CN101949944A - Triiodothyronine quantitative detection kit and preparation method thereof - Google Patents
Triiodothyronine quantitative detection kit and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a triiodothyronine quantitative detection kit which comprises magnetic particle suspension coated with diiodothyronine-gelatin, a triiodothyronine series calibrator, a triiodothyronine antibody labelled by horseradish peroxidase, a dissociation agent, a chemiluminescent substrate A, a chemiluminescent substrate B and wash concentrate. The invention further discloses a preparation method of the kit. The invention has the advantage that a chemical structural analogue of hormone is coated by a method for coating an antigen analogue with a labelled antibody, and the analogue and the detected hormone have identical immune correlation, thus realizing specific binding of the analogue and the antibody of the hormone; but the binding capability of the analogue with thyroid-binding protein (THBP) is significantly lowered, thus reducing influence of the binding protein on a measuring system to a large extent. The kit has greatly improved sensitivity and precision, greatly enlarged detection range, greatly shortened reaction time, enhanced product properties and lowered product cost.
Description
Technical field
The present invention relates to external immune detection, especially relate to a kind of trilute detection by quantitative kit, the invention still further relates to the preparation method of this kit.
Background technology
(3,5,3 '-trilute is that a kind of molecular weight is 651 iodotyrosine T3) to trilute, and the same with thyroxine (T4) is a kind of important thyroid hormone.Iodine and tyrosine are the primary raw materials of synthetic thyroid hormone, thyroglobulin is the place and the raw material of iodate, and the iodate of tyrosine and the coupling of iodotyrosine are all carried out at this, and T3, T4 are secreted into blood during the hydrolysis thyroglobulin, 90% is T4 in the secretion, and 10% is T3.20% T3 is directly synthetic by thyroid gland in the blood, and other 80% T3 sloughs 1 molecular iodine by T4 to generate, and take off iodine position difference and also can form anti-T3, but anti-T3 does not have biologically active.The half life period of T3 is 1.5 days, and 20% in the elimination of periphery tissue metabolism, and 80% through taking off iodine metabolism.
The amount of T3 only is about 1/60 of T4 in the serum, but its biological activity ratio T4 is big 5 times, so the mensuration of T3 is very meaningful to diagnosis dysthyroid and monitoring thyroid gland technical ability.To hyperthyroid patient, the level of total T3 of Most patients and total T4 all raises.But in some case, hyperthyroidism only is (the T3 type hyperthyroidism disease) that causes of increasing owing to T3, therefore to the normal hyperthyroid patient of fT4, suggestion detects T3, especially for fT4 and all normal patient of TSH (general patient Chang Shouxian detects fT4 and TSH), more should further detect T3.The T3 level raise and T4 normally to be also shown in the Thyreoidine function normal and suffer from the patient of functional autonomy thyroid disease (Graves illness in eye), also can be subclinical type thyroid function decline person's a kind of compensatory phenomenon, simultaneously with the TSH secretion increasing.
A large amount of experimental datas show that measuring serum T 3 is to judge one of first-selected index of hyperthyroidism, particularly T3 toxaemia patient, and serum T 4 concentration are normal, and T3 obviously raises.Therefore, measuring serum T 3 is important indicators of clinical diagnosis thyroid function.In general, hyperthyroidism disease human serum T3 value can obviously raise.In most hyperthyroidism cases, the rising of serum T 3 and serum T 4 raise and parallel; And the T3 toxication also claims in the T3 hyperthyroidism, and serum T 4 values are in normal range, and only T3 raises.Common clinically to some patient before developing into typical thyrotoxicosis, raise the stage earlier through serum T 3 levels, illustrate that to measure serum T 3 more meaningful for diagnosing hyperthyroidism to measure serum T 4.This external hyperthyroidism disease people carries out in radioiodine or the drug therapy process, and serum T 4 is reduced to normally, and serum T 3 still is higher than normally, recovers normal until clinical thyroid function, and serum T 3 sides reduce to normal level; Can be used as the reliability index of judging curative effect again so measure serum T 3.
From detecting on the principle, the method of detection trilute commonly used mainly is a competition law clinically at present, two kinds of detection methods that comprise the detection method of labelled antigen (coated antibody) and labelled antibody (envelope antigen), external reagent mostly is the method for labelled antigen, and internal reagent mostly is the method for labelled antibody.
On detection technique, past, to exempt from be that the earlier T 3, T4 of representative measured kit and be subjected to methodological restriction to put, and there are very big drawback in its sensitivity and antijamming capability wretched insufficiency, basically, withdraw from the market, use more be Enzyme-multiplied immune technique and chemiluminescence at present.It is continue Enzyme-multiplied immune technique and the emerging technology that grows up after putting immune technology the eighties that chemiluminescence rises in eighties of last century, because its high sensitivity, high specific, while method is easy, quick, the mark bond is stable, characteristics such as "dead" isotope damage and pollution have obtained develop rapidly in recent years.Immunity magnetic particle technology is an emerging technology in recent years, it is to utilize the magnetic solid phase particle of Polymer Synthesizing certain particle size size to do carrier, carrier surface is modified with the chemical functional group of some, can by method bags such as chemical coupling by on have specificity affinity immunologic active material (antigen or antibody), have that velocity of separation is fast, efficient is high, favorable repeatability, reaction all equate plurality of advantages.
Summary of the invention
The object of the present invention is to provide a kind of trilute detection by quantitative kit, this kit combines the enzyme-catalyzed chemical luminescence technology with magnetic separation technique, with low cost, and easy to be quick, the result is accurate; Another object of the present invention also is to provide the preparation method of this kit.
For achieving the above object, the present invention can take following technical proposals:
Trilute detection by quantitative kit of the present invention comprises magnetic particle suspension, trilute series calibration object, the trilute antibody of horseradish peroxidase-labeled, the agent of dissociating, luminous substrate A liquid, the luminous substrate B liquid that is coated with diiodo-thyronine-gelatin and concentrates washing lotion.
In the described magnetic particle suspension that is coated with diiodo-thyronine-gelatin, the magnetic particle particle diameter that is adopted is 0.8-1.5um, and the carboxyl reactive group that concentration is 20-30ueq/g is contained on the surface.
Described trilute series calibration object adopts and removes hormone serum is matrix, and the pure product of adding trilute are formulated.
The trilute antibody of described horseradish peroxidase-labeled is that horseradish peroxidase is connected with mouse-anti trilute monoclonal antibody.
The described agent of dissociating is for containing the Tris-NaCl damping fluid (three (methylol) aminomethane-hydrochloride buffer) of 1-anilino--8-naphthalene sulfonic acids (ANS).
Described luminous substrate A liquid is made up of enzyme-catalyzed chemical luminescence substrate, reinforcing agent and amino acid; Luminous substrate B liquid is made up of amino acid oxidase and stabilizing agent.
Described concentrated washing lotion is the phosphate buffer (PBS damping fluid) that is added with stabilizing agent and surfactant.
The preparation method of trilute detection by quantitative kit of the present invention comprises the steps:
The first step is coated with the preparation of the magnetic particle suspension of diiodo-thyronine-gelatin
Measure the carboxyl magnetic particle according to use, under acid condition (PH4.5-PH5.5) activates with 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) and N-hydroxy-succinamide (NHS), after activation is finished, add magnetic field, leave standstill magnetic particle and liquid are separated, supernatant discarded, the unnecessary activator of PBS damping fluid flush away of the 0.01M of usefulness PH7.6; Add an amount of diiodo-thyronine-gelatin, making its concentration is the 0.5ul/mg magnetic particle, (PH7.4-PH8.4) concussion reaction under weak basic condition; Reaction adds magnetic field after finishing, and leaves standstill to make magnetic particle and liquid separate supernatant discarded, PBS damping fluid with the 0.01M that contains 1% bovine serum albumin(BSA) seals, after sealing finishes, add confining liquid to preserve magnetic particle, the ultimate density that makes magnetic particle is 0.5mg/ml; This magnetic particle suspension places the 2-8 degree to preserve, in order to using;
Second step, the preparation of trilute series calibration object
The pure product of trilute are mixed with the hormone serum that goes that contains 0.1%-0.2%NaN3 (antiseptic) and 0.15%-0.25%PC300 (antiseptic) to indicate concentration be a series of calibration objects of 0ng/ml, 0.5ng/ml, 1ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml;
The 3rd step, the preparation of the trilute antibody of horseradish peroxidase-labeled
With activator EDC (1-(3-dimethylamino-propyl)-3-ethyl carbodiimide) amino on the horseradish peroxidase is activated, add mouse-anti trilute monoclonal antibody then, making the carboxyl on the antibody is amide compound with the amino condensation that activated, and has both got the trilute enzyme labelled antibody after the dialysis; In the good solution of mark, geometric ratio adds glycerine-20 degree and preserves standby;
The 4th step, the preparation of the agent of dissociating
Get purified water, Tris, NaCl, Proclin300, ANS and be mixed with the agent of dissociating: purified water 1000ml, Tri s 6.05g, NaCl 8.5g, Procl in3002ml, ANS 1-3g according to following ratio;
The 5th step, the preparation of luminous substrate A liquid and luminous substrate B liquid
Luminous substrate A liquid: get 0.2M Tris-Hcl, 0.15mM Luminol (luminol), 0.59mM Hydroxycoumarin, the 0.59mM gallic acid is formulated;
Luminous substrate B liquid: it is formulated to get 0.2M acetate-acetate buffering, 0.85mM amino acid oxidase, 0.008tween20,0.5mM DTPA (diethylene triamine pentacetic acid (DTPA)), 0.12mM vitamin C.
In the 6th step, concentrate the preparation of washing lotion
Get NaH2PO42H2O, Na2HPO412H2O, NaCl, Tween20, distilled water is formulated according to following ratio: NaH2PO42H2O 4.6g, Na2HPO412H2O 62.32g, NaCl 175.6g, Tween202-10ml, distilled water 1000ml.
The invention has the advantages that the method that has adopted labelled antibody envelope antigen analog, different with conventional tag with the immunoassay of parahormone, this method bag is by a kind of chemical constitution analog of hormone, and this analog has equal immunogenicity with the hormone of surveying, therefore can carry out specificity with the antibody of this hormone combines, but greatly reduce with the binding ability of TBP (THBP), reduce to finish the influence of hop protein to a great extent measuring system.
Main innovation part of the present invention is:
1, this kit combines chemiluminescence with immune magnetic particle, a kind of reaction system near homogeneous phase is provided, and adopted the single stage method reaction pattern, make that detecting performance improves (sensitivity, accuracy, sensing range etc.) greatly, reaction time shortens greatly (from beginning application of sample to testing result, time is less than 25min, obviously faster than similar kit);
2, invented a kind of new diiodo-thyronine-gelatin and the coupling method of magnetic particle, this method coupling efficiency is higher, in conjunction with firm, and process stabilizing, when enhancing product performance, greatly reduce cost of products;
3, the calibration object in the kit, enzyme conjugates, the agent of dissociating, concentrate washing lotion, and the luminous substrate prescription all are optimization formulas under this reaction system, imitate the phase and detect performance for the use of this kit powerful guarantee is provided.
Description of drawings
Fig. 1 is the typical curve Line Chart of kit of the present invention.
Fig. 2 is kit of the present invention and like product clinical control figure.
Embodiment
Trilute detection by quantitative kit of the present invention, comprise and be coated with trilute analogue (diiodo-thyronine, T2)-the magnetic particle suspension of gelatin, the magnetic particle particle diameter that adopts in this suspension is about 0.8-1.5um, the carboxyl reactive group that concentration is 20-30ueq/g is contained on the surface, adopt activator that its carboxyl is activated, the amino with diiodo-thyronine-gelatin is connected then, forms stable peptide bond; It is matrix that hormone serum is removed in employing, adds the formulated trilute series calibration object of the pure product of trilute; The trilute antibody of the horseradish peroxidase-labeled that horseradish peroxidase is connected with mouse-anti trilute monoclonal antibody; Contain the agent of dissociating of the Tris-NaCl damping fluid of ANS; The luminous substrate A liquid of forming by enzyme-catalyzed chemical luminescence substrate, reinforcing agent and amino acid; The luminous substrate B liquid of being made up of amino acid oxidase and stabilizing agent and contain stabilizing agent and the PBS damping fluid of surfactant, this damping fluid need carry out 20 times of dilutions before using.
The preparation method of trilute detection by quantitative kit of the present invention comprises the steps:
The first step is coated with the preparation of the magnetic particle suspension of diiodo-thyronine-gelatin
Measure an amount of carboxyl magnetic particle according to use, under acid condition (PH4.5-PH5.5) activates with excessive EDC and NHS, the activation damping fluid is MES (2-(N-morpholino) ethyl sulfonic acid) damping fluid of 0.05M-0.1M, soak time 30min, after activation is finished, add magnetic field, leave standstill 5min magnetic particle and liquid are separated, supernatant discarded is washed the unnecessary activator of flush away twice with the PBS damping fluid of the 0.01M of PH7.6; Add an amount of diiodo-thyronine-gelatin, making its concentration is the 0.5ul/mg magnetic particle, (PH7.4-PH8.4) concussion reaction 1h in the PBS damping fluid; Reaction adds magnetic field after finishing, leaving standstill 5min separates magnetic particle and liquid, supernatant discarded, PBS damping fluid with the 0.01M that contains 1% bovine serum albumin(BSA) seals, seal repeatedly 5 times, each 10min is after sealing finishes, add an amount of confining liquid to preserve magnetic particle, the ultimate density that makes magnetic particle is 0.5mg/ml; Place the 2-8 degree to preserve this magnetic particle suspension, in order to using; This method of attachment efficient is higher, has reduced the use amount of coating antigen material to a great extent, has reduced the cost of this kit, and this method of attachment simultaneously is comparatively stable, and differences between batches are less.
Second step, the preparation of trilute series calibration object
Trilute standard items according to the national drug biological products assay institute, the pure product of trilute are mixed with the hormone serum that goes that contains NaN3 (0.1%-0.2%) and PC300 (0.15%-0.25%) to indicate concentration be a series of calibration objects of 0ng/ml, 0.5ng/ml, 1ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml, the bottle cap color is followed successively by white, yellow, green, blue, purple, black; Because this calibration object adopts and removes hormone serum is matrix, makes the reactive more consistent of calibration object and sample to have reduced matrix effect to a great extent; This calibration object is formulated according to the national standard product simultaneously, has guaranteed the accuracy of measurement result; This calibration object is liquid in addition, need not to redissolve, and is easy to use.
The 3rd step, the preparation of the trilute antibody of horseradish peroxidase-labeled
With activator EDC the amino on the horseradish peroxidase is activated, add mouse-anti trilute monoclonal antibody then, making the carboxyl on the antibody is amide compound with the amino condensation that activated, and has both got the trilute enzyme labelled antibody after the dialysis; In the good solution of mark, geometric ratio adds glycerine-20 degree and preserves standby; Above-mentioned labeling method is carbodiimide (EDC) method, soon the carboxyl on the mouse-anti trilute monoclonal antibody and the amino of horseradish peroxidase (HRP) molecule are amide compound through the effect condensation of EDC, have both got the trilute enzyme labelled antibody after the dialysis.After testing, this enzyme labeling thing working concentration in use is 1: 10000--1: 20000.
The 4th step, the preparation of the agent of dissociating
Get purified water, Tris, NaCl, Proclin300, ANS and be mixed with the agent of dissociating: purified water 1000ml, Tris 6.05g, NaCl 8.5g, Proclin3002ml, ANS1-3g according to following ratio.The purpose of agent of dissociating be with the trilute in the serum sample from disintegrating down in conjunction with albumen, be the amount of the total trilute in the serum with what guarantee that this system detects.
The 5th step, the preparation of luminous substrate A liquid and luminous substrate B liquid
Luminous substrate A liquid: get 0.2M Tris-Hcl, 0.15mM Luminol, 0.59mM Hydroxycoumarin, the 0.59mM gallic acid is formulated;
Luminous substrate B liquid: it is formulated to get 0.2M acetate-acetate buffering, 0.85mM amino acid oxidase, 0.008tween20,0.5mM DTPA, 0.12mM vitamin C.
In the 6th step, concentrate the preparation of washing lotion
Get NaH2PO42H2O, Na2HPO412H2O, NaCl, Tween20, distilled water is formulated according to following ratio: NaH2PO42H2O 4.6g, Na2HPO412H2O 62.32g, NaCl 175.6g, Tween202-10ml, distilled water 1000ml.
The use running program of kit of the present invention is as follows:
1, sample collection
Adopt correct medical technology to collect serum (sample of significant hemolysis or piarhemia can not be used for measuring), the sample after the collection is placed in room temperature can not be above 8 hours; If in 8 hours, do not detect in the refrigerator that sample need be positioned over 2-8 ℃; If need to preserve more than 72 hours or transportation, then should be frozen in below-20 ℃, avoid multigelation.Return to room temperature before the use, shake mixing gently.
2, prepare before the experiment
1. get 1 bottle of concentrated washing lotion and carry out 20 times of dilutions with distilled water;
2. constant temperature oven or water-bath temperature are transferred to 37 ℃, treat to use behind the temperature stabilization;
3. with the abundant mixing of magnetic particle suspension to there not being the naked eyes visible precipitate.
3, experimental technique
1. take out a certain amount of reaction vessel, numbering.Add 50ul calibration object/quality-control product/clinical sample according to requirement of experiment;
2. shake up the magnetic particle suspension, every hole adds 20 μ l respectively;
3. every hole adds the agent 50 μ l that dissociate, enzyme labeling thing 50 μ l respectively;
4. solution in the reaction vessel is mixed 37 ℃ of incubations 15 minutes;
5. use magnetic to separate and washing facility, magnetic particle in the reaction vessel is washed 5 times with washing lotion;
6. the reaction vessel after will washing fully vibrates magnetic particle is scattered;
7. every hole adds luminous substrate A and each 50 μ l of luminous substrate B, and the lucifuge room temperature reaction is 5 minutes behind the vibration mixing;
8. chemiluminescence detector detects luminous intensity;
9. adopting four parameter fitting modes, is X-axis with the calibration object concentration value, is Y-axis with the calibration object luminous intensity values, sets up calibration curve.Return the corresponding concentration value of calculation according to the luminous intensity values of sample to be tested.
Kit of the present invention is identified according to methodology, can be reached following index:
Typical curve linearity: R is greater than 0.999 (as shown in Figure 1),
Minimum detectability: less than 0.1ng/ml
Accuracy: variation and batch variation are all less than 10% (seeing Table 1) between variation in analyzing, analysis
Table 1. accuracy tables of data
A), analyze in and analyze between the tables of data that makes a variation
B), batch variation tables of data
| Q1 | Q2 | Q3 | |
| First | 0.57 | 1.48 | 2.70 |
| Second batch | 0.51 | 1.56 | 2.54 |
| The 3rd batch | 0.49 | 1.51 | 2.65 |
| Variation (CV%) | 8.54 | 2.53 | 3.14 |
Specificity: with the T4 cross reacting rate of 500ng/ml less than 0.1%; With the rT3 cross reacting rate of 2000ng/ml less than 0.01% (seeing Table 2)
Table 2. specific data table
Accuracy: return a series of calibration objects of calculating this kit with the national standard product, return the ratio mean value of calculating concentration and indicating concentration and between 0.9-1.1, (see Table 3)
Table 3. accuracy tables of data
Contrast with like product: this kit and the T3 kit (euzymelinked immunosorbent assay (ELISA)) of internationally renowned brand Biocheck are measured 124 parts of clinical serum samples simultaneously, and the good relationship of the two measurement result, coefficient R were 0.986 (as shown in Figure 2).
Claims (8)
1. trilute detection by quantitative kit is characterized in that: it comprises and is coated with the diiodo-thyronine--magnetic particle suspension, trilute series calibration object, the trilute antibody of horseradish peroxidase-labeled, the agent of dissociating, luminous substrate A liquid, the luminous substrate B liquid of gelatin and concentrate washing lotion.
2. trilute detection by quantitative kit according to claim 1; it is characterized in that: the described diiodo-thyronine that is coated with--in the magnetic particle suspension of gelatin; the magnetic particle particle diameter that is adopted is 0.8-1.5um, and the carboxyl reactive group that concentration is 20-30ueq/g is contained on the surface.
3. trilute detection by quantitative kit according to claim 1 is characterized in that: described trilute series calibration object adopts and removes hormone serum is matrix, and the pure product of adding trilute are formulated.
4. trilute detection by quantitative kit according to claim 1, it is characterized in that: the trilute antibody of described horseradish peroxidase-labeled is that horseradish peroxidase is connected with mouse-anti trilute monoclonal antibody.
5. trilute detection by quantitative kit according to claim 1 is characterized in that: the described agent of dissociating is the Tris-NaCl damping fluid that contains 1-anilino--8-naphthalene sulfonic acids.
6. trilute detection by quantitative kit according to claim 1 is characterized in that: described luminous substrate A liquid is made up of enzyme-catalyzed chemical luminescence substrate, reinforcing agent and amino acid; Luminous substrate B liquid is made up of amino acid oxidase and stabilizing agent.
7. trilute detection by quantitative kit according to claim 1 is characterized in that: described concentrated washing lotion is the phosphate buffer that contains stabilizing agent and surfactant.
8. the preparation method of trilute detection by quantitative kit according to claim 1, it is characterized in that: it comprises the steps:
The first step is coated with the preparation of the magnetic particle suspension of diiodo-thyronine-gelatin
Measure the carboxyl magnetic particle according to use, under acid condition, activate, after activation is finished, add magnetic field, leave standstill magnetic particle and liquid are separated, supernatant discarded, the unnecessary activator of PBS damping fluid flush away of the 0.01M of usefulness PH7.6 with EDC and NHS; Add an amount of diiodo-thyronine-gelatin, making its concentration is the 0.5ul/mg magnetic particle, concussion reaction under weak basic condition; Reaction adds magnetic field after finishing, and leaves standstill to make magnetic particle and liquid separate supernatant discarded, PBS damping fluid with the 0.01M that contains 1% bovine serum albumin(BSA) seals, after sealing finishes, add confining liquid to preserve magnetic particle, the ultimate density that makes magnetic particle is 0.5mg/ml; This magnetic particle suspension places the 2-8 degree to preserve, in order to using;
Second step, the preparation of trilute series calibration object
With contain 0.1%-0.2%NaN3 and 0.15%-0.25%PC300 go hormone serum with the pure product of trilute be mixed with indicate concentration be 0ng/ml, 0.5ng/ml, 1ng/ml, 2.5ng/ml, 5ng/ml, 10ng/ml-serial calibration object;
The 3rd step, the preparation of the trilute antibody of horseradish peroxidase-labeled
With activator 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide the amino on the horseradish peroxidase is activated, add mouse-anti trilute monoclonal antibody then, making the carboxyl on the antibody is amide compound with the amino condensation that activated, and has both got the trilute enzyme labelled antibody after the dialysis; In the good solution of mark, geometric ratio adds glycerine-20 degree and preserves standby;
The 4th step, the preparation of the agent of dissociating
Get purified water, Tris, NaCl, Procl in300, the agent ANS that dissociates and be mixed with the agent of dissociating according to following ratio: purified water 1000ml, Tris 6.05g, NaCl 8.5g, Proclin3002ml, agent ANS1-3g dissociates;
The 5th step, the preparation of luminous substrate A liquid and luminous substrate B liquid
Luminous substrate A liquid: get 0.2M Tris-Hcl, 0.15mM Luminol, 0.59mM Hydroxycoumarin, the 0.59mM gallic acid is formulated;
Luminous substrate B liquid: it is formulated to get 0.2M acetate-acetate buffering, 0.85mM amino acid oxidase, 0.008tween20,0.5mM diethylene triamine pentacetic acid (DTPA), 0.12mM vitamin C;
In the 6th step, concentrate the preparation of washing lotion
Get NaH2PO42H2O, Na2HPO412H2O, NaCl, Tween20, distilled water is formulated according to following ratio: NaH2PO42H2O 4.6g, Na2HPO412H2O 62.32g, NaCl 175.6g, Tween202-10ml, distilled water 1000ml.
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