heteroplasmon 3 chemiluminescence immunoassay detection
Test kit and preparation method thereof.
Background technology
Hepatocarcinoma is one of the most common malignant tumor, and its grade malignancy is high, and progress is fast, poor prognosis, aggressive
By force, Most patients one, it is found that be late period, loses the best period for the treatment of.AFP is still most widely used liver now
The tumor markers of cancer diagnosis, but its Sensitivity and Specificity is the most undesirable, it is impossible to fully meet clinical needs.AFP-L3 is near
The hepatic carcinoma mark of latest find over Nian.At present, AFP-L3 has been that the ratio independent AFP detection generally acknowledged in the world is more excellent
Tumor markers, and be that hepatoma carcinoma cell is peculiar, be approved by the fda in the United States for the clinical diagnosis of hepatocarcinoma in 2005.
The common methods of Clinical detection AFP-L3 has enzyme linked immunosorbent assay, radio immunoassay, enzymatic chemistry at present
Luminescence method, but in place of these methods all also exist some shortcomings.
One, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay (ELISA) is widely used, but the method there is also following weak point::
(1) use 12 × 8 types, 6 × 8 types, 8 × 12 types or complete plate 96 hole Special micro porous plate as antigen coated apparatus and
Reaction vessel, can only be divided into 12 batches, 6 batches, 8 batches or imposite first use in use, it is impossible to carry out independent, single
The detection of part；
(2) reagent type used by quantitative determination is more, and each detectable will contain with reagent bottle, and often
Being required for changing imbibition nozzle when using a kind of reagent to be filled into respectively in the micropore of microwell plate, not only reagent bottle kind is many, adds
The operation of note reagent is the most loaded down with trivial details；
(3) lack the corresponding mark to detection information, just can only be will appreciate that by the mark checking test kit external packing box
Or know product batch number and the effect duration information of detectable, and the information known is uncontrolled during detection, has
The biggest randomness；
(4) detectable is in open space during detection, easily causes the cross-contamination between various reagent
And affect the accuracy of testing result；
(5) detection process uses manual operations, and the dosage of reagent or sample is not bery accurate, operating process the most loaded down with trivial details and
Complexity, is susceptible to bust, and accuracy and the precision of testing result are poor；
(6) in the quantity configuration and use of detection project reagent set, it is item number × 48/96 person-portion, the need to
Detect 10 projects, then configuration and the use number of reagent must be 10 × 48/96 person-portions, if the most a sample needs to detect 10
Individual different project, it is also desirable to the reagent of configuration 10 × 48/96 person-portions, the shortcoming that there is inadequate economical rationality.
Two, radio immunoassay
Radioimmunoassay method radioactive pollution is big, load procedure is loaded down with trivial details, operation is complicated, operating time length, mensuration knot
The weak points such as fruit is unstable, the reagent holding time is short, test kit operating automation degree is low, necessary instrument is expensive.
Two, chemoluminescence method
Chemoluminescence method can be divided into direct chemiluminescence and enzyme-catalyzed chemical luminescence by principle of luminosity.
Enzyme-catalyzed chemical luminescence mainly has horseradish peroxidase (HRP) and alkali phosphatase two kinds, but has certain office
Sex-limited, horseradish peroxidase major defect is: luminol, also can be by H in the case of not having horseradish peroxidase2O2
Aoxidizing self luminous, background is of a relatively high, affects signal to noise ratio, and kinetics is complicated, and influence factor is many, and result is not sufficiently stable,
Obtain highly sensitive and plateau length substrate to be not easy.Alkali phosphatase major defect is: substrate reach plateau time
Between long, substrate cost is high, causes testing cost high, patient burden's weight.
Acridinium ester is compared enzyme-catalyzed chemical luminescence as the direct chemiluminescence of label and is had detailed advantage, mainly shows
: reaction need not catalyst, as long as alkaline environment can be carried out, is swift in response, and background luminescence is low, and signal to noise ratio is high, disturb because of
Element is few, and reagent stability is good, can be with two-point calibration, and system is simple, exciting liquid low cost, acridinium ester easily and protein bind, and
After connection, photon productivity does not reduces.
Summary of the invention
Based on this, it is necessary to provide the human a-fetoprotein heteroplasmon 3 chemiluminescence immunoassay inspection that a kind of detection sensitivity is higher
Test agent box and preparation method thereof.
A kind of human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit, including: human a-fetoprotein heteroplasmon 3 is single
The coated carboxylated magnetic particle of clonal antibody and the chemiluminescent labels of inhibin labeling of monoclonal antibody.
In the described coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody, described human a-fetoprotein
Heteroplasmon 3 monoclonal antibody is 1:25～35 with the ratio of described carboxylated magnetic particle.
In the chemiluminescent labels of described inhibin labeling of monoclonal antibody, described human a-fetoprotein heteroplasmon 3 Dan Ke
Grand antibody is 50:1～10 with the ratio of described chemiluminescent labels.
The particle diameter of described carboxylated magnetic particle is 0.05 μm～1 μm.
Described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
Described human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit, also includes Chemoluminescent substrate, institute
State Chemoluminescent substrate and include A liquid and B liquid.
Described A liquid is H2O2Solution, described B liquid is NaOH solution.
Described human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit, also includes human a-fetoprotein heteroplasmon 3
Calibration product.
Described human a-fetoprotein heteroplasmon 3 calibrate product be concentration be respectively 0 μ g/L, 10 μ g/L, 50 μ g/L, 200 μ g/L,
The solution of the human a-fetoprotein heteroplasmon 3 of 1000 μ g/L and 2000 μ g/L.
The preparation method of a kind of above-mentioned human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit, including as follows
Step:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC water
Solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into human a-fetoprotein heteroplasmon 3 monoclonal antibody, under room temperature
Suspendible 2h～10h, Magneto separate is resuspended with Tris buffer after removing supernatant, obtains human a-fetoprotein heteroplasmon 3 monoclonal antibody
Coated carboxylated magnetic particle；And
Take human a-fetoprotein heteroplasmon 3 monoclonal antibody, mix after adding carbonate buffer solution, be subsequently adding chemiluminescence
Mix after label, remove impurity after lucifuge reaction 1h～2h, the chemiluminescence mark of the element labeling of monoclonal antibody that is inhibited under room temperature
Note thing.
This human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit can divide with Full-automatic chemiluminescence immunity
Analyzer is detection instrument, completes the detection this human a-fetoprotein heteroplasmon 3 chemiluminescence immunoassay inspection of human a-fetoprotein heteroplasmon 3
Test agent box, through experiment, its detection sensitivity reaches 1 μ g/L, relative to the detection side of traditional human a-fetoprotein heteroplasmon 3
Method sensitivity at least improves 10 times, the accuracy of detection of this human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit
Higher.
Accompanying drawing explanation
Fig. 1 is the preparation method of human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit of an embodiment
Flow chart；
Fig. 2 is human a-fetoprotein heteroplasmon 3 canonical plotting that embodiment 3 obtains.
Detailed description of the invention
Understandable for enabling the above-mentioned purpose of the present invention, feature and advantage to become apparent from, real with concrete below in conjunction with the accompanying drawings
Execute example the detailed description of the invention of the present invention is described in detail.Elaborate a lot of detail in the following description so that
Fully understand the present invention.But the present invention can implement to be much different from alternate manner described here, art technology
Personnel can do similar improvement in the case of intension of the present invention, and therefore the present invention is not embodied as by following public
Restriction.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit of one embodiment, including: human a-fetoprotein is different
The coated carboxylated magnetic particle of plastid 3 monoclonal antibody and the chemiluminescent labels of inhibin labeling of monoclonal antibody.
Preferably, in the coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody, human a-fetoprotein is different
Plastid 3 monoclonal antibody is 1:25～35 with the ratio of carboxylated magnetic particle.
Preferably, in the chemiluminescent labels of inhibin labeling of monoclonal antibody, human a-fetoprotein heteroplasmon 3 monoclonal
Antibody is 50:1～10 with the ratio of chemiluminescent labels.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
In other examples, above-mentioned human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit also including
Learn luminous substrate liquid.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten
Liquid.
In other examples, above-mentioned human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit also includes people
Alpha-fetoprotein variant 3 calibrates product.
It is that concentration is respectively 0 μ g/L, 10 μ g/L, 50 μ g/L, 200 μ g/L, 1000 μ that human a-fetoprotein heteroplasmon 3 calibrates product
The solution of the human a-fetoprotein heteroplasmon 3 of g/L and 2000 μ g/L.
Concrete, human a-fetoprotein heteroplasmon 3 is calibrated product and standard substance buffer can be used human a-fetoprotein heteroplasmon 3
Being configured to concentration, to be respectively the human a-fetoprotein of 0 μ g/L, 10 μ g/L, 50 μ g/L, 200 μ g/L, 1000 μ g/L and 2000 μ g/L different
The solution of plastid 3.
This human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit detects for human a-fetoprotein heteroplasmon 3
Time, utilize Full-automatic chemiluminescence immunoassay analysis meter that human a-fetoprotein heteroplasmon 3 is calibrated product and detect, draw standard bent
Line, is built in computer software；Then test actual sample, calculate concentration of specimens according to sample luminous value；Finally to people's first tire egg
White heteroplasmon 3 automatic chemiluminescence immunoassay system carries out the evaluation of performance (sensitivity, linear, precision, interference).
This human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit can divide with Full-automatic chemiluminescence immunity
Analyzer is detection instrument, completes the detection this human a-fetoprotein heteroplasmon 3 chemiluminescence immunoassay inspection of human a-fetoprotein heteroplasmon 3
Test agent box, through experiment, its detection sensitivity reaches 1 μ g/L, relative to the detection side of traditional human a-fetoprotein heteroplasmon 3
Method sensitivity at least improves 10 times, the accuracy of detection of this human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit
Higher.
Additionally, this human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit also has the advantage that:
1, selection acridinium ester is as marker material, and is applied to chemiluminescence immunoassay system, and this luminescence system is straight
Connecing chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, this reaction need not the participation of enzyme, more cost-effective；
2, select the chemiluminescence immunoassay system range of linearity width of acridinium ester, 1 μ g/L～1000 μ g/L can be reached, and
The inspection range of linearity of the detection method of traditional human a-fetoprotein heteroplasmon 3 is 10 μ g/L～1000 μ g/L；
3, acridinium ester chemiluminescent immunoassay system repeatability is high, and in batch and difference between batch is all within 5%, this is other
Chemiluminescence immunoassay system is unapproachable；
4, chemiluminescence immunoassay system has realized the quantitative of sample, by built-in standard curve to test software, only
Need test sample just can directly obtain the concentration value of sample；
5, chemiluminescence immunoassay system can realize the interpolation of full-automation, reagent and sample has instrument to complete entirely,
Operate easier, decrease artificial error.
The preparation method of above-mentioned human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit as shown in Figure 1, including
Following steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC water
Solution, the surface carboxyl groups of the magnetic particle of activated carboxyl, it is subsequently added into human a-fetoprotein heteroplasmon 3 monoclonal antibody, under room temperature
Suspendible 2h～10h, Magneto separate is resuspended with Tris buffer after removing supernatant, obtains human a-fetoprotein heteroplasmon 3 monoclonal antibody
Coated carboxylated magnetic particle.
The concentration of MES (2-(N-morpholine) ethyl sulfonic acid) buffer be 0.02M, pH be 5.5.
The concentration of Tris buffer is 0.1M and is 8.0 containing 2%BSA, pH.
The concentration of EDC (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimides) aqueous solution is 10mg/mL～20mg/
ML, EDC are 0.05:0.1～1 with the ratio of carboxylated magnetic particle.
Preferably, in the coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody, human a-fetoprotein is different
Plastid 3 monoclonal antibody is 1:25～35 with the ratio of carboxylated magnetic particle.
Preferably, the particle diameter of carboxylated magnetic particle is 0.05 μm～1 μm.
Take human a-fetoprotein heteroplasmon 3 monoclonal antibody, mix after adding carbonate buffer solution, be subsequently adding chemiluminescence
Mix after label, remove impurity after lucifuge reaction 1h～2h, the chemiluminescence mark of the element labeling of monoclonal antibody that is inhibited under room temperature
Note thing.
Carbonate buffer solution concentration be 0.1M, pH be 9.0～9.5,
The operation of remove impurity is centrifugal desalting column desalination, and concrete operations are: the most respectively with pure water and TBS buffer (40mM
Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0) process centrifugal desalting column, it is eventually adding the human a-fetoprotein obtained heterogeneous
The solution of the coated carboxylated magnetic particle of body 3 monoclonal antibody, finally collects the liquid in centrifuge tube.
Preferably, in the chemiluminescent labels of inhibin labeling of monoclonal antibody, human a-fetoprotein heteroplasmon 3 monoclonal
Antibody is 50:1～10 with the ratio of chemiluminescent labels.
Chemiluminescent labels can be luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.Wherein, chemiluminescence
Label is preferably acridinium ester.
The coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody obtained and inhibin monoclonal anti
The chemiluminescent labels combination of body tag i.e. can get above-mentioned human a-fetoprotein heteroplasmon 3 chemiluminescence immunoassay detectable
Box.
This human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit is in use, in addition it is also necessary at the bottom of chemiluminescence
Thing liquid and human a-fetoprotein heteroplasmon 3 calibrate product.
Chemoluminescent substrate and human a-fetoprotein heteroplasmon 3 are calibrated product and can be prepared voluntarily and obtain.
Chemoluminescent substrate includes A liquid and B liquid.A liquid can be H2O2Solution, B liquid can be NaOH solution.
In the present embodiment, A liquid be concentration be the H of 0.1mol/L2O2Solution, B liquid be concentration be that the NaOH of 0.25mol/L is molten
Liquid.
Concrete, human a-fetoprotein heteroplasmon 3 is calibrated product and standard substance buffer can be used human a-fetoprotein heteroplasmon 3
Being configured to concentration, to be respectively the human a-fetoprotein of 0 μ g/L, 10 μ g/L, 50 μ g/L, 200 μ g/L, 1000 μ g/L and 2000 μ g/L different
The solution of plastid 3.
The preparation method of this human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit is simple and convenient, prepares
The detection sensitivity of human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit is higher, has a good application prospect.
It it is below specific embodiment.
Embodiment 1: the preparation of human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit
(1) preparation of the coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody:
Take containing the carboxylated magnetic particle (MagnaBind that 50mg particle diameter is 0.05 μm～1 μmTM, article No. 21353) suspend
Liquid, Magneto separate removes supernatant, is that 5.5MES buffer is resuspended with 0.02M, pH, and the EDC adding 10mg/mL newly configured for 1mL is water-soluble
Liquid, activated magnetic beads surface carboxyl groups, add 4mg human a-fetoprotein heteroplasmon 3 monoclonal antibody (biorbyt, article No. orb48780),
Suspendible 6h under room temperature, Magneto separate, remove supernatant, be resuspended to 1mg/ with the Tris buffer that the 0.1M containing 2%BSA, pH are 8.0
ML, obtains the coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody, every bottle of 5mL subpackage be stored in 4 DEG C standby
With.
(2) preparation of the acridinium ester of inhibin labeling of monoclonal antibody:
Take human a-fetoprotein heteroplasmon 3 monoclonal antibody that 50 μ L concentration are 25mg/mL, add 150 μ L concentration be 0.1M,
PH is the carbonate buffer solution of 9.0～9.5, mixing, is subsequently adding the acridinium ester solution mixing that 1.5 μ L concentration are 5mg/mL, room
The lower lucifuge reaction of temperature, takes out after 1.5h, is centrifuged desalting column desalting processing with the zeba of 2mL, the most respectively with pure in desalination processes
Water purification and TBS buffer process, and are eventually adding the acridinium ester solution of the inhibin labeling of monoclonal antibody obtained, collect from
Liquid in heart pipe is in control the acridinium ester of inhibin labeling of monoclonal antibody to preserving, every bottle of 5mL subpackage be stored in 4 DEG C standby
With.
(3) human a-fetoprotein heteroplasmon 3 calibrates the preparation of product:
With standard substance buffer (40mM Tris-HCl, 0.5%BSA, 1%NaCl, pH 8.0), human a-fetoprotein is heterogeneous
It is 0 μ g/L, 50pg/mL, 200pg/mL, 500pg/mL, 1000pg/mL and 1600pg/mL that body 3 is configured to concentration, every bottle of 0.5mL
Subpackage lyophilizing, 4 DEG C save backup.
Embodiment 2: human a-fetoprotein heteroplasmon 3 chemical luminous immune detection method
It is detection instrument with Full-automatic chemiluminescence immunoassay analysis meter (YHLO, article No. iFlash3000), methodology pattern
Be sequentially added into the sample of 50 μ L for double antibody sandwich method, i.e. instrument, human a-fetoprotein heteroplasmon 3 monoclonal antibody of 50 μ L is coated
Carboxylated magnetic particle and human a-fetoprotein heteroplasmon 3 treatment fluid of 50 μ L, after reaction 20min, then add people's first of 50 μ L
The coated acridinium ester of fetoprotein heteroplasmon 3, after reaction 20min, carries out Magneto separate, and reactant mixture is sent into darkroom by instrument, depends on
Secondary addition luminous substrate A liquid (H2O2) and B liquid (NaOH) carry out luminescence-producing reaction, finally record luminous value.
Embodiment 3: human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit performance evaluation
Use the method in embodiment 2 that human a-fetoprotein heteroplasmon 3 is calibrated product to detect, obtain drawing standard curve
As shown in Figure 2.
Then to then testing actual sample, concentration of specimens is calculated according to sample luminous value.
The detection of sensitivity:
With reference to CLSI EP17-A file recommendation experimental program, calculate the detection of human a-fetoprotein heteroplasmon 3 chemiluminescence immunoassay
The sensitivity of test kit, the sensitivity tried to achieve is 1pg/mL.
Linear detection:
It is that 0 μ g/L, 10 μ g/L, 50 μ g/L, 200 μ g/L, 1000 μ g/L and 2000 μ g/L standard substance do linear point to concentration
Analysis, calculates linearly dependent coefficient, r=0.9996, it addition, linear to human a-fetoprotein heteroplasmon 3 sample detection of this test kit
Scope is 1 μ g/L～1000 μ g/L.
Precision measures:
Taking concentration is 10 μ g/L and two people's alpha-fetoprotein variant 3 samples of 500 μ g/L, and each concentration of each sample is respectively done
3 parallel, detects with three batches of test kits, calculates test kit and criticizes interior and difference between batch, and result shows that this test kit is criticized interior and criticizes
Between difference be respectively less than 5%.
Interference is tested:
Take pooled serum to add chaff interference respectively and include: conjugated bilirubin, unconjugated bilirubin, hemoglobin, ascorbic acid,
Glyceride, adding proportion is carried out according to 1:20, the survey of pooled serum after measuring pooled serum respectively and with the addition of various chaff interference
Value, calculates deviation therebetween, with ± 10% as tolerance interval.Result shows, interference all reaches the files-designated of NCCLS
Standard, can be used for the accurate evaluation of clinical laboratory's human a-fetoprotein heteroplasmon 3 situation.
Embodiment 4, the contrast experiment of human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit
Be 0 μ g/L by chemical luminescence detection method and traditional enzyme linked immunosorbent assay to concentration respectively, 10 μ g/L, 50 μ
Human a-fetoprotein heteroplasmon 3 sample of g/L, 200 μ g/L, 1000 μ g/L and 2000 μ g/L detects, and two kinds of method detections are sensitive
Degree is compared, and data are as shown in the table:
As can be seen from the above table, the sensitivity of chemical luminescence detection method relatively enzyme linked immunosorbent assay improves about 10 times.
Embodiment described above only have expressed the several embodiments of the present invention, and it describes more concrete and detailed, but also
Therefore the restriction to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that, for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, it is also possible to make some deformation and improvement, these broadly fall into the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be as the criterion with claims.
Claims (10)
1. human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit, it is characterised in that including: human a-fetoprotein
The coated carboxylated magnetic particle of heteroplasmon 3 monoclonal antibody and the chemiluminescent labels of inhibin labeling of monoclonal antibody.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that
In the described coated carboxylated magnetic particle of human a-fetoprotein heteroplasmon 3 monoclonal antibody, described human a-fetoprotein heteroplasmon 3 is single
Clonal antibody is 1:25～35 with the ratio of described carboxylated magnetic particle.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that
In the chemiluminescent labels of described inhibin labeling of monoclonal antibody, described human a-fetoprotein heteroplasmon 3 monoclonal antibody with
The ratio of described chemiluminescent labels is 50:1～10.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that
The particle diameter of described carboxylated magnetic particle is 0.05 μm～1 μm.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that
Described chemiluminescent labels is luminol, different luminol, tris (bipyridine) ruthenium or acridinium ester.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that
Also include that Chemoluminescent substrate, described Chemoluminescent substrate include A liquid and B liquid.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 6, it is characterised in that
Described A liquid is H2O2Solution, described B liquid is NaOH solution.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 1, it is characterised in that
Also include that human a-fetoprotein heteroplasmon 3 calibrates product.
Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit the most according to claim 8, it is characterised in that
Described human a-fetoprotein heteroplasmon 3 calibrate product be concentration be respectively 0 μ g/L, 10 μ g/L, 50 μ g/L, 200 μ g/L, 1000 μ g/L and
The solution of the human a-fetoprotein heteroplasmon 3 of 2000 μ g/L.
10. one kind is detected examination according to human a-fetoprotein heteroplasmon 3 chemiluminescence immunoassay according to any one of claim 1～9
The preparation method of agent box, it is characterised in that comprise the steps:
Taking the suspension of carboxylated magnetic particle, Magneto separate is resuspended with MES buffer after removing supernatant, is subsequently added into EDC aqueous solution,
The surface carboxyl groups of the magnetic particle of activated carboxyl, is subsequently added into human a-fetoprotein heteroplasmon 3 monoclonal antibody, suspendible 2h under room temperature
～10h, Magneto separate is resuspended with Tris buffer after removing supernatant, obtains human a-fetoprotein heteroplasmon 3 monoclonal antibody coated
Carboxylated magnetic particle；And
Take human a-fetoprotein heteroplasmon 3 monoclonal antibody, mix after adding carbonate buffer solution, be subsequently adding chemiluminescent labeling
Mix after thing, remove impurity after lucifuge reaction 1h～2h, the chemiluminescent labels of the element labeling of monoclonal antibody that is inhibited under room temperature.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610505988.6A CN106198998A (en) | 2016-06-30 | 2016-06-30 | Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit and preparation method thereof |
| PCT/CN2017/080401 WO2018000897A1 (en) | 2016-06-30 | 2017-04-13 | Human alpha-fetoprotein isomer 3 chemiluminescence immunodetection kit and preparation method therefor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610505988.6A CN106198998A (en) | 2016-06-30 | 2016-06-30 | Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit and preparation method thereof |
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| CN106198998A true CN106198998A (en) | 2016-12-07 |
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| CN201610505988.6A Pending CN106198998A (en) | 2016-06-30 | 2016-06-30 | Human a-fetoprotein heteroplasmon 3 chemiluminescence immune detection reagent kit and preparation method thereof |
Country Status (2)
| Country | Link |
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| CN (1) | CN106198998A (en) |
| WO (1) | WO2018000897A1 (en) |
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| WO2018000897A1 (en) | 2018-01-04 |
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