CN102095869A - Thyroid function detection protein chip and kit thereof - Google Patents
Thyroid function detection protein chip and kit thereof Download PDFInfo
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- CN102095869A CN102095869A CN2009102003836A CN200910200383A CN102095869A CN 102095869 A CN102095869 A CN 102095869A CN 2009102003836 A CN2009102003836 A CN 2009102003836A CN 200910200383 A CN200910200383 A CN 200910200383A CN 102095869 A CN102095869 A CN 102095869A
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Abstract
The invention discloses a thyroid function detection protein chip and a kit thereof. The kit comprises the thyroid function detection protein chip, wherein the chip is coated with a free triiodothyronine (FT3) antibody, a free thyroxine (FT4) antibody, a triiodothyronine (T3) antibody, a thyroxine (T4) antibody and a thyroid stimulating hormone (TSH) antibody. The kit can quantitatively detect the content of FT3, FT4, T3, T4 and TSH in patient serum, and is used for the auxiliary diagnosis of thyroid dysfunction diseases. Compared with the conventional enzyme linked immunosorbent assay (ELISA) technology, the chemiluminescence immunoassay keeps high specificity of the ELISA technology, stability and reliability of a detection result, and convenience of operation, and can improve detection sensitivity; and various indexes can be detected simultaneously, so time and cost are greatly saved.
Description
Technical field
The invention belongs to external clinical examination and chemiluminescence immunoassay law technology field, be specifically related to a kind of thyroid function that is used for and detect protein chip and kit thereof.
Background technology
Thyroid gland is the incretory of human body maximum, can secrete thyroxine (T4), trilute hormones such as (T3).These hormones are subjected to the adjusting by the thyroid-stimulating hormone (TSH) of adenohypophysis secretion, and to protein, fat, sugar, the vitamin of human body, growing of the metabolism of water and salt and human body plays crucial effect.Dysthyroid is the most common person in the endocrine system disease, and is still easily failed to pinpoint a disease in diagnosis so far.According to finding in the generaI investigation of the U.S. to 11 city 4.6 ten thousand people, there are the various dysthyroids of not diagnosed out in 11% the people of having an appointment.Dysthyroid can be divided into hyperthyroidism (hyperthyroidism is called for short hyperthyroidism) and hypothyroidism (hypothyroidism is called for short first and subtracts).
Antithyrotropic hormone (TSH) is the major hormone by the adjusting thyroid function of the S emiocytosis in the adenohypophysis basocyte, half life period 60min.It is a kind of glycoprotein, and molecular weight is 28000.Adenohypophysis TSH is pulsed and discharges, and once fluctuation appears in every 2h～4h, and changing diurnal periodicity is that early morning is high and low in the afternoon.It can promote the iodate of tyrosine and the condensation of iodotyrosine, promotes intracellular rna and protein to comprise synthesizing of thyroglobulin etc.Excessive thyroxine is that TSH secretes the most important physiology inhibition factor, and TSH concentration reduces strong prompting thyroid function and takes place hyperfunction.Because making it not only can distinguish thyroid function, the raising of TSH detection technique sensitivity lowers, the diagnosis hyperthyroidism, subclinical type thyroid function lowers or hyperthyroidism, even can estimate outpatient service and non-thyroid disease inpatient thyroid function situation.
Trilute (T3) molecular weight is 651, and about 65% is directly produced by thyroid gland, and 35%T4 takes off iodine in peripheral tissues and forms.In peripheral blood, 99.6% combines with thyroid binding globulin (TBG).T3 is the strongest thyroid hormone of known present biologically active, its biologically active is 3 times～5 times of T4, and the T3 that generally will not combine with TBG is called free T3 clinically, i.e. free triiodothyronine (FT3), have only free T3 to have metabolic activity, unconjugated then do not have.FT3 can be by cell membrane and receptors bind performance physiological effect, thus it be thyroid hormone generation physiological effect real active part, can reflect thyroid functional status and other influences more definitely to function of human body.In most hyperthyroidism cases, the rising of serum T 3 and T4 parallels, and during the T3 toxication only T3 raise, T4 is in normal range.Hyperthyroidism patient is in carrying out radioactivity or drug therapy process, and serum T 4 is reduced to normally, and T3 still is higher than normally, recovers normal until clinical thyroid function, and serum T 3 is just reduced to normally, so T3 can be used as the reliability index of judging curative effect again.In some disease, during as cirrhosis, T3 may reduce, but as long as thyroid function is normal, T4 is then in normal range.Therefore, as the index of estimating hypothyroidism, it is reliable not as T4 to measure T3.
Thyroxine (T4) is the thyroid hormone of and secretion synthetic by follicular epithelial cell, and the half life period is 7d, and its structure is made up of a tyrosine residue and a phenol ring, iodine atoms more than only being with the difference of T3.T4 go into behind the blood about 99.6% with combine protein combination, in conjunction with being reversible equilibrium state between T4 and the free thyroxine (FT4), having only free T4 just can enter target cell and receptors bind and bring into play its physiological function.Hyperthyroidism, iatrogenic or toxic hyperthyroidism T4 and FT4 raise; Subacute thyroiditis or chronic lymphocytic thyroiditis T4, FT4 one property crossed rising; TBG raises due to a variety of causes, and as estrogen, hepatitis etc. also make T4 raise but FT4 does not raise; Mental disease and some non-thyroid disease patients, only a few people T4 can occur and raise, but there is no the hyperthyroidism shape, and it is normal that protopathy is alleviated back T4 recovery.When thyroid function lowered, T4 reduced, and the reduction of T4 is prior to the reduction of T3, and after thyroid gland shortage or congenital dysplasia and thyroid gland were cut entirely, serum T T4 reduced.In the low case of first due to a variety of causes, FT4 all significantly is lower than normal range, but after pregnant 36 weeks, FT4 is the normal physiological reduction.
(Chemiluminescence Immunoassay CLIA) is the main means that detect the thyroid function index of correlation on the present home market for enzyme linked immunosorbent assay (ELISA) and chemiluminescence immune assay.But because ELISA method complex operation, consuming time longer, and sensing range and sensitivity progressively are in the stage that is eliminated at present not as CLIA.Though the CLIA testing result is accurate, highly sensitive, but because domestic present technology is not mature enough, main external electrochemiluminescence (the electrochemiluminescence immunoassay that still uses, robotization immune detection instrument such as ECLIA), its operation system sealing, must be equipped with expensive instrument and special-purpose kit, cost is high and can only carry out single index detection (can detect many index simultaneously when general hospital is detected thyroid function), is difficult at home promoting.
Therefore need on the market that a kind of cost is lower, sensing range is wide, can save detection time, improve the thyroid function class testing product of detection efficiency.
Summary of the invention
The present invention will provide a kind of protein chip and kit thereof that thyroid function detects that be specifically designed to.Solve single, the detection cost height of thyroid function testing product detectability in the market, and do not have integrated shortcoming.
Technical scheme of the present invention is as follows: provide a kind of thyroid function to detect protein chip, comprise substrate and the reacting hole that is positioned on the substrate, solid phase carrier is arranged at the bottom of described reacting hole, and is coated with the microarray of two or more antibody in free triiodothyronine antibody, trip thyroxine antibody, trilute antibody, thyroxine antibody, the thyrotropic hormone antibody on solid phase carrier.
Described reacting hole is made up of two classes, and a class reacting hole is coated with free triiodothyronine antibody, trip thyroxine antibody, and another kind of reacting hole is coated with trilute antibody, thyroxine antibody, thyrotropic hormone antibody.
Described two class reacting holes are arranged on the substrate by 1: 1 quantity.
Described all kinds of reacting hole quantity is 4-30.
Described free triiodothyronine antibody, trip thyroxine antibody, trilute antibody, thyroxine antibody, thyrotropic hormone antibody respectively have 2-8 point of sample on solid phase carrier, also be coated with a location reference point in each reacting hole.
Described solid phase carrier is NC film or PDVF film.
The present invention also provides a kind of thyroid function to detect protein chip kit, and it comprises that the described thyroid function of claim 1 detects protein chip.
Also comprise: enzyme mark working fluid and detection liquid, negative control, positive reference substance.
Described enzyme mark working fluid is made up of antibody, antigen or the antigen analogues of HRP mark, and described detection liquid is made up of shiner and two kinds of reagent of hydrogen peroxide.
Described shiner is luminol, different luminol or derivatives thereof.
The present invention with present hospital the thyroid function project of normal detection concentrate on the chip, can be simultaneously 5 indexs of same serum be detected.This invention can big time saver and resource, improves detection efficiency, reduces and detect cost.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor LaboratoryPress, 1989) condition described in, or the condition of advising according to manufacturer.
Embodiment 1: a kind of thyroid function detects the preparation of protein chip
Its step mainly is: (1) prepares the matrix nitrocellulose filter; (2) design chips is used FT3 antibody and the FT4 antibody antibody as the bag quilt in the odd column reacting hole, use T3 antibody, T4 antibody and the TSH antibody antibody as the bag quilt in the even column reacting hole; (3) spotting needle from 384 orifice plate sucking-off antibody speckings on nitrocellulose filter; (4) assembling nitrocellulose filter, and seal, dry, packing, preserve.
The above-mentioned methods of making protein chips that is used to detect thyroid function specifically may further comprise the steps:
1. thyroid function detects determining of related antigen antibody etc.:
Be used to prepare thyroid function and detect the purchase of protein chip detection index antigen from Fitzgerald;
FT3, FT4 analog are bought from Sigma-aldrich;
The detection antibody that is adopted is bought the life from Meridian.
2. point sample prepares the film chip that tool detects the index coating
With Gesim point sample instrument specking, on nitrocellulose filter, latticed form is 10 * 5 rectangular arrays to spotting needle from 384 orifice plate sucking-off antibody speckings, and when spot diameter is 0.1mm, the some horizontal spacing is 0.6mm, and the size of antibody array is 6mm * 5mm; Pad pasting behind the point sample, sealing; At last with chip drying, packing back in 4 ℃ of preservations.
3. protein chip assembling
With having the utensil of separating conversion zone conversion zone is fixed.Assembling mode is seen ZL032298900.
Embodiment 2: the preparation of kit
A kind of thyroid function detects protein chip kit, detects protein chip by thyroid function, standard items, and quality-control product, the sample dilution, enzyme mark working fluid, concentrated cleaning solution detects liquid and forms; Each several part is bottled, is fixed in the packing box with the papery template, does not limit modes of emplacement, supporting use during detection.Below be the preparation method of each several part:
1. thyroid function detection protein chip is 6 * 8 orifice plates, and as the sheet base, assembling mode is seen ZL032298900 by nitrocellulose filter.
2. standard items provide 12 pipes, every pipe 0.2ml.
With containing 3%BSA and 0.05% antiseptic (Proclin
TM300) phosphate buffer (pH 7.4) configuration, wherein the final concentration of T3, T4, FT3, FT4 and TSH standard items sees the following form:
3. quality-control product provides 4 bottles, every bottle of 0.2ml.
Quality-control product is divided into 4 concentration, is respectively quality-control product I, quality-control product II, quality-control product III, quality-control product IV.These four quality-control products are for containing the not synantigen mixed liquor of variable concentrations respectively.
Quality-control product is with containing 3%BSA and 0.05% antiseptic (Proclin
TM300) phosphate buffer (pH 7.4) preparation, following table shows the final concentration and the Quality Control boundary of T3, T4, FT3, FT4 and TSH quality-control product.
Above-mentioned quality-control product Quality Control boundary assay method is as follows:
Respectively by 2 experimenters according to standard test operation detection quality-control product, repeat 4 times/plate, every day, everyone repeated to survey 2 chip blocks, duplicate detection 10 days is calculated the average and the standard deviation of quality-control product respectively, the Quality Control boundary is X ± 15%.
4. sample dilution
By 3% BSA and 0.05% antiseptic (Proclin
TM300) phosphate buffer (pH 7.4) is formulated.Provide 1 bottle, 9ml.Be used for dilute serum sample (volume ratio 1: 1).
5. enzyme is marked working fluid
By 3%BSA and 0.05% antiseptic (Proclin
TM300) phosphate buffer (pH 7.4) wiring solution-forming, enzyme mark working fluid I contains T3, T4 antigen and the TSH monoclonal antibody of HRP mark; Enzyme mark working fluid II contains FT3, the FT4 analog of HRP mark, more than two kinds of enzymes mark working fluids all contain and detect antibody HRP-goat anti-rabbit igg.Provide 1.5ml, each 1 bottle.
6. concentrated cleaning solution (10 *)
Be phosphate buffer.Form by following method configuration: potassium dihydrogen phosphate 6.8g, sodium hydrogen phosphate 7.1g, TWEEN-2010ml is dissolved in the 1000ml purified water.It is pure that each chemical substance is analysis.Provide 50ml, 1 bottle.Dilute mixing before using with the 450ml deionized water.
7. detection liquid
Use PIERCE company
ELISA Femto Maximum Sensitivity Substrate is as supporting detection liquid.Each 1 bottle of A liquid, B liquid, every bottle of 0.6ml.
Embodiment 3: the application of protein chip and kit and detection
The application that is used to detect the protein chip of thyroid function of the present invention mainly adopts competition law and double antibodies sandwich method to detect index of correlation in the human blood.Method is that human serum is added on chip surface, adds enzyme mark working fluid again, directly reacts; Add to detect behind the liquid 1 minute, signal is also collected in scanning, and the concentration of antigen is linear in the signal intensity of sample point and the serum.
In the above-mentioned application that is used for detecting the protein chip kit of thyroid function, the concrete using method of its antigen-antibody reaction and detection is as follows:
(1) chip to specking antibody, positive control, negative control seals with 3%BSA solution, room temperature 2 hours, the non-specific site of sealing substrate surface;
(2) behind the drying confining liquid, room temperature was placed 4 hours, carried out drying;
(3) at the conversion zone of chip, add human serum sample, standard items, quality-control product; Every hole adds 100ul;
(4) add enzyme mark working fluid I at the radix row, add enzyme mark working fluid II, the every hole of 50ul/ at even column.Place 37 ℃ in isothermal reaction instrument, oscillating reactions 1 hour makes abundant reaction;
(5) after reaction finished, washing was 5 times on the reaction instrument, and the washing lotion amount is set to every hole 350ul.
(6) with PIERCE company
The matching used detection liquid A liquid of ELISA Femto Maximum Sensitivity Substrate and B liquid mix at 1: 1, and on each conversion zone, every array adds 60ul, carries out chemiluminescence on the adding chip;
(7) carry out chemiluminescent scanning and collect signal with the CCD detector, according to scanning result display analysis testing result.
Claims (10)
1. a thyroid function detects protein chip, comprise substrate and the reacting hole that is positioned on the substrate, it is characterized in that: solid phase carrier is arranged at the bottom of described reacting hole, and is coated with the microarray of two or more antibody in free triiodothyronine antibody, trip thyroxine antibody, trilute antibody, thyroxine antibody, the thyrotropic hormone antibody on solid phase carrier.
2. reaction plate as claimed in claim 1, it is characterized in that, described reacting hole is made up of two classes, one class reacting hole is coated with free triiodothyronine antibody, trip thyroxine antibody, and another kind of reacting hole is coated with trilute antibody, thyroxine antibody, thyrotropic hormone antibody.
3. reaction plate as claimed in claim 2 is characterized in that, described two class reacting holes are arranged on the substrate by 1: 1 quantity.
4. reaction plate as claimed in claim 3 is characterized in that, described all kinds of reacting hole quantity are 4-30.
5. reaction plate as claimed in claim 1 or 2, it is characterized in that, described free triiodothyronine antibody, trip thyroxine antibody, trilute antibody, thyroxine antibody, thyrotropic hormone antibody respectively have 2-8 point of sample on solid phase carrier, also be coated with a location reference point in each reacting hole.
6. reaction plate as claimed in claim 1 is characterized in that, described solid phase carrier is NC film or PDVF film.
7. a thyroid function detects protein chip kit, it is characterized in that, it comprises that the described thyroid function of claim 1 detects protein chip.
8. kit as claimed in claim 7 is characterized in that, also comprises: enzyme mark working fluid and detection liquid, negative control, positive reference substance.
9. kit as claimed in claim 8 is characterized in that, described enzyme mark working fluid is made up of antibody, antigen or the antigen analogues of HRP mark, and described detection liquid is made up of shiner and two kinds of reagent of hydrogen peroxide.
10. kit as claimed in claim 9 is characterized in that, described shiner is luminol, different luminol or derivatives thereof.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102003836A CN102095869A (en) | 2009-12-11 | 2009-12-11 | Thyroid function detection protein chip and kit thereof |
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|---|---|---|---|
| CN2009102003836A CN102095869A (en) | 2009-12-11 | 2009-12-11 | Thyroid function detection protein chip and kit thereof |
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| CN102095869A true CN102095869A (en) | 2011-06-15 |
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| CN2009102003836A Pending CN102095869A (en) | 2009-12-11 | 2009-12-11 | Thyroid function detection protein chip and kit thereof |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102338801A (en) * | 2011-08-05 | 2012-02-01 | 张灿 | High-sensitivity immunochip detection system and application method thereof |
| WO2013132461A1 (en) * | 2012-03-09 | 2013-09-12 | Basf Se | Means and methods for assessing hyperthyroidism |
| CN103499564A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | T4 chemiluminescent in-vitro diagnostic kit and application method thereof |
| CN103630695A (en) * | 2013-11-20 | 2014-03-12 | 中国人民解放军南京军区南京总医院 | Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum |
| CN104040338A (en) * | 2011-12-05 | 2014-09-10 | 奎斯特诊断投资公司 | Methods For Detecting Reverse Triiodothyronine By Mass Spectrometry |
| CN108267601A (en) * | 2018-01-19 | 2018-07-10 | 中国医学科学院阜外医院 | The system and kit of adverse events after a kind of prediction heart infarction |
| CN108387750A (en) * | 2018-02-08 | 2018-08-10 | 江苏三联生物工程有限公司 | A kind of chip and preparation method thereof of joint-detection F-T3, F-T4 and TSH |
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
| CN110286237A (en) * | 2019-06-29 | 2019-09-27 | 山东博科诊断科技有限公司 | Five chemiluminescence detection kits of first function |
| CN112904025A (en) * | 2021-02-02 | 2021-06-04 | 广东云曌医疗科技有限公司 | Chemiluminescence method-based determination kit and determination method thereof |
| WO2023102771A1 (en) * | 2021-12-08 | 2023-06-15 | Roche Diagnostics Gmbh | Test result level based analysis |
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2009
- 2009-12-11 CN CN2009102003836A patent/CN102095869A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102338801B (en) * | 2011-08-05 | 2013-11-20 | 嘉兴艾锐生物科技有限公司 | High-sensitivity immunochip detection system and application method thereof |
| CN102338801A (en) * | 2011-08-05 | 2012-02-01 | 张灿 | High-sensitivity immunochip detection system and application method thereof |
| CN104040338A (en) * | 2011-12-05 | 2014-09-10 | 奎斯特诊断投资公司 | Methods For Detecting Reverse Triiodothyronine By Mass Spectrometry |
| WO2013132461A1 (en) * | 2012-03-09 | 2013-09-12 | Basf Se | Means and methods for assessing hyperthyroidism |
| CN103499564B (en) * | 2013-09-06 | 2016-02-24 | 上海裕隆医学检验所股份有限公司 | The external diagnostic kit of T4 chemiluminescence and using method thereof |
| CN103499564A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | T4 chemiluminescent in-vitro diagnostic kit and application method thereof |
| CN103630695A (en) * | 2013-11-20 | 2014-03-12 | 中国人民解放军南京军区南京总医院 | Chemiluminiscence imaging immunoassay method for simultaneously measuring oxidized lipoprotein (a) and oxidized low-density lipoprotein of human serum |
| CN108267601A (en) * | 2018-01-19 | 2018-07-10 | 中国医学科学院阜外医院 | The system and kit of adverse events after a kind of prediction heart infarction |
| CN108387750A (en) * | 2018-02-08 | 2018-08-10 | 江苏三联生物工程有限公司 | A kind of chip and preparation method thereof of joint-detection F-T3, F-T4 and TSH |
| CN109799355A (en) * | 2019-02-06 | 2019-05-24 | 吉林双正生物工程有限公司 | First function five fluorescent microsphere joint-detection device and preparation method thereof |
| CN110286237A (en) * | 2019-06-29 | 2019-09-27 | 山东博科诊断科技有限公司 | Five chemiluminescence detection kits of first function |
| CN112904025A (en) * | 2021-02-02 | 2021-06-04 | 广东云曌医疗科技有限公司 | Chemiluminescence method-based determination kit and determination method thereof |
| WO2023102771A1 (en) * | 2021-12-08 | 2023-06-15 | Roche Diagnostics Gmbh | Test result level based analysis |
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Application publication date: 20110615 |