CN106645689A - Thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit and preparation method thereof - Google Patents
Thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit and preparation method thereof Download PDFInfo
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- CN106645689A CN106645689A CN201611018792.0A CN201611018792A CN106645689A CN 106645689 A CN106645689 A CN 106645689A CN 201611018792 A CN201611018792 A CN 201611018792A CN 106645689 A CN106645689 A CN 106645689A
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- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
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Abstract
The invention discloses a thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit. The thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit comprises thyroid-stimulating hormone receptor-enveloped magnetic particles, mouse anti-human IgG antibody-enveloped acridinium ester, a thyroid-stimulating hormone receptor antibody calibration product, pre-stimulation fluid and stimulation fluid. In addition, the invention also discloses a preparation method of the thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit. Compared with the existing kit, the thyroid-stimulating hormone receptor antibody chemiluminescent immunoassay kit has the characteristics of simplicity in operation, high sensitivity and the like.
Description
Technical field
The present invention relates to in-vitro diagnosis immunoassays field, specifically, the invention provides a kind of chemiluminescence immunoassay is surveyed
Determine thyrotrophin receptor antibody kit and preparation method thereof.
Background technology
AITD（Autoimmune thyroid disease, AITD）It is that one group of pathogenesis is complicated
Organ specific autoimmune's disease, including Graves is sick（Graves ' disease, GD）, Hashimoto thyroiditis
（Hashimoto ' s thyroiditis, HT）Deng.Thyroid stimulating hormone receptor (hTSHR) is the main target antigen of AITD,
Play an important role in the morbidity of AITD.HTSHR is the presence of human thyroid follicular epithelial cells（TEC）A kind of sugared egg on film
In vain, it and thyroid-stimulating hormone（TSH）With reference to after, growth, differentiation and its synthesis of TEC and the work(of release thyroid hormone are adjusted
Energy.On the basis of E＆H factor, body's immunity ANOMALOUS VARIATIONS cause AITD, hTSHR be AITD it is main itself
One of antigen, stimulates body to produce serum thyrotropin receptor antibody（TRAb）, the autoantibody belongs to heterogeneous antibody, including first
Shape gland blocking antibody（TSBAb）And thyroid stimulating antibody（TSAb）, the former is combined with TSHR specific sites and is prevented
The combination of TSH and TSHR and its performance of physiological action, cause atrophy of thyroid gland and function reduction, show as thyroid function
Go down（First subtracts）, it is more common in the later stage of HT；The latter simulate TSH functions, for a long time constantly stimulate thyroid hormone synthesis release and
Cause GD, show as hyperthyroidism.
At present the common methods of clinical assays thyrotrophin receptor antibody have enzyme linked immunosorbent assay, radio-immunity point
Analysis method, enzyme-catalyzed chemical luminescence method, but these methods all have some shortcomings part.
First, enzyme linked immunosorbent assay
Enzyme linked immunosorbent assay（ELISA）It is widely used, but the method there is also following weak points：
(1) using 12 × 8 types, 6 × 8 types, 8 × 12 types or the hole Special micro porous plate of complete plate 96 as antigen coat apparatus and anti-
Container is answered, 12 batches, 6 batches, 8 batches or whole plate first use can only be divided into when in use, it is impossible to carry out independent, single part
Measure；
(2) reagent type used is quantitative determined more, each measure reagent will be contained with reagent bottle, and often be made
It is required for changing imbibition nozzle to be filled into respectively in the micropore of microwell plate during with a kind of reagent, not only reagent bottle species is more, filling
The operation of reagent is also extremely loaded down with trivial details；
(3) lack the corresponding mark to determining information, can only just will appreciate that by checking the mark of kit external packing box or know
The product batch number and term of validity information for determining reagent is known, and the information known is uncontrolled in continuous mode, with very big
Randomness；
(4) reagent is determined in continuous mode in open space, easily cause the cross pollution between various reagents and shadow
Ring the accuracy of measurement result；
(5) dosage more than continuous mode using manual operations, reagent or sample is not bery accurate, and operating process is extremely loaded down with trivial details and multiple
It is miscellaneous, bust is susceptible to, the degree of accuracy of measurement result and precision are poor；
(6) item number × 48/96 person-portion is in the quantity configuration for determining Complete sets of a project reagent and using on, if necessary to survey
Fixed 10 projects, the then configuration of reagent and the use of number must be 10 × 48/96 person-portions, if only a sample needs to determine 10
Different project, it is also desirable to configure the reagent of 10 × 48/96 person-portions, haves the shortcomings that inadequate economical rationality.
2nd, radio immunoassay
Radioimmunoassay method radioactive pollution is big, load procedure is loaded down with trivial details, complex operation, operating time length, measurement result not
The stable, weak point such as the reagent holding time is short, kit operating automation degree is low, necessary instrument is expensive.
3rd, 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）With two kinds of alkaline phosphatase, but there is certain office
Sex-limited, horseradish peroxidase major defect is：Luminol, also can be by H in the case of existing without horseradish peroxidase2O2
Oxidation itself lights, and background is of a relatively high, affects signal to noise ratio, and kinetics is complicated, and influence factor is more, is as a result not sufficiently stable,
The substrate for obtaining sensitivity height and plateau length is not easy.Alkaline phosphatase major defect is：Substrate reach plateau when
Between long, substrate high cost, cause cost of determination high, patient burden's weight.
Acridinium ester compares enzyme-catalyzed chemical luminescence and has detailed advantage, main performance as the direct chemiluminescence of label
：Reaction does not need 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
Element is few, and reagent stability is good, can be simple with two-point calibration, system, exciting liquid low cost, acridinium ester easily and protein bind, and
Photon yield is not reduced after connection.
The content of the invention
At present thyrotrophin receptor antibody determination techniques are suffered from the drawback that：Cost of determination is high, it is low to determine sensitivity,
Determine that the range of linearity is narrow, reappearance is low, can not quantitatively, complex operation etc..
The present invention discloses that a kind of cost of determination is low, sensitivity is high, reappearance precisely in order to overcome the above shortcoming
High, thyrotrophin receptor antibody kit that can be quantitative, simple to operate and preparation method thereof.The method comprises the steps of firstly, preparing changing
Luminescence immunoassay kit is learned, is mainly included：The coated magnetic particle of thyrotropin receptor, rush mouse anti-human IgG antibodies' bag
The acridinium ester of quilt and thyrotrophin receptor antibody calibration product；Then using Full-automatic chemiluminescence immunoassay analysis meter to fixed
Mark product are measured, and draw calibration curve, are built in computer software, test actual sample, and according to sample luminous value sample is calculated
Concentration；Finally performance is carried out to thyrotrophin receptor antibody automatic chemiluminescence immunoassay system（Sensitivity, line
Property, precision, interference）Evaluation.
It is of the invention compared with current technology, with advantages below：
1st, the present invention selects acridinium ester as marker material, and is applied to chemiluminescence immunoassay system, and the luminescence system is
Directly chemiluminescence, compared with traditional enzyme-catalyzed chemical luminescence, the reaction does not need the participation of enzyme, more cost-effective；
2nd, the acridinium ester chemiluminescent immunoassay system that the present invention is selected determines sensitivity height, can reach 0.3IU/L, compares
The sensitivity of other thyrotrophin receptor antibody assay methods at least improves 6 times；
3rd, the acridinium ester chemiluminescent immunoassay system repeatability that the present invention is selected is high, in batch and difference between batch is within 5%,
This is that other chemiluminescence immunoassay systems are unapproachable；
4th, chemiluminescence immunoassay system of the invention has realized the quantitative of sample, soft to testing by built-in calibration curve
Part, only needs test sample to directly obtain the concentration value of sample；
5th, chemiluminescence immunoassay system of the invention has realized that the addition of full-automation, reagent and sample has instrument complete entirely
Into operation is easier, reduces artificial error.
Description of the drawings
Fig. 1 is the thyrotrophin receptor antibody canonical plotting that embodiment 3 is obtained.
Specific embodiment
Embodiment 1：Thyrotrophin receptor antibody chemiluminescence immunoassay kit preparation method
（1）It is prepared by the coated nanometer magnetic bead of thyrotropin receptor：
Take the magnetic particle of 50mg carboxylated（Particle diameter is 0.05-1 μm）Suspension, Magneto separate removes supernatant, uses 0.02 M, pH to be 5.5
MES buffer solutions are resuspended, add the EDC aqueous solution of the 10mg/mL of the new configurations of 0.5-2mL, activated magnetic beads surface carboxyl groups to add 3-5
Be suspended 2-10h under mg thyrotropin receptors, room temperature, Magneto separate, removes supernatant, is 8.0 with the 0.1M pH containing 2% BSA
Tris buffer solutions be resuspended to 1mg/mL, obtain the coated magnetic particle of thyrotropin receptor, every bottle of 5mL packing is stored in 4
It is DEG C standby.
（2）The preparation of the acridinium ester of mouse anti-human IgG antibodies mark：
The mouse anti-human IgG antibodies of 50 μ L 25mg/mL are taken, the carbonate buffer of 150 μ L 0.1-0.2 M pH 9.0-9.5 is added
Liquid, mixes, and the acridinium ester for being subsequently adding 1-2 μ L 5mg/mL is mixed, and lucifuge reaction under room temperature is taken out, with 2mL's after 1-2h
Zeba is centrifuged desalting column desalting processing, is processed with pure water and TBS buffer solutions respectively first in desalination processes, is eventually adding
The acridine ester solution of the mouse anti-human IgG antibodies mark for obtaining, the liquid in collection centrifuge tube to preservation is in control mouse anti-human igg and resists
The acridinium ester of body tag, per bottle of 5 mL packing be stored in 4 DEG C it is standby.
（3）Thyrotrophin receptor antibody calibrates the preparation of product：
Use standard items buffer solution（40 mM Tris-HCl, 0.5% BSA, 1% NaCl, pH 8.0）By thyrotropin receptor
Antibody is configured to concentration for 0.03IU/L, 4.1IU/L, 10.75IU/L, 24.5IU/L, 30.55IU/L, 38.56IU/L, per bottle
0.5mL packing is lyophilized, and 4 DEG C save backup.
（4）The preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 80 μ L mass fractions are 20% is sequentially added2O2), 1.0 grams of sodium azide, 1.5
Gram polysorbas20, shakes up rear lucifuge storage.
(5) preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.6 gram of NaOH, 0.5 gram of PC300,0.5g sodium azide, 1.5 grams of Triton are sequentially added
X-405, shakes up rear lucifuge storage.
Embodiment 2：Thyrotrophin receptor antibody chemiluminescent immunoassay method：
With Full-automatic chemiluminescence immunoassay analysis meter as tools for measurement, method of the present invention pattern is indirect method to the present invention, i.e.,
Instrument sequentially adds the coated magnetic particle of thyrotropin receptor of the sample of 40 μ L after pre-dilution, 50 μ L, reacts 20min
Afterwards, Magneto separate is carried out, the acridinium ester of the mouse anti-human IgG antibodies mark of 50 μ L is added, 20min is reacted, is cleaned again, sequentially added
50 μ L chemiluminescence preexciting liquid, 50 μ L chemiluminescences exciting liquids carry out luminescence-producing reaction, finally record luminous intensity, bent from standard
Line computation goes out the thyrotrophin receptor antibody content of sample.
Embodiment 3：Thyrotrophin receptor antibody chemiluminescence immunoassay kit performance evaluation
Determine curve and see accompanying drawing 1.
The measure of sensitivity：
With reference to CLSI EP17-A file recommendation experimental programs, thyrotrophin receptor antibody chemiluminescence immune assay is calculated
The sensitivity of kit, the sensitivity tried to achieve is 0.3IU/L.
Linear measure：
Concentration is done for 0.03IU/L, 7.72IU/L, 15.41IU/L, 23.1IU/L, 30.79IU/L, 38.48IU/L standard items
Linear analysis, calculates linearly dependent coefficient, r=0.9992, in addition, the kit is surveyed to thyrotrophin receptor antibody sample
The fixed range of linearity is 0.3IU/L-40IU/L.
Precision is determined：
Concentration is taken for two thyrotrophin receptor antibody samples of 5.98IU/L and 31.99IU/L, each sample each concentration
Respectively do 3 it is parallel, be measured with three batches of kits, calculate in kit batch and difference between batch, as a result show in the kit batch
And difference between batch is respectively less than 5%.
Interference is tested：
Taking pooled serum and adding chaff interference respectively includes：Hemoglobin, bilirubin, triglycerides, RF interference determine respectively mixing
Serum and the measured value of pooled serum after various chaff interferences is with the addition of, deviation therebetween is calculated, with ± 10% as acceptable model
Enclose.As a result show, interference reaches files of the NCCLS about standard, can be used for thyrotrophin receptor antibody situation
Accurate evaluation.
Embodiment 4：The Sensitivity comparison experiment point of thyrotrophin receptor antibody chemiluminescence immunoassay kit
It is that the calibration object or Sample dilution of 0IU/L enter to concentration not with chemical luminescent detecting method and traditional enzyme linked immunosorbent assay
Row is determined, and replication 20 times draws the RLU values of 20 measurement results（Relative light unit）, calculate its mean value（M）And standard
Difference（SD）, M+2SD is drawn, the luminous value is substituted into calibration curve and is calculated corresponding concentration value.Using chemical luminescent detecting
The concentration value that method is obtained is 0.3IU/L, relative to traditional IU/L of enzyme linked immunosorbent assay LDL 2, is improve about
6 times.
Claims (10)
1. a kind of thyrotrophin receptor antibody chemiluminescence immunoassay kit, the kit includes：TH
The coated nanometer magnetic microsphere of hormone receptor, chemiluminescent labels, Chemoluminescent substrate, thyrotrophin receptor antibody are fixed
Mark product.
2. kit according to claim 1, it is characterised in that the coated solid phase carrier of the thyrotropin receptor
For magnetic particle.
3. kit according to claim 1, it is characterised in that the coated solid phase carrier of the thyrotropin receptor
For tosyl magnetic bead, particle diameter is 0.05-1 μm of magnetic particle.
4. kit according to claim 1, it is characterised in that the chemiluminescent labels are acridinium ester, acridinium ester
Sulfonamide, acridinium ester toluenesulfonamide, acridinium ester are to methylsulfonamides, acridinium ester trimethyl fluoride sulfonyl amine.
5. kit according to claim 1, it is characterised in that the preferred acridinium ester of the chemiluminescent labels.
6. kit according to claim 1, it is characterised in that the Chemoluminescent substrate is excited including chemiluminescence
Liquid, chemiluminescence preexciting liquid.
7. kit according to claim 1, it is characterised in that the chemiluminescence preexciting liquid is mass fraction
0.005% ~ 0.5% hydrogen peroxide (H2O2) solution, exciting liquid is the NaOH (NaOH) of 0.005mol/L ~ 0.025mol/L
Solution.
8. kit according to claim 1, it is characterised in that the thyrotrophin receptor antibody calibration product are use
Standard items buffer solution by thyrotrophin receptor antibody be configured to concentration for 0.03 IU/L, 4.1 IU/L, 10.75 IU/L,
24.5 IU/L, 30.55 IU/L, 38.56 IU/L, 4 DEG C save backup.
9. kit according to claim 1, it is characterised in that the preparation method of the kit, it is characterised in that bag
Include preparation, preparation, the chemiluminescence of the acridinium ester of mouse anti-human IgG antibodies mark of the coated magnetic particle of thyrotropin receptor
The preparation of product is calibrated in the preparation of substrate solution, thyrotrophin receptor antibody.
10. according to claim 1 and claim 9 kit preparation method, it is characterised in that comprise the following steps：
1）The preparation of the coated magnetic particle of thyrotropin receptor：
Tosyl nanometer magnetic bead suspension is taken, Magneto separate removes supernatant, and MES buffer solutions are resuspended, add the EDC aqueous solution, activation
Magnetic bead surfaces carboxyl, adds thyrotropin receptor, and be suspended 2-10h under room temperature, Magneto separate, removes supernatant, Tris buffer solutions
It is resuspended, obtain the coated magnetic particle of thyrotropin receptor；Optionally, tosyl nanometer magnetic bead it is a diameter of 0.1 μm ~
2.0μm；MES buffer concentrations are 10mM ~ 100mM, pH 5.5 ~ 8.5；
2）The preparation of the acridinium ester of mouse anti-human IgG antibodies mark：
Mouse anti-human IgG antibodies are taken, carbonate buffer solution is added, is mixed, be subsequently adding acridinium ester mixing, lucifuge reaction under room temperature,
Take out after 1-2h, desalting column desalting processing be centrifuged, processed with pure water and TBS buffer solutions respectively first in desalination processes,
The acridine ester solution of the mouse anti-human IgG antibodies mark for obtaining is eventually adding, the liquid collected in centrifuge tube is in control mouse to preservation
The acridinium ester of anti-human IgG antibodies' mark；
3）Thyrotrophin receptor antibody calibrates the preparation of product：
With standard items buffer solution by thyrotrophin receptor antibody be configured to concentration for 0.03 IU/L, 4.1 IU/L, 10.75
IU/L, 24.5 IU/L, 30.55 IU/L, 38.56 IU/L, 4 DEG C save backup；
4）The preparation of chemiluminescence preexciting liquid：
1.0 liters of purified waters are measured, the hydrogen peroxide (H that 0.5 ~ 100uL mass fractions are 20% is sequentially added2O2), 0.5 ~ 5 gram of anti-corrosion
Agent, 0.5 ~ 5 gram of surfactant, shake up rear lucifuge storage；Optionally, preservative be commercialization sodium azide, PC300, surface
Activating agent is polysorbas20, Tween 80, Triton X-100, Triton X-405；
5）The preparation of chemiluminescence exciting liquid：
1.0 liters of purified waters are measured, 0.2 ~ 1 gram of NaOH, 0.5 ~ 5 gram of preservative, 0.5 ~ 5 gram of surfactant is sequentially added,
Shake up rear lucifuge storage；Optionally, preservative be commercialization sodium azide, PC300, surfactant be polysorbas20, Tween 80,
Triton X-100、Triton X-405。
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| CN201620676832 | 2016-06-30 | ||
| CN201620676832X | 2016-06-30 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108548932A (en) * | 2018-05-09 | 2018-09-18 | 南京岚煜生物科技有限公司 | Thyrotropic hormone TSH kits and preparation based on micro-fluidic chip and detection method |
| 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 |
| CN111103432A (en) * | 2020-01-07 | 2020-05-05 | 郑州安图生物工程股份有限公司 | Freeze-drying auxiliary preparation for thyroid stimulating hormone receptor and freeze-drying method |
| 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 |
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| CN101692085A (en) * | 2009-09-11 | 2010-04-07 | 天津医科大学总医院 | Method for quantitatively detecting TRAb plate-type fluorescent enzyme immunity and application thereof |
| CN103954779A (en) * | 2014-03-12 | 2014-07-30 | 长春迪瑞医疗科技股份有限公司 | Testosterone detection reagent based on microparticle chemiluminescence immunoassay technology |
| CN104628843A (en) * | 2015-02-10 | 2015-05-20 | 深圳市新产业生物医学工程股份有限公司 | Thyroid-stimulating hormone receptor protein, gene sequence and kit thereof |
| CN104897901A (en) * | 2015-05-12 | 2015-09-09 | 西安金磁纳米生物技术有限公司 | Goldmag particle-based acridinium ester chemiluminescence immunological detection method of HE4 |
| CN105652018A (en) * | 2016-02-04 | 2016-06-08 | 广州科方生物技术有限公司 | C-peptide quantitative determination kit |
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| CN101692085A (en) * | 2009-09-11 | 2010-04-07 | 天津医科大学总医院 | Method for quantitatively detecting TRAb plate-type fluorescent enzyme immunity and application thereof |
| CN103954779A (en) * | 2014-03-12 | 2014-07-30 | 长春迪瑞医疗科技股份有限公司 | Testosterone detection reagent based on microparticle chemiluminescence immunoassay technology |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108548932A (en) * | 2018-05-09 | 2018-09-18 | 南京岚煜生物科技有限公司 | Thyrotropic hormone TSH kits and preparation based on micro-fluidic chip and detection method |
| 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 |
| CN111103432A (en) * | 2020-01-07 | 2020-05-05 | 郑州安图生物工程股份有限公司 | Freeze-drying auxiliary preparation for thyroid stimulating hormone receptor and freeze-drying method |
| 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 |
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Application publication date: 20170510 |