CN1924580A - Chemical luminescent analysis reagent kid for quantitatively detecting trilute - Google Patents
Chemical luminescent analysis reagent kid for quantitatively detecting trilute Download PDFInfo
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- CN1924580A CN1924580A CN 200510017941 CN200510017941A CN1924580A CN 1924580 A CN1924580 A CN 1924580A CN 200510017941 CN200510017941 CN 200510017941 CN 200510017941 A CN200510017941 A CN 200510017941A CN 1924580 A CN1924580 A CN 1924580A
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- trilute
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Abstract
This invention relates to quantitatively triiodothyronine chemical light analysis agent box, which belongs to clinical blood immune testing technique field and comprises the following parts: non-transparent polyphony lacetylene board with the gland antigen, prostate antigen series standard product, enzyme mark gland specific antigen, lighting bottom object A liquid composed of effective light agent, compound strengthening agent and amino acid and B liquid composed of amino acid oxidase and stabilizer.
Description
Technical field
The invention belongs to the clinical blood technical field of immunoassay, particularly a kind of kit that detects trilute (T3) content with the enzyme-catalyzed chemical luminescence standard measure.
Background technology
Human thyroglobulin is an ingredient important in the internal system, and thyroid hormone participates in many important physical reactions.They grow at human body, cellular metabolism and hormone in vivo balance, and the growth of keeping metabolism, skeletal muscle and each tract is all played important regulatory role.Thyroxine in the blood circulation (T4) and 3,5,3 '-thyronine (T3), the overwhelming majority combines with plasma proteins-thyroid binding globulin (TBG).T3 concentration is far below T4, but it has stronger physiologically active, so the concentration of trilute (T3) is to estimate the important parameter of thyroid function in the serum.It is an important means that T3 detects in the diagnosis thyroid disease, and it can find that many T4 are normal and TG companion hyperthyroidism patient that T3 increases.T3 raises and T4 is normal often through the tendency of treatment patient hyperthyroidism recurrence, though T4 is lower than normally, and the T3 value normally, shows that patient's thyroid function has recovered normal.
T3 detects the monitoring that can be used for treating phase hyperthyroidism patient and stop to carry out antithyroid drug treatment patient, and it is for differentiating that thyroid function is normal and the hyperthyroidism state is especially valuable.Except that hyperthyroidism, in women's pregnancy period and oral contraceptive and estrin treatment phase, the T3 level also can raise, and the TBG level is similar to the T4 level to a certain extent.Equally, reduce TBG concentration, the T3 level reduces, yet these T3 levels change, can not be as the correct reflection of thyroid function state.
The correct T3 level that detects is judged and to help the doctor to get rid of non-thyroid disease all significant diagnosis, the curative effect of thyroid disease.Detecting at present that T3 institute generally adopts, is the radioimmunoassay method that grows up from the sixties mostly still.Because it must use radioactively labelled substance, the checkout equipment complexity must be measured testing result with special radioactivity seeker.Simultaneously, operating personnel are also existed radioactive contamination and injury, need special protection and washer.In addition, because of the radioactive nuclide moment of employed radioactively labelled substance is all decaying, its retention period is not long, has brought inconvenience to use.
Summary of the invention
The object of the invention is to provide a kind of trilute chemical luminescent analysis reagent kid, and it is little, highly sensitive to utilize this kit to detect pollution.
For reaching above-mentioned purpose, the present invention adopts following technical scheme: detection by quantitative trilute chemiluminescence analysis kit, mainly form by the opaque polystyrene board that is coated with anti-trilute antibody, trilute series standard product, enzyme labeling trilute, luminous substrate A liquid and luminous substrate B liquid, luminous substrate A liquid is made into by efficient luminous agent, composite fortifier and amino acid, and luminous substrate B liquid is made into by amino acid oxidase and stabilizing agent.
The opaque polystyrene board that is coated with anti-trilute antibody is for being coated with the opaque polystyrene board of anti-trilute monoclonal antibody; Trilute series standard product are that matrix, the pure product of adding trilute are formulated to go hormone serum or analysis buffer; The enzyme labeling trilute is the trilute of horseradish peroxidase-labeled, available trilute enzyme diluted before using; Luminous substrate is a HRP-luminol luminescent system.
The opaque polystyrene board that is coated with anti-trilute antibody is the opaque polystyrene board that is coated with the anti-trilute antibody of rabbit; Analysis buffer is formed PH7.4 by sodium chloride and 3% bovine serum albumin(BSA) of 0.01M sodium hydrogen phosphate-sodium dihydrogen phosphate, 0.05M; Trilute enzyme dilution is made up of phosphate buffer and 1% bovine serum albumin(BSA), contains 0.8 ‰ ANS; The working concentration of horseradish peroxidase-labeled trilute is 1: 500000-1000000.
The opaque polystyrene board that is coated with the anti-trilute antibody of rabbit adopts the anti-method of bridge, and excessive bag is wrapped by anti-trilute monoclonal antibody more indirectly by sheep anti-mouse igg earlier; Luminous substrate A liquid is made up of following ingredients: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M; Luminous substrate B liquid is made up of following ingredients: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA 0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M.
Among the present invention, the opaque polystyrene board that is coated with anti-trilute antibody can adopt the opaque and white microwell plate in 48 holes or 96 holes, is reaction plate.The T3 monoclonal antibody that used antibody adopts conventional quadroma technology to prepare, after adopting sad-ammonium sulfate salting-out process that the antibody for preparing is carried out purifying, carry out Screening and Identification again, it is higher and T4, the little monoclonal antibody of T3 cross reaction be used for detecting to select affinity costant, standby in-20 ℃ of preservations.Used T3 series standard product, can spend hormone serum is matrix, the pure product of T3 that adopt SIGMA to buy are as the criterion to dilute with the national standard product and form, and are serial dried frozen aquatic products, redissolve with distilled water before using.Used enzyme labeling T3 adopts carbodiimide (EDC) method to carry out, and being about to the carboxyl of T3 molecule and the amino of horseradish peroxidase (HRP) molecule is amide compound through the effect condensation of EDC, and dialysis is removed unlabelled free T3 and promptly got T3 enzyme labeling thing.In the good T3-HRP solution of mark, it is standby in-20 ℃ of preservations that geometric ratio adds glycerine.Test shows that the working concentration that used enzyme mark T3 uses in the detectable is 1: 500000-1000000.The luminous substrate system adopts the efficient stable enzyme-catalyzed chemical luminescence substrate system, and A liquid is: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M, and pH 9.4; B liquid: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA 0.5mM, vitamin C 0.12Mm, acetate-acetate buffer 0.2M, pH 6.5.
The chemiluminescence immune analysis method that the present invention adopts is to utilize the horseradish peroxidase enzyme catalytic luminous substrate, and then luminous substrate generation chemical reaction and discharge lot of energy produces the excited state intermediate.This excited state intermediate when it gets back to stable ground state, can be launched photon simultaneously, utilizes the luminous signal surveying instrument can the measuring light quantum yield, and the amount of the test substance in this quantum yield of luminscence and the sample is directly proportional.Can set up the content of test substance in typical curve and the calculation sample thus.
The present invention adopts the enzyme-catalyzed chemical luminescence method to set up T3 detection by quantitative kit, adopts the method for wrapping the antibody of being limited the quantity of indirectly, can save the consumption of specific antibody (anti-T3 antibody) on the one hand, has increased bag on the other hand by the homogeneity of plate.And, detection signal is strengthened greatly owing to adopted chemiluminescent method, making as a result, reappearance improves greatly.
The use running program of detection kit of the present invention is as follows:
(1) prepares before the experiment
1, all reagent are returned to room temperature (half an hour approximately), the whole operation environment should be controlled at 20 to 25 ℃;
2, constant temperature oven or water-bath are transferred to temperature of reaction;
3, the liquid standard product can directly use; The solid standard items will fully dissolve the back on request to be used;
4, prepare T3 enzyme conjugates reagent, the T3 enzyme conjugates concentrate of 0.1ml is joined in the T3 enzyme dilution of 1ml (dilution in 1: 10), and mix.The enzyme amount depends on detection limit, instant joining;
5, luminous substrate A, B liquid equal proportion are mixed to this test volume required (every hole needs 100 μ l, can be needed mixed luminous substrate 1ml to mix by plate according to every bag, and the rest may be inferred);
6, the solid washing lotion fully being dissolved the back with 500ml distilled water uses.
(2) experimental technique:
1, the capillary strip with institute's expense is placed on the support;
2, in the reaction micropore, add 50 μ l standard items or sample to be measured (asking accurate application of sample) successively;
3, every hole adds 100 μ l enzyme conjugates, light shaking micropore support, fully mixing;
4, behind the sealing film, under 24 ± 2 ℃ of conditions, incubation 60 minutes;
5, each hole liquid is abandoned to the greatest extent, washed plate five times (or wash with washing the plate machine washing), after each flushing moisture is abandoned to the greatest extent, after the last flushing residual moisture in the microwell plate is patted dry, note being sure not wiping and react the micropore inwall with washing lotion;
6, every hole adds mixed substrate 100 μ l, room temperature reaction 5 minutes.
7, chemiluminescence detector detects luminous intensity values, calculates measurement result.
8, being horizontal ordinate (X-axis) with the standard items concentration value, is ordinate (Y-axis) with the standard items luminous intensity values, sets up typical curve, calculates measurement result.
It is shorter to measure the used time with detection kit of the present invention by said procedure, and a collection of mensuration generally only needed to finish in one hour, fast convenient.
Through experimental results demonstrate, the methodology appraising datum of the above-mentioned detection kit of the present invention when being used for the trilute assay can reach following index:
Sensitivity---minimum detectable activity is 0.15ng/ml;
Standard curve range---0-10ng/ml;
Precision---average 6.5% (n=10) of precision in analyzing, precision average out to 10.2% (n=10) between analysis far above national standard, illustrates that kit of the present invention has good repeatability in test experience;
Accuracy---the recovery that known T3 concentration serum is spent behind the hormone serum doubling dilution is 95%-105%.
Experiment shows, uses the present invention the trilute in the human serum is measured, and the sample that only need take a morsel can carry out, to the detected person without any injury.Simultaneously, in These parameters, all be better than enzyme-linked immune detection method, also do not have the shortcomings such as radioactive contamination, term of validity weak point, complicated operation of radioimmunology simultaneously because the present invention has adopted chemiluminescent EIA enzyme immunoassay detection method.Another advantage of the present invention is to adopt the anti-method p-poly-phenyl of bridge vinyl plate to wrap quilt, has not only reduced the package amount of specific antibody, and has increased the homogeneity of bag quilt.Therefore, the present invention provides a kind of more accurate, method easily and efficiently that detects trilute for clinical, can satisfy clinical needs better.
Embodiment
Detection by quantitative trilute chemiluminescence analysis kit, mainly be coated with the opaque polystyrene board of the anti-trilute antibody of rabbit by (1), (2) be matrix, the formulated trilute series standard product of the adding pure product of trilute to go hormone serum or analysis buffer, (3) trilute of horseradish peroxidase-labeled, (4) luminous substrate A liquid and (5) luminous substrate B liquid are formed., luminous substrate A liquid is made into by efficient luminous agent, composite fortifier and amino acid, and luminous substrate B liquid is made into by amino acid oxidase and stabilizing agent.
The opaque polystyrene board that is coated with the anti-trilute antibody of rabbit adopts the anti-method bag of bridge quilt, and excessive bag is wrapped by anti-trilute monoclonal antibody more indirectly by sheep anti-mouse igg earlier; Available trilute enzyme diluted before the trilute of horseradish peroxidase-labeled uses, dilution is made up of phosphate buffer and 1% bovine serum albumin(BSA), contains 0.8 ‰ ANS; The working concentration of horseradish peroxidase-labeled trilute is 1: 500000.
When with analysis buffer preparation trilute series standard product, analysis buffer is formed PH7.4 by sodium chloride and 3% bovine serum albumin(BSA) of 0.01M sodium hydrogen phosphate-sodium dihydrogen phosphate, 0.05M.
Luminous substrate A liquid is made up of following ingredients: luminol 0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M; Luminous substrate B liquid is made up of following ingredients: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA 0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M.
Claims (4)
1, detection by quantitative trilute chemiluminescence analysis kit, it is characterized in that, mainly form by the opaque polystyrene board that is coated with anti-trilute antibody, trilute series standard product, enzyme labeling trilute, luminous substrate A liquid and luminous substrate B liquid, luminous substrate A liquid is made into by efficient luminous agent, composite fortifier and amino acid, and luminous substrate B liquid is made into by amino acid oxidase and stabilizing agent.
2, kit as claimed in claim 1 is characterized in that, the opaque polystyrene board that is coated with anti-trilute antibody is for being coated with the opaque polystyrene board of anti-trilute monoclonal antibody; Trilute series standard product are that matrix, the pure product of adding trilute are formulated to go hormone serum or analysis buffer; The enzyme labeling trilute is the trilute of horseradish peroxidase-labeled, available trilute enzyme diluted before using; Luminous substrate is a HRP-luminol luminescent system.
3, kit as claimed in claim 2 is characterized in that, the opaque polystyrene board that is coated with anti-trilute antibody is the opaque polystyrene board that is coated with the anti-trilute antibody of rabbit; Analysis buffer is formed PH7.4 by sodium chloride and 3% bovine serum albumin(BSA) of 0.01M sodium hydrogen phosphate-sodium dihydrogen phosphate, 0.05M; Trilute enzyme dilution is made up of phosphate buffer and 1% bovine serum albumin(BSA), contains 0.8 ‰ ANS; The working concentration of horseradish peroxidase-labeled trilute is 1: 500000-1000000.
4, kit as claimed in claim 3 is characterized in that, the opaque polystyrene board that is coated with the anti-trilute antibody of rabbit adopts the anti-method of bridge, and excessive bag is wrapped by anti-trilute monoclonal antibody more indirectly by sheep anti-mouse igg earlier; Luminous substrate A liquid is made up of following ingredients: luminol0.15mM, Hydroxycoumarin 0.59mM, gallic acid 0.35mM, Tris-Hcl damping fluid 0.2M; Luminous substrate B liquid is made up of following ingredients: amino acid oxidase 0.85mM, Tween-20 0.8% (V/V), DTPA 0.5mM, vitamin C 0.12mM, acetate-acetate buffer 0.2M.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2005100179417A CN1924580B (en) | 2005-08-30 | 2005-08-30 | Chemical luminescent analysis reagent kid for quantitatively detecting trilute |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2005100179417A CN1924580B (en) | 2005-08-30 | 2005-08-30 | Chemical luminescent analysis reagent kid for quantitatively detecting trilute |
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| CN1924580A true CN1924580A (en) | 2007-03-07 |
| CN1924580B CN1924580B (en) | 2010-04-14 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033623A (en) * | 2012-12-10 | 2013-04-10 | 天津市协和医药科技集团有限公司 | Human M2 type pyruvate kinase chemiluminescence immune assay kit and preparation method |
| CN103293145A (en) * | 2013-05-16 | 2013-09-11 | 赫利森（厦门）生物科技有限公司 | Chemiluminescence reagent |
| CN106053785A (en) * | 2016-05-18 | 2016-10-26 | 北京北方生物技术研究所有限公司 | Solid antibody pre-coating method for competitive chemiluminescent immunoreactions |
| CN110988367A (en) * | 2019-11-18 | 2020-04-10 | 迈克生物股份有限公司 | Storage agent, calibrator for detecting LH and detection kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1311053C (en) * | 1998-12-24 | 2007-04-18 | 中国科学院长春应用化学研究所 | Intensifier for enzymatically chemical luminous reaction |
-
2005
- 2005-08-30 CN CN2005100179417A patent/CN1924580B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103033623A (en) * | 2012-12-10 | 2013-04-10 | 天津市协和医药科技集团有限公司 | Human M2 type pyruvate kinase chemiluminescence immune assay kit and preparation method |
| CN103293145A (en) * | 2013-05-16 | 2013-09-11 | 赫利森（厦门）生物科技有限公司 | Chemiluminescence reagent |
| CN103293145B (en) * | 2013-05-16 | 2016-08-10 | 赫利森（厦门）生物科技有限公司 | A kind of chemical illuminating reagent |
| CN106053785A (en) * | 2016-05-18 | 2016-10-26 | 北京北方生物技术研究所有限公司 | Solid antibody pre-coating method for competitive chemiluminescent immunoreactions |
| CN110988367A (en) * | 2019-11-18 | 2020-04-10 | 迈克生物股份有限公司 | Storage agent, calibrator for detecting LH and detection kit |
| CN110988367B (en) * | 2019-11-18 | 2024-02-09 | 迈克生物股份有限公司 | Storage agent, calibrator for detecting LH and detection kit |
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| CN1924580B (en) | 2010-04-14 |
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Owner name: ZHENGZHOU AUTOBIO ENGINEERING CO., LTD. Free format text: FORMER NAME: ZHENGZHOU AUTOBIO DIAGNOSTICS CO., LTD. |
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Address after: 450016, No. fifth, No. 87, No. 1, main street, Zhengzhou economic and Technological Development Zone, Henan Patentee after: Autobio Diagnostics Co., Ltd. Address before: 450016, No. fifth, No. 87, No. 1, main street, Zhengzhou economic and Technological Development Zone, Henan Patentee before: Zhengzhou Autobio Diagnostics Co., Ltd. |