1,1.0mg DSS (disuccinimidyl suberate) is dissolved among the 50ul DMSO, promptly concentration is 20mg/ml; Get in the 0.1mol/L PB damping fluid that the anti-TT4 monoclonal antibody of 2mg (day strong bio-pharmaceuticals (Tianjin) company limited, concentration is 1.89mg/ml) is dissolved in PH 9.5 to cumulative volume be 1ml;

2, calculate the input amount of DSS, calculate according to following formula: (antibody quality/16000) * 10 * 368/C_DSS), C wherein_DSSRefer to the amount of substance concentration mol/L of DSS;

3, the DSS with liquid-transfering gun absorption respective volume joins in the antibody-solutions of step 1, puts room temperature 90min;

4, with step 3 antibody-solutions join put into then in the Centricon-10 concentration tube in the sigma2-16k high speed freezing centrifuge under the centrifugal force of 3000g, concentrate about 30min to volume be 0.5ml;

5, get the 0.5ml magnetic bead, magnetic bead concentration is 0.5g/ml, and described magnetic bead diameter adds in the 5ml reaction cup between 0.9 μ m, puts into special-purpose test tube rack, draws supernatant through magnet absorption after 2 minutes;

6, add 1.5ml PH9.5 0.1mol/L PB, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times at every turn; The antibody-solutions that step 4 is obtained joins in the magnetic bead, and room temperature reaction is 4 hours behind the mixing, keeps the mixing state;

7, the TRIS solution 37 that adds 0.3ml 1mol/L is spent 15 minutes, and wherein the application of sample amount of TRIS adds 0.15mlTRIS for 1mg antibody;

8, add 1.5ml PH 7.2 0.1mol/L PB at every turn clean the magnetic bead of mark, mixing 30 seconds is put on the shelf, removes supernatant, repetitive operation 3 times;

9, preserve liquid with the 100ml magnetic bead and change magnetic bead over to the 125ml vial, be 0.05% TT4 magnetic separation agent; It is 0.1%BSA that magnetic bead is preserved formula of liquid, 0.05% Tween-20,0.02%NaN3,20% ethanol, 4 ℃ of preservations.

10, the magnetic separation agent that step 9 is obtained is with the ratio mixing of magnetic bead buffer solution according to 1: 1;

11, post label and store in 2-8 ℃ of freezer, the magnetic separation agent term of validity is 12 months;

Embodiment 2

One, enzyme reaction thing diluent preparing working specification: prescription is seen table 2, and 1L is an example with preparation:

1, gets Tris 6.06g, NaCl 13.0g, Zncl₂0.05g, Proclin-300 0.2ml and MgCl₂0.05g in flask; In flask, add the 800ml purified water then, fully stir, reagent is dissolved fully;

2, transfer PH with HCl or NaOH, measurement makes PH in the 7.35-7.45 scope;

3, taking by weighing BSA 3g pours in the said vesse;

4, be settled to 1000ml at last, survey pH value, scope promptly meets the requirements between 7.35-7.45, filters with the 0.2um filter;

After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;

Enzyme reaction thing dilution table 2

Two, the coupling of alkaline phosphatase ALP and TT4 antigen

1, getting TT4 antigen 1 mg is positioned in the 1ml glass tube;

5, with the solution of step 4 with PD-10 post purifying, collect refined solution, add enzyme reaction thing dilution according to 1: 5000 volume, mix, promptly get the enzyme reaction thing.

Embodiment 3

Increased response agent formulation operations rules: prescription is seen (table 3), and 1L is an example with preparation:

1, take by weighing TRIS (trishydroxymethylaminomethane, molecular formula: (HOCH2) 3CNH2) 1.56g and NaCl 4.23g are in the 1L container; With pipettor with Proclin-300 measure 0.2ml in the beaker of a small amount of purified water fully the dissolving after, pour in the above-mentioned 1L container;

2, measure the 800ml purified water in the 1L container with graduated cylinder, fully stir, until dissolving fully; Transfer PH with HCL or NaOH, measure its scope between 7.35-7.45;

3, take by weighing Mak33 0.9g in above-mentioned 1L container;

4, last constant volume 1000ml after the dissolving, surveys pH value fully, and scope promptly meets the requirements between 7.35-7.45, filters with the 0.2um filter; After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;

The preparation (table 3) of increased response agent

Embodiment 4

The dilution prescription is seen (table 4), and 1L is an example with preparation:

1, takes by weighing NaCl9.0g and BSA 60g in the container of 1L;

The preparation (table 4) of dilution

Embodiment 5

The preparation of calibration object and quality-control product:

1, peak: maximum concentration point is X, and impact point concentration is A, B, and C, D, E, F, when preparing the solution of V volume, the volume that needs the adding raw material is for being respectively: table 5

Concentration	Add calibration object dilution volume	Add the X volume
			A	V-A*V/X	A*V/X
B	V-B*V/X	B*V/X
			C	V-C*V/X	C*V/X
D	V-D*V/X	D*V/X
			E	V-E*V/X	E*V/X
OF	V-F*V/X	F*V/X

2, to be mixed with concentration point with dilution (concrete prescription see embodiment 4) be 20,40,80,150 to total thyroxin (TT4) quantitative determination reagent kit TT4 calibration object raw material (purchase in AMG Co., concentration is 10mg/ml), 300ng/ml; It is 40 that quality-control product uses the concentration point of diluent preparing, 150ng/ml.

3, fully the dissolving after, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months.

Embodiment 6:

Cleaning concentrate formulation operations rules: prescription is seen (table 6), and 1L is an example with preparation:

1, takes by weighing TRIS 12.54g and NaCl 325.6g in the 1L container;

2, take by weighing 5g Tween-20 adds suitable quantity of water it is dissolved fully in the 100ml container after, pour in the said vesse;

5, transfer PH with HCL or NaOH, measure its scope between 7.35-7.45;

6, last constant volume 1000ml surveys pH value, and scope promptly meets the requirements between 7.35-7.45, and filter with the 0.2um filter dissolving back fully; After having filtered, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months;

The preparation (table 6) of cleaning concentrate

Embodiment 7

Substrate solution formulation operations rules: prescription is seen (table 7), and 1L is an example with preparation:

2, measure the 600ml purified water in the 1L beaker with graduated cylinder, fully stir, until dissolving fully; Transfer PH with HCl or NaOH, measure its scope between 7.95-8.05;

3, behind the adding 250ml Lumi-Phos 480, filter collection filtrating with the 0.2um filter, be settled to 1000ml with purified water, behind the mixing, post label and store in 2-8 ℃ of freezer, the term of validity is 12 months.

The preparation (table 7) of chemical luminous substrate

Method of testing of the present invention is following

1, adds 15 μ l TT4 calibration objects, quality-control product, sample to be measured to corresponding test tube bottom.

2, add 30 μ l enzyme reaction things to each test tube.

3, add 30 μ l increased response agent to each test tube.

4, add 30 μ l magnetic separation agents to each test tube.

5, cover test tube with plastic sheeting, the multitube vortex mixer behind the tube shaken frame 30s, is put 37 ℃ of water-baths 30 minutes gently.

6, the test tube frame linking is put to magnetic separator, guarantees that every test tube all contacts with separator surface, precipitates 2 minutes.The separation vessel that reverses is slowly poured out supernatant, is placed on the test tube of reversing on the filter paper together with separation vessel, firmly bounces the separation vessel bottom to remove all drops that are bonded on the tube wall.

7, the cleaning fluid concentrate with 20 times of purified water dilutions after, add cleaning fluid after the 200 μ l dilution to each test tube, put the mixing 30s that vibrates gently on the multitube vortex mixer.Should avoid the application of sample dynamics excessive and cause magnetic bead to spill during application of sample.Mixing is wanted thoroughly.

8, repeating step is 6,7,6 one times.

9, added in 200 μ l substrate solution to the test tubes mixing 3 seconds, detect with ready luminous detection appearance rapidly.

10, as running into high value HOOK sample, for fear of high value HOOK effect occurring, the suggestion clinician selects suitable extension rate that sample is diluted according to all the other test indexs.

Clinical testing:

1, detects data

Sample picks up from the normal health check-up of 1200 examples, blood donor.The sample physical examination result does not all have liver, brain, kidney, disease of digestive tract, does not have blood transfusion and major operation history in half a year, and the women is not in the gestational period and lactation.Measured value is carried out statistical analysis, draw normal serum term of reference: 49-129ng/ml.The testing result of the kit of producing with external renowned company is compared, and kit of the present invention is more accurate, is accurate to 2 significant digits.

Notebook data is only for reference, and different regions, Different Individual and employing distinct methods detect, and measured TT4 level also can be different, advises the range of normal value of each laboratory foundation oneself.The TT4 value that can not only draw with this method is made diagnosis, only as the intermediate data reference role, should combine clinical other analyses result, comprises patient's concrete condition and treatment situation.TT4 concentration value and this kit measurement result in the sample that obtains through additive method do not have direct comparability.The sample that exceeds the kit measurement scope, system can't provide definite numerical value.Measure its definite result like desire, measure again after the suggestion dilution.The testing result of this kit only supplies clinical reference, can not be separately as the foundation of making a definite diagnosis or get rid of case, and for reaching diagnostic purpose, this testing result will be used in combination with clinical examination, medical history and other inspection.These article can be used for the mensuration of serum sample, and the reliability that is used for other body fluid samples TT4 concentration determination is fully confirmed.

2, kit performance index of the present invention

Sensitivity for analysis is defined as: to the mensuration of 20 times zero calibration objects, get its mean deviation of 2 times, its pairing concentration on typical curve is sensitivity for analysis; Sensitivity for analysis: 5ng/mL; Accuracy: CV%≤15.0%; The range of linearity: 5-300ng/ml; Linear coefficient: r>=0.9900; Specificity:

Cross reaction situation with main analog:

Claims

1. the quantitative determination reagent kit of a total thyroxin TT4 is characterized in that, this kit comprises TT4 magnetic separation agent, enzyme reaction thing, increased response agent, dilution, TT4 calibration object, TT4 quality-control product, cleaning fluid concentrate and substrate solution.

2. kit as claimed in claim 1 is characterized in that, described TT4 magnetic separation agent contains the magnetic microsphere that is marked with anti-TT4 monoclonal antibody;

Described enzyme reaction thing is the TT4 antigen that contains alkali phosphatase enzyme mark;

Described increased response agent is the damping fluid that contains Tris;

Said dilution is the solution that contains BSA;

Described calibration object and quality-control product are the BSA solution that contains a certain amount of TT4 antigen;

Said cleaning fluid concentrate is the damping fluid that contains TWEEN-20 and Proclin-300;

3. let alone a described kit like claim 1-2, it is characterized in that, each component makes according to the method for being prepared as follows in the said kit:

One, the preparation process of magnetic separation agent

One), magnetic bead buffer solution formulation operations step, the preparation 1L:

1, takes by weighing TRIS 4.58g and NaCl6.81g in the 1L container, take by weighing 0.96g TWEEN-20 adds suitable quantity of water it is dissolved fully in the 20ml container after, pour in the above-mentioned 1L container;

3, regulate PH, PH is between 7.95-8.05 in control;

4, taking by weighing BSA 3g pours in the above-mentioned 1L container;

5, be settled to 1000ml at last, promptly get with the filtration of 0.2um filter;

Two), the preparation process of magnetic separation agent

2, draw in the antibody-solutions that above-mentioned disuccinimidyl suberate joins step 1 with liquid-transfering gun, put room temperature 90min;

3, step 2 antibody-solutions is joined in the concentration tube, put into then in the high speed freezing centrifuge that centrifugal 30min is concentrated into 0.5ml under 3000g;

4, get the 0.5ml magnetic bead, add in the 5ml reaction cup, draw supernatant through magnet absorption after 2 minutes, add 1.5ml PH9.50.1mol/L PB then, mixing 30 seconds adds the antibody-solutions that step 3 obtains then, and room temperature reaction is 4 hours behind the mixing;

5, add 37 ℃ of the TRIS solution 15 minutes of 0.3ml 1mol/L, add 1.5ml PH 7.2 0.1mol/L PB then and clean the magnetic bead of mark, mixing 30 seconds;

6, preserve liquid with the 100ml magnetic bead and change magnetic bead over to vial;

7, solution that step 6 is obtained and above-mentioned magnetic bead buffer solution promptly get the magnetic separation agent in claim 1 or the 2 said kits according to 1: 1 volume ratio mixing;

Two, the preparation process of enzyme reaction thing

One), enzyme reaction thing diluent preparing operation steps, the preparation 1L:

2, transfer PH, PH is in the 7.35-7.45 scope in control;

3, taking by weighing BSA 3g pours in the above-mentioned beaker;

4, be settled to 1000ml at last, promptly get with the filtration of 0.2um filter;

Two), the coupling of alkaline phosphatase ALP and TT4 antigen

1, gets TT4 antigen;

4, the mol ratio that adds the ALP of 1mol according to 5mol antigen is added ALP toward step 3 solution in, adding 1mlPH then is that 7.4 concentration are the PB damping fluid of 0.1M, places 37 degree constant temperature ovens to react 3 hours;

5, with the solution of step 4 with PD-10 post purifying, collect refined solution, add above-mentioned steps one according to 1: 5000 volume ratio) the enzyme reaction thing dilution that obtains, mix, promptly get the enzyme reaction thing.

Three, the preparation process of increased response agent, preparation 1L:

1, get TRIS1.56g and NaCl 4.23g in the 1L container, get 0.2ml Proclin-300 in the beaker of 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;

2, get the 800ml purified water in above-mentioned 1L container, fully stir, transfer PH between 7.35-7.45 until dissolving fully;

3, take by weighing Mak33 0.9g in above-mentioned 1L container; Last constant volume 1000ml after the dissolving, promptly gets with the filtration of 0.2um filter fully;

Four, the preparation process of dilution, preparation 1L:

1, takes by weighing NaCl9.0g and BSA 60g in the container of 1L;

2, get 0.5ml Proclin-300 and add the dissolving of 10ml purified water, pour in the container of above-mentioned 1L;

3, last constant volume 1000ml after the dissolving, promptly gets with the filtration of 0.2um filter fully;

Five, the preparation of TT4 calibration object and TT4 quality-control product:

The concentration of TT4 antigen is respectively 20,40,80,150 in the TT4 calibration object, 300ng/ml; The concentration of TT4 antigen is respectively 40 in the TT4 quality-control product, 150ng/ml;

Six, the preparation process of cleaning fluid concentrate, preparation 1L:

1, gets TRIS 12.54g and NaCl 325.6g in the 1L container;

2, get 5g Tween-20 and in the 100ml container, add 20ml water it is dissolved fully after, pour in the above-mentioned 1L container;

3, get 0.2ml Proclin-300 in the beaker that fills the 10ml purified water fully the dissolving after, pour in the above-mentioned 1L container;

4, get the 800ml purified water in above-mentioned 1L container, fully stir, until dissolving fully;

5, transfer PH, control its scope between 7.35-7.45;

6, last constant volume 1000ml, the dissolving back is filtered with the 0.2um filter and is promptly got fully;

Seven, the preparation process of substrate solution, preparation 1L:

1, gets TRIS 2.35g, NaCl 6.41g, Na₂SO₃0.002g and Proclin-300 0.2ml is in the 1L beaker;

2, get the 600ml purified water in the 1L beaker, fully stir, transfer PH between 7.95-8.05 until dissolving fully;

3, add 250ml Lumi-Phos 480, filter with the 0.2um filter and collect filtrating, be settled to 1000ml, promptly get behind the mixing with purified water.

4. let alone a described kit like claim 1-3, it is characterized in that, the method for application of said kit is following:

1), adds 15 μ l TT4 calibration objects, TT4 quality-control product, sample to be measured to corresponding test tube bottom;

2), add 30 μ l enzyme reaction things to each test tube;

3), add 30 μ l increased response agent to each test tube;

4), add 30 μ l magnetic separation agents to each test tube;

5), cover test tube with plastic sheeting, the multitube vortex mixer behind the tube shaken frame 30s, is put 37 ℃ of water-baths 30 minutes gently;

6) put to magnetic separator, then, guarantee that every test tube all contacts with separator surface, precipitate 2 minutes, the reversing separation vessel is poured out supernatant;

7), the cleaning fluid concentrate with 20 times of purified water dilutions after, add cleaning fluid after the 200 μ l dilution to each test tube, put the mixing 30s that vibrates gently on the multitube vortex mixer;

8), added in 200 μ l substrate solution to the test tubes mixing 3 seconds, the luminous detection appearance detects.

5. kit as claimed in claim 4 is characterized in that, when said sample to be measured is high value HOOK sample, uses dilution that sample is diluted as required.