CN109001472A - Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method - Google Patents
Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method Download PDFInfo
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Abstract
The present invention relates to a kind of human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application methods, belong to immunoassay technical field of medical detection.Human thyrotropin receptor irritation, inhibition, neutrality antibody, the technical problem of detection sensitivity, accuracy difference cannot be distinguished in the immunoassay method for solving existing detection human thyrotropin receptor antibody.Kit of the present invention uses Streptavidin-biotin system, and the combination ratio of Streptavidin and biotin is 1:4, so that signal is further amplified, improves reagent sensitivity.Enhance the specificity of detection by changing labelled antibody and antigen, specific detection thyrotropin receptor irritation antibody, for clinical diagnosis provide it is more accurate, have more targetedly external diagnosis reagent.Kit of the invention uses the reaction pattern of one-step method competition law, the content of thyrotropin receptor irritation antibody in quantitative detection human serum, it is ensured that the sensitivity of detection, specificity.
Description
Technical field
The present invention relates to a kind of immunoassay technical field of medical detection, and in particular to a kind of human thyrotropin receptor is anti-
Body chemiluminescence detection kit and preparation method thereof and application method, for thyrotropin receptor irritation antibody in human body
Quantitative detection.
Background technique
Thyrotropin receptor autoantibody (TRAb) is one group of polyclonal antibody, has heterogeneity, there are mainly three types of:
(1) thyroid stimulating antibody (TSAb), analog thyrotropic hormone (TSH) effect stimulation thyroid cell function and
Growth, causes hyperthyroidism and Thyroid Gland Swell.(2) thyroid gland retardancy antibody (TBAb), have retardance TSH or
TSAb can cause to add and subtract to thyroid excitation.(3) neutrality autoantibody, with thyrotropin receptor
(TSHR) TSHR is neither activated after combining nor blocks TSAb to the activation of TSHR.
Graves disease is a kind of organ specific autoimmune's property disease, in the active stage blood circulation of disease, is existed certainly
Body antibody and the T cell of activation.Irritation antibody TSAb in TRAb in conjunction with tsh receptor after stimulate thyroid gland, cause first shape
Gland hyperfunction.Clinically the degree of determination Graves thyroid gland of TRAb has early diagnosis meaning, and specificity can reach 90%
More than in addition 100%, that is, do not suffer from this disease the measurement overwhelming majority of person TRAb as the positive.It detects Graves patient treatment and answers
Heat condition has important guiding effect to clinical treatment management.TRAb concentration is normal during Graves disease Anti-thyroid Medicine
Decline.TRAb low concentration or disappearance may prompt remission after drug therapy, it may be considered that stopped treatment.
Trimestral TRAb measurement after pregnancy period.TRAb is IgG class antibody, can pass through placenta and cause newborn's thyroid gland disease
Disease.Then baby may suffer from hyperthyroidism to pregnant woman TSAb high, and newborn TSAb high may suffer from hyperthyroidism, therefore have the trouble of thyroid disease history
It is extremely important for assessment newborn's thyroid disease degree of danger that person measures TRAb during pregnancy.
In addition, clinically to the detection of TSAb, there are also other significances: initial patient's positive rate of not controlling is greater than 80%, because
This has important value to the diagnosis for the GD that is not true to type；For the identification with other type hyperthyroidisms；Detecting GD family numbers of patients, whether there is or not itself to exempt from
The tendency of epidemic disease thyroid disease.
Immunoassay method currently used for detecting human thyrotropin receptor antibody mainly has enzyme-linked immunosorbent assay, changes
Learn luminescent immunoassay.That there are sensitivity is low for enzyme-linked immunosorbent assay, and the range of linearity is narrow, is not easy to realize the methods of full-automation
Learn limiting factor.Chemiluminescence immunoassay is a kind of immune detection skill to grow up on the basis of enzyme-linked immunosorbent assay
Art has high sensitivity, the detection range of linearity wide, easy to operate, the advantages such as the degree of automation height.Chemiluminescence immunoassay point at present
Factorial its have many advantages, such as above-mentioned be widely used.
However, the interference component as contained in test serum sample is more, to a certain degree in actual immune detection
On affect the sensitivity and accuracy of detection.From complicated blood serum sample, quick separating, being purified into purpose determinand is to face
The great difficult problem that bed in-vitro diagnosis faces.
Summary of the invention
The invention solves the immunoassay methods for detecting human thyrotropin receptor antibody in the prior art cannot be distinguished
Human thyrotropin receptor irritation, inhibition, neutrality antibody, the technical problem of detection sensitivity, accuracy difference, provide one
Kind human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method.
In order to solve the above-mentioned technical problem, technical solution of the present invention is specific as follows:
A kind of human thyrotropin receptor antibody chemical luminescence detection kit, comprising: calibration object, quality-control product, R1 reagent,
R2 reagent and R3 reagent；
The R1 reagent is the magnetic bead solution for being coated with Streptavidin；
The R2 reagent is diluted with the 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH
The thyrotropin receptor irritation antibody-solutions of acridinium ester label；
The R3 reagent is diluted with the 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH
The thyrotropin receptor albumen n end solution of biotin labeling.
In the above-mentioned technical solutions, the magnetic bead concentration of the magnetic bead solution for being coated with Streptavidin is 0.05%-
0.1%.
In the above-mentioned technical solutions, promote first shape in the thyrotropin receptor irritation antibody-solutions of the acridinium ester label
The molar ratio of adrenoceptor irritation antibody and acridinium ester is 1:3-1:15.
In the above-mentioned technical solutions, thyroid in the thyrotropin receptor albumen n end solution of the biotin labeling
The molar ratio of plain receptor protein N-terminal and biotin is 1:3-1:15.
A kind of preparation method of human thyrotropin receptor antibody chemical luminescence detection kit, comprising the following steps:
1) calibration object is prepared with the human thyrotropin irritation antibody of human serum matrix；
2) quality-control product is prepared with the human thyrotropin irritation antibody of human serum matrix；
3) preparation of R1 reagent
After Streptavidin MagneSphere being diluted with magnetic bead dilution buffer, be prepared the magnetic bead for being coated with Streptavidin
Solution, as R1 reagent；
4) preparation of R2 reagent
Thyrotropin receptor irritation antibodies buffer is replaced into PBS buffer solution using ultra-filtration centrifuge tube, a word used for translation is added
Pyridine ester, mixes well, after being placed at room temperature for 2-4 hours, AKTA protein purification instrument purification tag antibody, and the acridinium ester mark that will be collected into
The thyrotropin receptor irritation antibody of note is buffered with the 100mM-400mM bistris for being 6.0-8.0 containing 1%BSA, pH
Liquid dilution, as R2 reagent；
5) preparation of R3 reagent
Using ultra-filtration centrifuge tube by thyrotropin receptor albumen n end buffer exchange be PBS buffer solution, be added biology
Element mixes well, after being placed at room temperature for 2-4 hours, AKTA protein purification instrument purification tag antibody, and the biotin labeling that will be collected into
Thyrotropin receptor albumen n end it is dilute with the 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH
It releases, as R3 reagent；
6) calibration object, quality-control product, R1 reagent, R2 reagent and R3 reagent are dispensed, finished product is assembled into.
In the above-mentioned technical solutions, the magnetic bead concentration that the magnetic bead solution of Streptavidin is coated with described in step 3) is
0.05%-0.1%.
In the above-mentioned technical solutions, the molar ratio of the antibody of thyrotropin receptor irritation described in step 4) and acridinium ester
For 1:3-1:15.
In the above-mentioned technical solutions, the molar ratio of thyrotropin receptor albumen n end described in step 5) and biotin is
1:3-1:15。
In the above-mentioned technical solutions, step 4) and the concentration of R2 reagent described in step 5) and R3 reagent are 0.05-1.0
μg/mL。
A kind of application method of human thyrotropin receptor antibody chemical luminescence detection kit, comprising the following steps:
Sample to be tested and R2 reagent are incubated for 10min, R3 and R1 reagent is added and is incubated for 10min, exciting liquid note is added in washing
It records relative luminous intensity (RLU).
The beneficial effects of the present invention are:
Human thyrotropin receptor antibody chemical luminescence detection kit provided by the invention is anti-using a step competition law
Mode is answered, the content of thyrotropin receptor irritation antibody in quantitative detection human serum, it is ensured that the sensitivity of detection, special
Property.Every Testing index of the invention reaches the analysis level of similar import reagent box.
Human thyrotropin receptor antibody chemical luminescence detection kit provided by the invention uses Streptavidin-biology
Ferritic system, compared with existing magnetic bead system, the combination ratio of Streptavidin and biotin is 1:4, so that signal is further put
Greatly, reagent sensitivity is improved.By changing labelled antibody and antigen, compared with original detection technique, specific detection promotees first shape
Adrenoceptor irritation antibody, enhances the specificity of detection, provides for clinical diagnosis more accurate, more targetedly external
Diagnostic reagent.
Detailed description of the invention
Invention is further described in detail with reference to the accompanying drawings and detailed description.
Fig. 1 is to be surveyed using the detection calibration product examine of the present inventor's serum thyrotropin receptor antibody chemiluminescence detection kit
The standard curve of relative light unit, abscissa are concentration, and unit mIU/mL, ordinate is luminous value.
Fig. 2 be the present inventor's serum thyrotropin receptor antibody chemiluminescence detection kit serum testing result with
The correlation of Roche company kit test result, abscissa X are Roche company kit sample measured value, concentration mIU/
ML, ordinate y are kit sample value prepared by embodiment 1, concentration unit mIU/mL.
Specific embodiment
Invention thought of the invention are as follows: the present invention uses Streptavidin-biotin system, has signal amplification,
Further increase reagent sensitivity.Magnetic bead immunoassay technology is led to using the micron order magnetic bead of Polymer Synthesizing as carrier
Cross chemical coupling coating Streptavidin, biotin labelled antibodies, by Streptavidin so that immune multiple in conjunction with biotin
Object is closed to connect with magnetic bead.By externally-applied magnetic field, cleaning separation unbonded material, have separating rate fast, the features such as high specificity,
The sensitivity and specificity of reagent can effectively be promoted.Based on this, the present invention provides a kind of human thyrotropin receptor antibody
Chemiluminescence detection kit.Kit measurement principle of the invention is competition law.Sample and biotin labelled antibodies R3 solvent
Common to be incubated for 10min, addition acridinium ester label antibody R2 reagent and the magnetic bead solution R1 reagent for being coated with Streptavidin are common
It is incubated for 10min, sample and acridinium ester label antibody and biotin labelled antibodies forms immune complex, pass through biotin and strepto-
Avidin is integrated to magnetic bead.By magnetic separator separating immune complexes, cleaning solution washes away unbonded antibody.It is excited by soda acid
Liquid excites acridinium ester to shine, and records relative luminous intensity (RLU), in the linear range, the content of TRAb and opposite hair in sample
Luminous intensity is inversely proportional.
The present invention is described in detail with reference to the accompanying drawing.
The present invention provides a kind of human thyrotropin receptor antibody chemical luminescence detection kit, comprising: calibration object, Quality Control
Product, R1 reagent, R2 reagent and R3 reagent；The R1 reagent is the magnetic bead solution for being coated with Streptavidin；The R2 reagent is
With the thyroid for the diluted acridinium ester label of 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH
Plain receptor for stimulating antibody-solutions；The R3 reagent is with the 100mM-400mM for being 6.0-8.0 containing 1%BSA, pH
The thyrotropin receptor albumen n end solution of the diluted biotin labeling of bistris buffer.It is preferred that described be coated with strepto- parent
Magnetic bead concentration with the magnetic bead solution of element is 0.05%-0.1%.It is preferred that the thyrotropin receptor of the acridinium ester label stimulates
Property antibody-solutions in the molar ratio of thyrotropin receptor irritation antibody and acridinium ester be 1:3-1:15.It is preferred that the biotin
The molar ratio of thyrotropin receptor albumen n end and biotin is 1:3- in the thyrotropin receptor albumen n end solution of label
1:15。
The present invention also provides a kind of preparation methods of human thyrotropin receptor antibody chemical luminescence detection kit, including
Following steps:
1) calibration object is prepared with the human thyrotropin irritation antibody of human serum matrix；
2) quality-control product is prepared with the human thyrotropin irritation antibody of human serum matrix；
3) preparation of R1 reagent
After being diluted Streptavidin MagneSphere with magnetic bead dilution buffer, it is 0.05%-0.1% that magnetic bead concentration, which is prepared,
The magnetic bead solution for being coated with Streptavidin, as R1 reagent；
4) preparation of R2 reagent
Thyrotropin receptor irritation antibodies buffer is replaced into 50mM- using the aperture 50KDa ultra-filtration centrifuge tube
200mM PBS buffer solution is added acridinium ester, mixes well, after being placed at room temperature for 2-4 hours, AKTA protein purification instrument purification tag
The thyrotropin receptor irritation antibody for the acridinium ester label being collected into containing 1%BSA, pH is 6.0-8.0's by antibody
It is 0.05-1.0 μ g/mL, as R2 reagent that 100mM-400mM bistris buffer, which is diluted to concentration,；The thyroid-stimulating hormone
The molar ratio of receptor for stimulating antibody and acridinium ester is 1:3-1:15；
5) preparation of R3 reagent
Using the aperture 10KDa ultra-filtration centrifuge tube by thyrotropin receptor albumen n end buffer exchange be 50mM-200mM
PBS buffer solution is added biotin, mixes well, and after being placed at room temperature for 2-4 hours, AKTA protein purification instrument purification tag antibody will
The thyrotropin receptor albumen n end for the biotin labeling being collected into is with the 100mM- for being 6.0-8.0 containing 1%BSA, pH
It is 0.05-1.0 μ g/mL, as R3 reagent that 400mM bistris buffer, which is diluted to concentration,；The thyrotropin receptor egg
The molar ratio of white N-terminal and biotin is 1:3-1:15；
6) calibration object, quality-control product, R1 reagent, R2 reagent and R3 reagent are dispensed, finished product is assembled into.
The present invention also provides a kind of application methods of human thyrotropin receptor antibody chemical luminescence detection kit, including
Following steps:
Sample to be tested and R2 reagent are incubated for 10min, R3 and R1 reagent is added and is incubated for 10min, exciting liquid note is added in washing
It records relative luminous intensity (RLU), in a certain range, content and the relative luminous intensity of TRAb is inversely proportional.
Embodiment 1 prepares TRAb chemiluminescence detection kit of the invention
One, the preparation of calibration object
TRAb is diluted to calibration object with the 100mM PBS buffer solution containing 20% human serum, be distributed into 0mIU/mL,
0.3mIU/mL、2.5mIU/mL、5mIU/mL、10mIU/mL、20mIU/mL、40mIU/mL。
Two, the preparation of quality-control product
TRAb antibody is diluted to quality-control product with the 100mM PBS buffer solution containing 20% human serum, being distributed into concentration is
The two o'clock quality-control product of 1mIU/mL, 25mIU/mL.
Three, it is coated with the preparation of the magnetic bead solution R1 reagent of Streptavidin
It is 0.1% that Streptavidin MagneSphere, which is diluted to magnetic bead concentration, with magnetic bead dilution buffer, 4 DEG C of preservations.
Four, the preparation of the thyrotropin receptor irritation antibody R2 reagent of acridinium ester label
500 μ g of thyrotropin receptor irritation antibody is added in 50KDa ultra-filtration centrifuge tube, the 100mM of 500 μ L is added
PBS buffer solution, 10000rpm are centrifuged 10min, are repeated 3 times, and are acridinium ester label buffer by buffer exchange in antibody.It collects
Solution after displacement, by thyrotropin receptor irritation antibody: acridinium ester is added in acridinium ester=1:3 molar ratio, stands 2h.It selects
The full-automatic protein purification instrument of GE company AKTA purifies the thyrotropin receptor irritation antibody-solutions of acridinium ester label,
Purified solution is collected, with 1%BSA is contained, purified antibodies are formulated as by the 100mM bistris buffer that pH is 6.0
The R2 reagent of 0.05 μ g/mL, 4 DEG C of preservations.
Five, the preparation of the thyrotropin receptor albumen n end R3 reagent of biotin labeling
500 μ g of thyrotropin receptor albumen n end is added in 10KDa ultra-filtration centrifuge tube, the 100mM of 500 μ L is added
PBS buffer solution, 10000rpm are centrifuged 10min, are repeated 3 times, and are biotin labeling buffer by buffer exchange in antibody.It collects
Solution after displacement, by thyrotropin receptor albumen n end: biotin is added in biotin=1:15 molar ratio, stands 2h.Select GE
The full-automatic protein purification instrument of company AKTA purifies biotin labelled antibodies solution, collects purified solution, with containing 1%
Purified antibodies are formulated as the R3 reagent of 0.5 μ g/mL, 4 DEG C of preservations by the 100mM Bistris buffer that BSA, PH are 6.0.
Six, semi-finished product and finished product composition
The packing of above-mentioned steps products obtained therefrom is semi-finished product, extracts three parts out and passes through specificity, accuracy, sensitivity, stabilization
After property assay approval, then with quality-control product it is assembled into human thyrotropin receptor antibody chemical luminescence kit.
Seven, reagent preparation box is examined and determine according to the manufacture and vertification regulation of this field routine, is as a result seen below
Table 1:
To sum up, in research process of the invention, the present invention has carried out screening and quality control to raw material used first
It makes, the research of concentration, purity, affinity including antibody, antibody activity after acridinium ester and biotin labeling.Then to acridinium ester
And biotin labeling system is studied, and is tested using not isolabeling ratio and label buffer, by testing repeatedly
And comparative experiments, eventually find method simplicity, high, at low cost, reliable in quality the labeling method of yield.
In addition, the present invention also explores the reaction pattern of kit and condition.By different reaction pattern and
Reaction time comparative experiments has finally determined that this kit is incubated for using one step two of competition law, and has finally been determined that reagent is incubated for
Time is 10min, shortens detection time, improves detection efficiency.
The methodology of the TRAb chemiluminescence detection kit of the invention of embodiment 2 is examined and determine
The kit prepared in embodiment 1 is examined and determine according to professional standard in this field and vertification regulation, as a result such as
Under:
1. the kit range of linearity measures
By the kit prepared in embodiment 1 wherein 1 batch, measurement range of linearity sample 0mIU/mL, 0.3mIU/mL,
2.5mIU/mL, 5mIU/mL, 10mIU/mL, 20mIU/mL, 40mIU/mL show that the range of linearity is 0.3-40mIU/mL, related
Coefficients R 2=0.9999.
The measurement of the one kit range of linearity of table
| It measures calibration object concentration (mIU/mL) | Relative light intensity (RLU) |
| 0 | 142648 |
| 0.3 | 125453 |
| 2.5 | 65633 |
| 5 | 38415 |
| 10 | 18752 |
| 20 | 10247 |
| 40 | 5410 |
2. the measurement of kit sensitivity
By the kit prepared in embodiment 1 wherein 1 batch, 20 zero-dose samples are measured, calculate average value (M) and standard
Poor (SD), obtains M+2SD, carries out two o'clock regression fit according to the concentration-RLU between zero-dose calibration object and adjacent calibration object and obtains
Linear function out brings the RLU of M+2SD into equation, show that respective concentration value is kit sensitivity 0.148mIU/mL.
(1) first luminous value
First point of shine mean value M=142733, SD=4296.8, M-2SD=134140
(2) second point luminous value
First point and the matched curve of second point line, sensitivity=0.148mIU/mL of calculating.
3. kit specificity experiments
By a large amount of repeated experiments, kit index of the invention is as follows:
Detection range: 0.15-40mIU/mL；
Sensitivity: minimum detection limit is not higher than 0.148mIU/mL；
Specificity: being 0.001%, follicle-stimulating hormone (FSH) cross reaction with thyroid-stimulating hormone (TSH) cross reacting rate
Rate is 0.001%, lutropin (LH) cross reacting rate is 0.002%, human chorionic's glandular hormone (HCG) cross reacting rate
It is 0.005% for 0.001%, thyroglobulin antibody (Anti-TG) cross reacting rate, anti-first shape Peroxidase Antibody
(Anti-TPO) cross reacting rate is 0.005%.
The kit of the invention of embodiment 3 is compared with external kit clinical sample measured value
Clinical serum is detected simultaneously with the kit and Roche company kit prepared in embodiment 1.Its testing result is shown in
Fig. 2.Using the result that Roche kit measures as abscissa X, concentration mIU/mL, using the result that measures of the present invention as indulging
Coordinate y, concentration unit mIU/mL make regression equation, dependent equation are as follows: y=1.009x+0.353, related coefficient 0.999.
Be statistically analyzed the result shows that, kit of the invention and external kit clinical correlation are good.
Embodiment 4 prepares TRAb chemiluminescence detection kit of the invention
One, the preparation of calibration object
TRAb is diluted to calibration object with the 100mM PBS buffer solution containing 20% human serum, be distributed into 0mIU/mL,
0.3mIU/mL、2.5mIU/mL、5mIU/mL、10mIU/mL、20mIU/mL、40mIU/mL。
Two, the preparation of quality-control product
TRAb antibody is diluted to quality-control product with the 100mM PBS buffer solution containing 20% human serum, being distributed into concentration is
The two o'clock quality-control product of 1mIU/mL, 25mIU/mL.
Three, it is coated with the preparation of the magnetic bead solution R1 reagent of Streptavidin
It is 0.05% that Streptavidin MagneSphere, which is diluted to magnetic bead concentration, with magnetic bead dilution buffer, 4 DEG C of preservations.
Four, the preparation of the thyrotropin receptor irritation antibody R2 reagent of acridinium ester label
500 μ g of thyrotropin receptor irritation antibody is added in 50KDa ultra-filtration centrifuge tube, the 100mM of 500 μ L is added
PBS buffer solution, 10000rpm are centrifuged 10min, are repeated 3 times, and are acridinium ester label buffer by buffer exchange in antibody.It collects
Solution after displacement, by thyrotropin receptor irritation antibody: acridinium ester is added in acridinium ester=1:15 molar ratio, stands 2h.Choosing
It is carried out with thyrotropin receptor irritation antibody-solutions of the full-automatic protein purification instrument of GE company AKTA to acridinium ester label pure
Change, collect purified solution, with 1%BSA is contained, purified antibodies are formulated as by the 400mM bistris buffer that pH is 8.0
The R2 reagent of 0.05 μ g/mL, 4 DEG C of preservations.
Five, the preparation of the thyrotropin receptor albumen n end R3 reagent of biotin labeling
500 μ g of thyrotropin receptor albumen n end is added in 10KDa ultra-filtration centrifuge tube, the 100mM of 500 μ L is added
PBS buffer solution, 10000rpm are centrifuged 10min, are repeated 3 times, and are biotin labeling buffer by buffer exchange in antibody.It collects
Solution after displacement, by thyrotropin receptor albumen n end: biotin is added in biotin=1:3 molar ratio, stands 2h.Select GE
The full-automatic protein purification instrument of company AKTA purifies biotin labelled antibodies solution, collects purified solution, with containing 1%
Purified antibodies are formulated as the R3 reagent of 0.5 μ g/mL, 4 DEG C of preservations by the 400mM Bistris buffer that BSA, PH are 8.0.
Human thyrotropin receptor antibody chemical luminescence detection kit manufactured in the present embodiment is using a step competition law
Reaction pattern, the content of thyrotropin receptor irritation antibody in quantitative detection human serum, it is ensured that the sensitivity of detection, spy
It is anisotropic.Every Testing index of the invention reaches the analysis level of similar import reagent box.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of human thyrotropin receptor antibody chemical luminescence detection kit characterized by comprising calibration object, Quality Control
Product, R1 reagent, R2 reagent and R3 reagent；
The R1 reagent is the magnetic bead solution for being coated with Streptavidin；
The R2 reagent is with the diluted acridine of 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH
The thyrotropin receptor irritation antibody-solutions of ester label；
The R3 reagent is with the diluted biology of 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH
The thyrotropin receptor albumen n end solution of element label.
2. human thyrotropin receptor antibody chemical luminescence detection kit according to claim 1, which is characterized in that institute
Stating and being coated with the magnetic bead concentration of the magnetic bead solution of Streptavidin is 0.05%-0.1%.
3. human thyrotropin receptor antibody chemical luminescence detection kit according to claim 1, which is characterized in that institute
State thyrotropin receptor irritation antibody and acridinium ester in the thyrotropin receptor irritation antibody-solutions of acridinium ester label
Molar ratio be 1:3-1:15.
4. human thyrotropin receptor antibody chemical luminescence detection kit according to claim 1, which is characterized in that institute
State mole of thyrotropin receptor albumen n end and biotin in the thyrotropin receptor albumen n end solution of biotin labeling
Than for 1:3-1:15.
5. the system of human thyrotropin receptor antibody chemical luminescence detection kit described in a kind of claim 1-4 any one
Preparation Method, which comprises the following steps:
1) calibration object is prepared with the human thyrotropin irritation antibody of human serum matrix；
2) quality-control product is prepared with the human thyrotropin irritation antibody of human serum matrix；
3) preparation of R1 reagent
After Streptavidin MagneSphere is diluted with magnetic bead dilution buffer, be prepared be coated with Streptavidin magnetic bead it is molten
Liquid, as R1 reagent；
4) preparation of R2 reagent
Thyrotropin receptor irritation antibodies buffer is replaced into PBS buffer solution using ultra-filtration centrifuge tube, acridinium ester is added,
It mixes well, after being placed at room temperature for 2-4 hours, AKTA protein purification instrument purification tag antibody, by the acridinium ester label being collected into
Thyrotropin receptor irritation antibody is dilute with the 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH
It releases, as R2 reagent；
5) preparation of R3 reagent
Using ultra-filtration centrifuge tube by thyrotropin receptor albumen n end buffer exchange be PBS buffer solution, be added biotin, fill
Divide and mixes, after being placed at room temperature for 2-4 hours, AKTA protein purification instrument purification tag antibody, by the rush for the biotin labeling being collected into
Pth receptor albumen n end is diluted with the 100mM-400mM bistris buffer for being 6.0-8.0 containing 1%BSA, pH, i.e.,
For R3 reagent；
6) calibration object, quality-control product, R1 reagent, R2 reagent and R3 reagent are dispensed, finished product is assembled into.
6. the preparation method of human thyrotropin receptor antibody chemical luminescence detection kit according to claim 5,
It is characterized in that, the magnetic bead concentration that the magnetic bead solution of Streptavidin is coated with described in step 3) is 0.05%-0.1%.
7. the preparation method of human thyrotropin receptor antibody chemical luminescence detection kit according to claim 5,
It is characterized in that, the molar ratio of the antibody of thyrotropin receptor irritation described in step 4) and acridinium ester is 1:3-1:15.
8. the preparation method of human thyrotropin receptor antibody chemical luminescence detection kit according to claim 5,
It is characterized in that, the molar ratio of thyrotropin receptor albumen n end described in step 5) and biotin is 1:3-1:15.
9. the preparation method of human thyrotropin receptor antibody chemical luminescence detection kit according to claim 5,
It is characterized in that, the concentration of R2 reagent described in step 4) and step 5) and R3 reagent is 0.05-1.0 μ g/mL.
10. human thyrotropin receptor antibody chemical luminescence detection kit described in a kind of claim 1-4 any one
Application method, which comprises the following steps:
Sample to be tested and R2 reagent are incubated for 10min, R3 and R1 reagent is added and is incubated for 10min, washing is added exciting liquid and records phase
To luminous intensity.
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| CN201810880155.7A CN109001472A (en) | 2018-08-03 | 2018-08-03 | Human thyrotropin receptor antibody chemical luminescence detection kit and preparation method thereof and application method |
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| CN110579593A (en) * | 2019-09-17 | 2019-12-17 | 郑州安图生物工程股份有限公司 | kit for detecting concentration of stimulated thyroid stimulating hormone receptor antibody |
| CN111208309A (en) * | 2020-01-07 | 2020-05-29 | 郑州安图生物工程股份有限公司 | Dissolving solution and redissolving method of thyrotropin receptor freeze-dried product |
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| CN111208309A (en) * | 2020-01-07 | 2020-05-29 | 郑州安图生物工程股份有限公司 | Dissolving solution and redissolving method of thyrotropin receptor freeze-dried product |
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| CN112462063A (en) * | 2020-07-24 | 2021-03-09 | 迪瑞医疗科技股份有限公司 | Neuron cytoplasmic protein gene product 9.5 antibody colloidal gold detection reagent and preparation method thereof |
| WO2023103827A1 (en) * | 2021-12-07 | 2023-06-15 | 郑州安图生物工程股份有限公司 | Thyroid stimulating hormone receptor antigen reagent and thyroid stimulating hormone receptor antibody quantitative test kit |
| CN116819103A (en) * | 2023-08-28 | 2023-09-29 | 成都大熊猫繁育研究基地 | Panda TSH enzyme-linked immunosorbent assay method and monoclonal antibody |
| CN116819103B (en) * | 2023-08-28 | 2023-11-07 | 成都大熊猫繁育研究基地 | Panda TSH enzyme-linked immunosorbent assay method and monoclonal antibody |
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