CN102095882A - Chemiluminescence quantitative detection kit for free triiodothyronine - Google Patents
Chemiluminescence quantitative detection kit for free triiodothyronine Download PDFInfo
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- CN102095882A CN102095882A CN2009102003855A CN200910200385A CN102095882A CN 102095882 A CN102095882 A CN 102095882A CN 2009102003855 A CN2009102003855 A CN 2009102003855A CN 200910200385 A CN200910200385 A CN 200910200385A CN 102095882 A CN102095882 A CN 102095882A
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Abstract
The invention discloses a chemiluminescence quantitative detection kit for free triiodothyronine. The kit comprises a free triiodothyronine detection reaction plate, an enzyme conjugate, a luminescent substrate, a calibrator, a quality control material and washing concentrate, wherein the reaction plate is coated with a free triiodothyronine antibody. The kit can specifically and quantitatively detect the content of the free triiodothyronine 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 simultaneously.
Description
Technical field
The invention belongs to external clinical examination and chemiluminescence immunoassay law technology field, be specifically related to a kind of free triiodothyronine (FT3) chemical luminescent analysis reagent kid and related manufacturing processes and use-pattern of being used for.
Background technology
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).
Thyroid hormone be thyroxine (thyroxine, T4) and trilute (3,5,3 '-triiodothyronine, general designation T3), both are tyrosine and contain the iodine derivant.T3 in the blood plasma more than 99%, T4 all with the reversible combination of plasma proteins, (thyroxine binding globulin, TBG) combination also have partly and albumin, prelbumin combination for main and thyroxine-binding globulin.Only there are the T3 that accounts for blood plasma 0.1%-0.3% and the T4 of 0.02%-0.05% to dissociate, have only free T3, T4 just can enter target cell and play a role.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.So the level of FT3 more can really reflect thyroid functional status than T3.
(Chemiluminescence Immunoassay's chemiluminescence immune assay CLIA) came out in 1977, and first generation chemiluminescence immunoassay kit was succeeded in developing and put on market in 1985.Last century, the mid-1970s Arakawe reported first was carried out EIA enzyme immunoassay with luminous signal, utilized luminous chemical reaction to analyze the ultramicron material, especially for check ultramicron active substance in the clinical immunoassay.At present, this technology carries out the transition to clinical medical conventional sense means from breadboard rare technology.Chemiluminescence immune assay is that chemiluminescence or bioluminescence system are combined with immune response, is used to detect a kind of novel markings immunoassay of micro-antigen or antibody.It is similar with enzyme immunity (EIA) to radio-immunity (RIA) that it detects principle, difference be with luminescent substance as substrate, and directly measure with the luminous intensity of himself.
The chemiluminescent labeling immunoassay is the immune analysis method with direct labelled antigen of chemiluminescence agent or antibody.The chemiluminescent substance that is usually used in mark has the acridinium ester compounds--and acridinium ester (AE) is effective luminous marker, and it is by starting luminescence reagent (NaOH-H
2O
2) effect and luminous, strong direct luminously in a second, finish, luminous for glimmering fast.Also have with tris (bipyridine) ruthenium (Ru (byp)
3 2+) label is luminous substrate, comes the exciting light reaction with another reactant tripropyl amine (TPA) (TPA), produces efficient stable luminous electrochemiluminescence immunoassay continuously thereby carry out redox reaction repeatedly at electrode surface.These immune analysis methods all need the automatic measurer of high precision complexity, also are difficult at home at present realize.
Chemiluminescence enzyme immunoassay is to carry out immune response with enzyme labeling bioactivator (as the antigen or the antibody of enzyme labeling), enzyme on the immune response compound remakes and is used for luminous substrate, luminous under the signal reagent effect, carry out luminescence assays with the luminous signal analyzer.Marker enzyme commonly used at present is horseradish peroxidase (HRP) and alkaline phosphatase (ALP), and they have luminous substrate separately.The substrate that HRP is commonly used is luminol (the amino phthalylhydrazine of 3-, luminol) or derivatives thereof such as different luminol (the amino phthalylhydrazine of 4-) etc., the oxidation reaction of luminol is carried out in alkaline buffer, in the presence of peroxidase and active oxygen, generate the excited state intermediate, luminous when it gets back to ground state, its wavelength is 425nm.Luminous intensity depends on the concentration of enzyme in the enzyme immune reaction thing.If do not use reinforcing agent, the luminous of luminol system is essentially a little less than flash type and the signal, can strengthen luminous signal and can prolong the luminous duration greatly by adding some special reinforcing agent.
Chemiluminescence immune assay had both had the high sensitivity of radio-immunity, and (detection limit can reach 10
-15～10
-18Mol/L), have easy and simple to handle, the characteristics fast of enzyme linked immunological again, be easy to normalizing operation, and do not use harmful reagent in the test.The reagent maintenance phase is long, is applied to biology, medical research and clinical trial diagnostic work, becomes in the on-radiation immunoassay one of the most promising method.But luminous Enzyme Immunoassay Analyzer of external robotics and the kit that is complementary with it adopt closed system to be used more, cost an arm and a leg, and complicated operation is difficult for promoting at home.
Summary of the invention
The present invention combines chemiluminescence with immuno analytical method, a kind of kit that can detection by quantitative free triiodothyronine (FT3) is provided.
Technical solution of the present invention is as follows:
A kind of free triiodothyronine chemical luminescent analysis reagent kid comprises reaction plate, enzyme conjugates, and luminous substrate, calibration object, quality-control product, concentrated cleaning solution, described reaction plate are coated with anti-FT3 antibody.
Described calibration object is with the preparation of the pure product of FT3, and concentration is preferably 0,0.9,2.2,5.0,9.0,19.0pg/ml.
Described quality-control product is made up of quality-control product I and quality-control product II two parts with the pure product preparation of free triiodothyronine, and the concentration of quality-control product I is 0.6-4.5pg/ml, and the concentration of quality-control product II is 5.2-18.5pg/ml.
The concentration of described quality-control product I is preferably 1.37pg/ml, and the concentration of quality-control product II is preferably 7.1pg/ml.、
Described enzyme conjugates is the FT3 analog of HRP mark, described reaction plate material is opaque polystyrene or tygon, described luminous substrate is luminol, different luminol or derivatives thereof, and described concentrated cleaning solution is the neutral buffered liquid of pH7.0-pH8.0.
This kit has open system, can be applied to the chemiluminescence detector device of multiple different company, need not expensive special-purpose assorted instrument, and has easyly, and fast, stable detectability is easy to marketing at home.
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.
The preparation of embodiment 1:FT3 detection by quantitative kit
(1) the antibody sandwich plate of anti-FT3 preparation
A. wrap quilt: get 1mol/L Na
2HPO
477.4ml and 1mol/L NaH
2PO
422.6ml mixing, add deionized water be settled to 1000ml 10 times of coating buffers, face with ten times of preceding dilutions, add an amount of anti-FT3 monoclonal antibody (purchase is from fitzgerald) mixing, join then in the microwell plate plate hole, 100 μ l/ holes, 4 ℃ 16 hours;
B. sealing: discard coating buffer, on thieving paper paper, pat dry, add and contain 3%BSA and 0.05% antiseptic (Proclin
TM300) phosphate buffer (pH 7.4), 200 μ l/ holes, 37 ℃ 2 hours;
C. envelope: discard confining liquid, pat dry on thieving paper, took out in vacuum drying chamber under the room temperature 5 hours, carry out vacuum sealing bag immediately, check to have or not gas leakage, if any envelope again, labelled back is in 2-8 ℃ of preservation.
(2) preparation of enzyme labeling thing
With containing 2.5%BSA, 1.5% skimmed milk power and 0.05% antiseptic (Proclin
TM300) phosphate buffer (pH 7.4) preparation buffer system.
FT3 analog (buying from sigma-aldrich) is crosslinked with periodates oxidizing process and horseradish peroxidase.
(3) preparation of FT3 calibration object
With containing 3%BSA and 0.05% antiseptic (Proclin
TM300) phosphate buffer (pH 7.4) preparation buffer system.
With the pure product of FT3 preparation calibration object working fluid (the pure product of FT3 are bought from fitzgerald), be respectively 0,0.9,2.2,5.0,9.0,19.0pg/ml totally 6 working concentrations.
(4) preparation of FT3 quality-control product
Calf serum mixes with deionized water at 6: 4, and adds 0.05% antiseptic (Proclin
TM300), be mixed with the quality-control product damping fluid.
Prepare quality-control product working fluid (the pure product of FT3 are bought from fitzgerald) packing 1.37,7.1pg/ml with the pure product of FT3.
(5) preparation of chemical luminous substrate liquid
Use PIERCE company
ELISA Femto Maximum Sensitivity Substrate is as supporting detection liquid.Each 1 bottle of A liquid, B liquid.
The preparation of (6) 20 * concentrated cleaning solutions
Get NaCl 170g Tween-20 10ml and add water and be settled to 1L, face with 20 times of preceding dilutions.
(7) semi-manufacture and finished product are formed
Be semi-manufacture after the packing of above-mentioned steps products obtained therefrom.Inspect by random samples and be assembled into the kit finished product after qualified, finished product also need be inspected by random samples, just can dispatch from the factory after qualified.
The using method of embodiment 2:FT3 chemical luminescent analysis reagent kid
1. reagent and sample are prepared
(1) reagent is prepared
A. kit was put room temperature (18-26 ℃) balance 20 minutes.
B. from kit, take out concentrated cleaning solution, add with 1: 20 dilution back of fresh purified water and wash in the bottle for handling liquid toilet or cosmetic substance of plate machine.
(2) sample is prepared
Before using qualified serum to be measured or blood plasma were put room temperature (18-26 ℃) balance 20 minutes.
2. operation steps
A. taking out required FT3 antibody sandwich microwell plate places on the micropore frame
B. add calibration object 1-6, quality-control product 1-2 and sample to be tested, every hole 50 μ l.
C. add enzyme labeling thing 100 μ l/ holes.
D. softly shake mixing.
E. incubated at room is 1 hour.
F. discard waste liquid in the hole.
Hand washing: discard liquid in the hole, cleansing solution is filled with each hole, was left standstill 5 seconds, dries, and pats dry after repeating 5 times; Washing the plate machine washing washs: every hole 350 μ l, repeat 4 times, and pat dry after the washing.
G. add each 50 μ l of the every hole of luminous substrate A, B, fully mixing adds detected signal value in the Chemiluminescence Apparatus at once.
3. the calculating of experimental result
Can adopt graphing method or computing method to carry out calculating as a result
Graphing method: the lg value with the standard items antigenic content is horizontal ordinate (X), with its corresponding signal value is that ordinate (Y) is drawn out typical curve, the signal value that records according to the detection sample is found corresponding antigen content lg value on typical curve then, calculates antigenic content again.
Computing method: the lg value with the standard items antigenic content is independent variable (X), is dependent variable (Y) with its corresponding signal value, obtains regression equation, brings the signal value of sample to be measured into regression equation, tries to achieve corresponding antigen content.
Claims (5)
1. a free triiodothyronine chemical luminescent analysis reagent kid is characterized in that, comprises reaction plate, enzyme conjugates, and luminous substrate, calibration object, quality-control product, concentrated cleaning solution, described reaction plate is coated with free triiodothyronine antibody.
2. kit as claimed in claim 1 is characterized in that, described calibration object is with the preparation of the pure product of free triiodothyronine, and concentration is 0,0.9,2.2,5.0,9.0,19.0pg/ml.
3. kit as claimed in claim 1, it is characterized in that described quality-control product is made up of quality-control product I and quality-control product II two parts with the pure product preparation of free triiodothyronine, the concentration of quality-control product I is 0.6-4.5pg/ml, and the concentration of quality-control product II is 5.2-18.5pg/ml.
4. kit as claimed in claim 3 is characterized in that, the concentration of described quality-control product I is 1.37pg/ml, and the concentration of quality-control product II is 7.1pg/ml.
5. kit as claimed in claim 1, it is characterized in that, described enzyme conjugates is the free triiodothyronine analog of HRP mark, described reaction plate material is opaque polystyrene or tygon, described luminous substrate is luminol, different luminol or derivatives thereof, and described concentrated cleaning solution is the neutral buffered liquid of pH7.0-pH8.0.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102003855A CN102095882A (en) | 2009-12-11 | 2009-12-11 | Chemiluminescence quantitative detection kit for free triiodothyronine |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009102003855A CN102095882A (en) | 2009-12-11 | 2009-12-11 | Chemiluminescence quantitative detection kit for free triiodothyronine |
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| CN102095882A true CN102095882A (en) | 2011-06-15 |
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| CN2009102003855A Pending CN102095882A (en) | 2009-12-11 | 2009-12-11 | Chemiluminescence quantitative detection kit for free triiodothyronine |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102749455A (en) * | 2012-06-26 | 2012-10-24 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemilumineseent quantitative immunoassay of angiotensin II and preparation method thereof |
| CN103499566A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | T3 chemiluminescent in-vitro diagnostic kit and application method thereof |
| CN103499565A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | FT4 in-vitro diagnostic kit and application 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 |
| CN103499697A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | FT3 in-vitro diagnostic kit and application method thereof |
-
2009
- 2009-12-11 CN CN2009102003855A patent/CN102095882A/en active Pending
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102749455A (en) * | 2012-06-26 | 2012-10-24 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemilumineseent quantitative immunoassay of angiotensin II and preparation method thereof |
| CN102749455B (en) * | 2012-06-26 | 2014-12-10 | 博奥赛斯（天津）生物科技有限公司 | Kit for chemilumineseent quantitative immunoassay of angiotensin II and preparation method thereof |
| CN103499566A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | T3 chemiluminescent in-vitro diagnostic kit and application method thereof |
| CN103499565A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | FT4 in-vitro diagnostic kit and application 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 |
| CN103499697A (en) * | 2013-09-06 | 2014-01-08 | 上海裕隆医学检验所股份有限公司 | FT3 in-vitro diagnostic kit and application method thereof |
| CN103499697B (en) * | 2013-09-06 | 2015-12-23 | 上海裕隆医学检验所股份有限公司 | FT3 external diagnosis reagent case and using method thereof |
| CN103499566B (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 T3 chemiluminescence and using method thereof |
| 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 |
| CN103499565B (en) * | 2013-09-06 | 2016-02-24 | 上海裕隆医学检验所股份有限公司 | FT4 external diagnosis reagent case and using method thereof |
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Application publication date: 20110615 |