CN101949944B - Triiodothyronine quantitative detection kit - Google Patents
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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, greatlyenlarged 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 triiodothyronine quantitative detection kit, the invention still further relates to the preparation method of this kit.
Background technology
(T3 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 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 large 5 times of its biological activity ratio T4, 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 the equal normal patient (general patient Chang Shouxian detects fT4 and TSH) of 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 thyroid function decline person's a kind of compensatory phenomenon, simultaneously with the TSH secretion increasing.
A large amount of experimental datas show, 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, Patients with Hyperthyroidism serum T 3 values 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.Clinically common to some patient before developing into typical thyrotoxicosis, raise the stage through serum T 3 level first, illustrate that mensuration serum T 3 is measured serum T 4 for diagnosis of hyperthyroidism more meaningful.This external Patients with Hyperthyroidism carries out in radioiodine or the drug therapy process, and serum T 4 is down to normally, and serum T 3 still is higher than normally, until clinical thyroid function recovers normal, serum T 3 sides are down to normal level; Can be used as again the reliability index of judging curative effect therefore measure serum T 3.
From detecting on the principle, the method of detection trilute commonly used mainly is 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 is subjected to methodological restriction to put earlier T 3, the T4 mensuration kit of exempting from as representative, and there are very large drawback in its sensitivity and antijamming capability wretched insufficiency, basically, withdraw from the market, use at present more be Enzyme-multiplied immune technique and chemiluminescence.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, the characteristics such as "dead" isotope damage and pollution have obtained develop rapidly in recent years.Immunity magnetic particle technology is 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, the immunologic active material (antigen or antibody) that has specificity affinity on can be coated by methods such as chemical couplings has 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 triiodothyronine quantitative detection 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:
Triiodothyronine quantitative detection kit of the present invention, it comprises and is coated with T2--magnetic particle suspension, trilute series calibration object, the trilute antibody of horseradish peroxidase-labeled, the agent of dissociating, luminous substrate A liquid, luminous substrate B liquid and the concentrated washing lotion of gelatin; The preparation method of described kit comprises the steps:
The first step is coated with the preparation of the magnetic particle suspension of T2-gelatin
Measure the carboxyl magnetic particle according to use, activate under acid condition with EDC and 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 T2-gelatin, making its concentration is 0.5 μ l/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 containing 0.1%-0.2%NaN
3The trilute sterling is mixed with the hormone serum that goes of 0.15%-0.25% PC300 to indicate concentration be a series of calibration objects of 0ng/ml, 0.5 ng/ml, 1 ng/ml, 2.5 ng/ml, 5 ng/ml, 10 ng/ml;
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, then add mouse-anti trilute monoclonal antibody, 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 saves backup;
The 4th step, the preparation of the agent of dissociating
Get purified water, Tris, NaCl, Proclin300, 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, Proclin300 2ml, agent ANS 1-3g 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 acetic acid-acetate buffering, 0.85mM amino acid oxidase, 0.008 Tween20,0.5mM diethylene triamine pentacetic acid (DTPA), 0.12mM vitamin C;
The 6th step, the preparation of concentrated washing lotion
Get NaH
2PO
42H
2O, Na
2HPO
412H
2O, NaCl, Tween 20, distilled water are formulated according to following ratio: NaH
2PO
42H
2O 4.6g, Na
2HPO
412H
2O 62.32g, NaCl 175.6g, Tween 20 2-10ml, distilled water 1000ml.
In the magnetic particle suspension of the described T2 that is coated with--gelatin, the magnetic particle particle diameter that adopts is 0.8-1.5 μ m, and the carboxyl reactive group that concentration is 20-30 μ eq/g is contained on the surface.
The invention has the advantages that the method that has adopted labelled antibody envelope antigen analog, different with the immunoassay of parahormone from conventional tag, the method has been coated with a kind of chemical constitution analog of hormone, and this analog has equal immunogenicity with the hormone of surveying, therefore can carry out specific binding with the antibody of this hormone, but greatly reduce with the binding ability of TBP (THBP), reduce to a great extent to finish hop protein to the impact of 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, greatly improve (sensitivity, accuracy, sensing range etc.) so that detect performance, reaction time shortens greatly (from beginning to be loaded onto testing result, time is less than 25min, obviously faster than similar kit);
2, invented a kind of new T2-gelatin and the coupling method of magnetic particle, the 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, concentrated washing lotion, and the luminous substrate prescription all are optimization formulas under this reaction system, giving the use effect phase of this kit and detecting performance provides powerful guarantee.
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
Triiodothyronine quantitative detection kit of the present invention, comprise and be coated with trilute analogue (T2, T2)-the magnetic particle suspension of gelatin, the magnetic particle particle diameter that adopts in this suspension is about 0.8-1.5 μ m, the carboxyl reactive group that concentration is 20-30 μ eq/g is contained on the surface, adopt activator that its carboxyl is activated, then be connected with the amino of T2-gelatin, form stable peptide bond; It is matrix that hormone serum is removed in employing, adds the formulated trilute series calibration object of trilute sterling; 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 that is formed by enzyme-catalyzed chemical luminescence substrate, reinforcing agent and amino acid; The luminous substrate B liquid that is comprised 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 triiodothyronine quantitative detection kit of the present invention comprises the steps:
The first step is coated with the preparation of the magnetic particle suspension of T2-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 the MES(2-(N-morpholino of 0.05M-0.1M) ethyl sulfonic acid) damping fluid, soak time 30min, after activation is finished, add magnetic field, leave standstill 5 min 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 T2-gelatin, making its concentration is 0.5 μ l/mg magnetic particle, (pH7.4-pH8.4) concussion reaction 1h in the PBS damping fluid; Reaction adds magnetic field after finishing, leaving standstill 5 min separates magnetic particle and liquid, supernatant discarded, PBS damping fluid with the 0.01M that contains 1% bovine serum albumin(BSA) seals, repeatedly seal 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 to a great extent coated raw-material use amount, has reduced the cost of this kit, and simultaneously this method of attachment is comparatively stable, and differences between batches are less.
Second step, the preparation of trilute series calibration object
According to the trilute standard items of national drug biological products assay institute, with containing NaN
3(0.1%-0.2%) and PC300(0.15%-0.25%) the hormone serum that goes the trilute sterling is mixed with to indicate concentration be a series of calibration objects of 0ng/ml, 0.5 ng/ml, 1 ng/ml, 2.5 ng/ml, 5 ng/ml, 10 ng/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, so that calibration object and sample is reactive more consistent, has reduced to a great extent matrix effect; This calibration object is formulated according to national standard simultaneously, has guaranteed accuracy of measurement results; 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, then add mouse-anti trilute monoclonal antibody, 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 saves backup; 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 according to following ratio: purified water 1000ml, Tris 6.05g, NaCl 8.5g, Proclin300 2ml, ANS 1-3g.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 TT3 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 acetic acid-acetate buffering, 0.85mM amino acid oxidase, 0.008 Tween 20,0.5mM DTPA, 0.12mM vitamin C.
The 6th step, the preparation of concentrated washing lotion
Get NaH
2PO
42H
2O, Na
2HPO
412H
2O, NaCl, Tween 20, distilled water are formulated according to following ratio: NaH
2PO
42H
2O 4.6g, Na
2HPO
412H
2O 62.32g, NaCl 175.6g, Tween 20 2-10ml, 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 gently mixing.
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 ℃, behind temperature stabilization, use;
3. with the abundant mixing of magnetic particle suspension to without 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 respectively 20 μ l;
3. every hole adds respectively the agent 50 μ l that dissociate, enzyme labeling thing 50 μ l;
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. adopt four parameter fitting modes, take the calibration object concentration value as X-axis, take the calibration object luminous intensity values as Y-axis, set 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 is linear: R is greater than 0.999(as shown in Figure 1),
Minimum detectability: less than 0.1ng/ml
Accuracy: variation and batch variation all see Table 1 less than 10%(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
| First reagent | Second batch reagent | The 3rd batch of reagent | ||
| Quality Control mean value | Q1 | 0.63 | 0.51 | 0.46 |
| （ng/ml） | Q2 | 1.43 | 1.56 | 1.49 |
| Q3 | 2.57 | 2.75 | 2.46 | |
| Variation in analyzing | Q1 | 2.27 | 6.49 | 7.12 |
| (CV%) | Q2 | 3.39 | 2.41 | 3.76 |
| Q3 | 3.19 | 1.96 | 2.34 | |
| Make a variation between analysis | Q1 | 7.86 | 3.59 | 4.05 |
| (CV%) | Q2 | 6.44 | 2.97 | 6.81 |
| Q3 | 5.31 | 4.21 | 3.01 |
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%; See Table 2 with the rT3 cross reacting rate of 2000ng/ml less than 0.01%()
Table 2. specific data table
| ? | Measured value (ng/ml) | Cross reacting rate (%) |
| T4(500ng/ml) | 0.073 | 0.015 |
| r-T3(2000ng/ml) | 0.16 | 0.008 |
Accuracy: return a series of calibration objects of calculating this kit with national standard, 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, the good relationship of the two measurement result, coefficient R be 0.986(as shown in Figure 2).
Claims (2)
1. triiodothyronine quantitative detection kit, it comprises and is coated with T2--magnetic particle suspension, trilute series calibration object, the trilute antibody of horseradish peroxidase-labeled, the agent of dissociating, luminous substrate A liquid, luminous substrate B liquid and the concentrated washing lotion of gelatin; It is characterized in that: the preparation method of described kit comprises the steps:
The first step is coated with the preparation of the magnetic particle suspension of T2-gelatin
Measure the carboxyl magnetic particle according to use, activate under acid condition with EDC and 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 T2-gelatin, making its concentration is 0.5 μ l/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 containing 0.1%-0.2%NaN
3The trilute sterling is mixed with the hormone serum that goes of 0.15%-0.25% PC300 to indicate concentration be a series of calibration objects of 0ng/ml, 0.5 ng/ml, 1 ng/ml, 2.5 ng/ml, 5 ng/ml, 10 ng/ml;
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, then add mouse-anti trilute monoclonal antibody, 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 saves backup;
The 4th step, the preparation of the agent of dissociating
Get purified water, Tris, NaCl, Proclin300, 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, Proclin300 2ml, agent ANS 1-3g 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 acetic acid-acetate buffering, 0.85mM amino acid oxidase, 0.008 Tween20,0.5mM diethylene triamine pentacetic acid (DTPA), 0.12mM vitamin C;
The 6th step, the preparation of concentrated washing lotion
Get NaH
2PO
42H
2O, Na
2HPO
412H
2O, NaCl, Tween 20, distilled water are formulated according to following ratio: NaH
2PO
42H
2O 4.6g, Na
2HPO
412H
2O 62.32g, NaCl 175.6g, Tween 20 2-10ml, distilled water 1000ml.
2. triiodothyronine quantitative detection kit according to claim 1; it is characterized in that: in the magnetic particle suspension of the described T2 that is coated with--gelatin; the magnetic particle particle diameter that adopts is 0.8-1.5 μ m, and the carboxyl reactive group that concentration is 20-30 μ eq/g is contained on the surface.
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| US8669519B2 (en) * | 2011-12-05 | 2014-03-11 | Quest Diagnostics Investments, Inc. | Methods for detecting reverse triiodothyronine by mass spectrometry |
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