CN104165995A - Kit for detecting concentration of TNNI3K in blood as well as preparation method and application thereof - Google Patents
Kit for detecting concentration of TNNI3K in blood as well as preparation method and application thereof Download PDFInfo
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- CN104165995A CN104165995A CN201410392328.2A CN201410392328A CN104165995A CN 104165995 A CN104165995 A CN 104165995A CN 201410392328 A CN201410392328 A CN 201410392328A CN 104165995 A CN104165995 A CN 104165995A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6827—Total protein determination, e.g. albumin in urine
- G01N33/683—Total protein determination, e.g. albumin in urine involving metal ions
- G01N33/6836—Silver staining
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The invention discloses a kit for detecting the concentration of TNNI3K in blood as well as a preparation method and application thereof. The kit comprises a mouse anti-human TNNI3K monoclonal antibody aqueous solution with concentration of 0.1-3mu g/mL, a goat anti-mouse IgG (immunoglobulin G)(H+L) antibody aqueous solution with concentration of 1-20mu g/mL, mixed liquor of nano-gold and a goat anti-mouse IgG(H+L) antibody, PBS (phosphate buffered saline) of glycerol and a carrier with a small circle with depth and diameter of 0.5mm and 6mm respectively. The kit can be used for detecting the concentration of TNNI3K in blood, can detect 0.08ng of TNNI3K/spot at a minimum, consumes short time and is low in sample consumption.
Description
Technical field
the present invention relates to detection kit of the concentration of TNNI3K in a kind of blood and its preparation method and application.
Background technology
Ischemic heart disease many by comprise coronary artery obstruction that atherosclerotic lesion causes or narrow due to.Because ischemic heart disease has morbidity suddenly, the feature that mortality ratio is high, morbidity starts rear a few hours to 24 hour, and the early diagnosis in the time in 3 days to one week and corresponding treatment are the principal elements that determines its help rate height.In general, although according to the ischemic of cardiogram S-T section, raise and CPK, cardiac muscle troponin I, T (cTnI, cTnT), the indexs such as myoglobins (MB) rising can be made a definite diagnosis, still have a lot of cases be difficult to soon under with diagnosis.
Particularly measure serum myoglobin (MB) though can be used as the sensitiveest early stage index of acute myocardial infarction AMI (AMI) diagnosis, poor specificity, the diseases such as Skeletal muscle injury, wound, renal failure, all can cause its rising; Cardiac muscle troponin I, T (cTnI, cTnT), has relatively high specificity, but equally also can increase in some skeletal muscle disease blood.CTnI not only increases in acute myocardial infarction patients blood, at acute renal failure blood sample, also has rising.
Due to myocardial ischemia, the myocardium cell necrosis in various degree that myocardial infarction causes and leaking into causes in blood TNNI3K Enrichment in various degree in blood vessel.In blood, the detection of TNNI3K concentration mainly adopts enzyme linked immunosorbent assay to measure at present, but this detection method blood sample of growing, need to be more consuming time and the special technical matterss such as instrument.
Summary of the invention
One of of the present invention for solve the detection of TNNI3K concentration in above-mentioned blood consuming time long, need more blood sample and the special technical matterss such as instrument, and provide a kind of detection consuming time short, need blood sample amount few, do not need the detection kit of the concentration of TNNI3K in the blood of special instrument.
The preparation method of the detection kit of the concentration of TNNI3K in a kind of blood that two of object of the present invention is to provide above-mentioned.
The method that three of object of the present invention is to utilize the detection kit of the concentration of TNNI3K in above-mentioned blood to detect the concentration of TNNI3K in blood.
technical scheme of the present invention
A detection kit for the concentration of TNNI3K in blood, contains following component:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1～3 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1～20 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.2～0.24;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with the carrier of ringlet
Described carrier is polyvinylidene fluoride film, nitrocellulose filter, polystyrene film or the cellulose acetate membrane of the porous of aperture≤0.25 μ m;
The diameter of described ringlet is that 6mm, the degree of depth are 0.5mm.
In above-mentioned a kind of blood, the preparation method of the detection kit of the concentration of TNNI3K, specifically comprises the steps:
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, being made into concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1～3 μ g/mL;
(2), with distilled water, sheep anti mouse lgG (H+L) antibody is dissolved, being made into concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1～20 μ g/mL;
(3), the preparation of the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
The trisodium citrate aqueous solution that is 1% with mass percent concentration and mass percent concentration are that 0.01% aqueous solution of chloraurate calculates by volume, be that mass percent concentration is 1% trisodium citrate aqueous solution: mass percent concentration is that 0.01% aqueous solution of chloraurate is after the ratio of 1.5mL:100mL is mixed, stirring reaction to the reactant liquor of gained is claret, be chilled to room temperature, then it is 6.5～8.0 that the wet chemical that is 0.05～0.1mol/L by concentration regulates pH value, obtains nano-Au solution;
2., in step, 1. to add the concentration of step (2) gained in the nano-Au solution of gained be sheep anti mouse lgG (H+L) the antibody aqueous solution of 1～20 μ g/mL, obtains the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody;
In the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.2～0.24;
(4), the preparation of the PBS damping fluid of glycerine
It is in 7.4 sodium hydrogen phosphate aqueous solution, to mix and obtain the PBS damping fluid of glycerine that glycerine is joined to pH value, and wherein the concentration of volume percent of glycerine is 30%;
(5), with the preparation of carrier of ringlet
With card punch, on carrier, impress out ringlet;
Described carrier is polyvinylidene fluoride film, nitrocellulose filter, polystyrene film or the cellulose acetate membrane of the porous of aperture≤0.25 μ m;
The diameter of described ringlet is that 6mm, the degree of depth are 0.5mm.
The method of utilizing the detection kit of the concentration of TNNI3K in above-mentioned blood to detect the concentration of TNNI3K in blood, specifically comprises the steps:
(1), the PBS damping fluid of glycerine is added drop-wise to the ringlet of carrier from the front of carrier;
(2), until the PBS damping fluid of the glycerine of step (1), penetrate into after carrier, mouse-anti-human T NNI3K monoclonal antibody aqueous solution is added drop-wise to the ringlet of carrier from the front of carrier;
Until mouse-anti-human T NNI3K monoclonal antibody aqueous solution, penetrate into after carrier, blocking agent is added drop-wise to the ringlet of carrier from the front of carrier, after blocking agent penetrates into carrier, carrier is washed 3 times with washing lotion, distilled water successively;
Described blocking agent is that mass percent concentration is a kind of in the aqueous solution of bovine serum albumin(BSA), gelatin or milk of 2-10%;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
(3), on there is the plastic support of opening on both sides, put absorbent material, and then by the facing up of the carrier after step (2) washing, be placed on absorbent material;
Described absorbent material is one or more the potpourri in filter paper, absorbent cotton, sponge, water-absorption fiber;
(4), with distilled water by 6 times of diluting blood samples to be measured, by dilution after blood sample from the front of the carrier 1 of step (3) gained, be slowly added drop-wise to the ringlet of carrier, after blood sample penetrates into carrier;
By with dilution after the mixed liquor of the isopyknic nm of gold-sheep anti mouse of blood sample lgG (H+L) antibody from the front of carrier, slowly join the ringlet of carrier, mixed liquor until nm of gold-sheep anti mouse lgG (H+L) antibody penetrates into after carrier, carrier is washed 3 times with washing lotion, distilled water successively, then the silver-colored stain of 5 times of the blood sample volume for after dilution is joined rapidly the ringlet of carrier from the front of carrier, lucifuge reaction 1min, then washes away silver-colored stain with distilled water;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
Described silver-colored stain is the aqueous solution that contains silver nitrate and p-dihydroxy-benzene, and wherein the concentration of silver nitrate is that the concentration of 0.015mol/L and p-dihydroxy-benzene is 0.075mol/L;
According to the degree of depth that occurs the grey black spot colors of uniformity in ringlet, by colourimetry, can obtain the concentration of hemorrhage middle TNNI3K.
Beneficial effect of the present invention
The detection kit of the concentration of TNNI3K in a kind of blood of the present invention, owing to having adopted the carrier of mouse-anti-human T NNI3K monoclonal antibody high adsorption capacity, gold label silver stain technology, having replaced Static Adsorption with Dynamic Adsorption, thereby solved current employing enzyme linked immunosorbent assay, measure the detection method length consuming time of TNNI3K concentration in blood, need more blood sample and the special technical matterss such as instrument.
Utilize the detection kit of the concentration of TNNI3K in blood of the present invention to detect the TNNI3K in blood, it is consuming time shorter, from fixing mouse-anti-human T NNI3K monoclonal antibody, to going out result, only needs three minutes; Need the blood sample of use less, only need 0.33 μ L; Do not need the instrument detecting; Low energy detects 0.08ngTNNI3K/ point.
Accompanying drawing explanation
fig. 1 a,with the structural representation of carrier of ringlet, wherein 1 is carrier, 2 is the ringlet on carrier;
fig. 1 b,with ringlet carrier A-A to schematic diagram;
the front schematic view of Fig. 2, carrier 1;
fig. 3,the placement of plastic support 4, absorbent material 3 and carrier 1 is related to schematic diagram, is followed successively by plastic support 4, absorbent material 3 and carrier 1 from bottom to top;
fig. 4,there is the structural representation of the plastic support of opening on both sides.
Embodiment
Below by specific embodiment, also by reference to the accompanying drawings the present invention is further set forth, but do not limit the present invention.
embodiment 1
A detection kit for the concentration of TNNI3K in blood, comprising:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.2;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with the carrier of ringlet
Described with ringlet carrier structural representation as shown in Figure 1a, wherein 1 is carrier, 2 is the ringlet on carrier; Its A-A to schematic diagram as shown in Figure 1 b;
Described carrier 1 for aperture be the porous polyvinylidene fluoride film of 0.25 μ m;
The degree of depth and the diameter of described ringlet 2 are respectively 0.5mm and 6mm.
The preparation method of the detection kit of the concentration of TNNI3K in above-mentioned a kind of blood, concrete steps are as follows:
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, being made into concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1 μ g/mL, and step is as follows:
By the mouse-anti-human T NNI3K monoclonal antibody of 1mg, in 50mL small beaker, adding distil water dissolves it, moves in the volumetric flask of 100mL, with distilled water, is settled to 100mL, and obtaining concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 10 μ g/mL;
The concentration of getting 0.25mL be the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 10 μ g/mL in 25mL volumetric flask, with distilled water, be settled to 25mL, obtaining concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1 μ g/mL;
(2), with distilled water, sheep anti mouse lgG (H+L) antibody is dissolved, being made into concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1 μ g/mL, and step is as follows:
By sheep anti mouse lgG (H+L) antibody of 1mg, in 50mL small beaker, adding distil water dissolves it, moves in the volumetric flask of 100mL, with distilled water, is settled to 100mL, and obtaining concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 10 μ g/mL;
Sheep anti mouse lgG (H+L) antibody-solutions of getting 2.5mL concentration and be 10 μ g/mL, in 25mL volumetric flask, is settled to 25mL with distilled water, and obtaining concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1 μ g/mL;
(3), the preparation of the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
With the trisodium citrate aqueous solution that the mass percent concentration of 1.5mL is 1%, be after 0.01% aqueous solution of chloraurate mixes with the mass percent concentration of 100ml, stirring reaction to the reactant liquor of gained is claret, be chilled to room temperature, then it is 6.5 that the wet chemical that is 0.05mol/L by concentration regulates pH value, then with distilled water, be settled to 100ml, obtain nano-Au solution;
2., in 1.31mL step, 1. adding the concentration of step (2) gained of 2.5mL in the nm of gold of gained is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1 μ g/mL, then by the sodium hydrogen phosphate aqueous solution that concentration is 0.01 mol/L, be settled to 25ml, obtain the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody;
In the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.2;
(4), the preparation of the PBS damping fluid of glycerine
It is in 7.4 sodium hydrogen phosphate aqueous solution, to mix and obtain the PBS damping fluid of glycerine that glycerine is joined to pH value, and wherein the concentration of volume percent of glycerine is 30%;
(5), with the preparation of carrier 1 of ringlet 2
With card punch, on carrier 1, impress out ringlet 2;
Wherein carrier 1 for aperture be the porous polyvinylidene fluoride film of 0.25 μ m;
Described ringlet 2, its degree of depth and diameter are respectively 0.5mm and 6mm.
Utilize the detection kit of the concentration of TNNI3K in above-mentioned blood to detect the concentration of TNNI3K in blood, this detection method, specifically comprises the steps:
(1), first, the PBS damping fluid of 10 μ L glycerine is added drop-wise to the ringlet 2 of carrier 1 from the front of carrier 1, the front of described carrier 1 as shown in Figure 2;
(2), the PBS damping fluid until the glycerine of step (1) penetrates into after carrier 1, the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that by 2 μ L concentration is 0.1 μ g/mL is added drop-wise to the ringlet 2 of carrier 1 from the front of the carrier 1 of step (1), after penetrating into carrier 1, the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that is 0.1 μ g/mL until concentration adds 2 μ L blocking agents, after blocking agent penetrates into carrier 1, carrier 1 is washed 3 times with washing lotion, distilled water successively;
Described blocking agent is that mass percent concentration is the aqueous solution of 2% bovine serum albumin(BSA);
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
(3), on having the plastic support 4 of opening, both sides put absorbent material 3, and then facing up the carrier 1 after step (2) processing, be placed on absorbent material 3, concrete placement schematic diagram as shown in Figure 3, is followed successively by plastic support 4, absorbent material 3 and carrier 1 from bottom to top;
Described both sides have opening plastic support 4 structural representation as shown in Figure 4;
Described absorbent material 3 is filter paper;
(4), then, with distilled water, by 6 times of diluting blood samples to be measured, the blood sample after 2 μ L dilutions is slowly dripped in the ringlet 2 of carrier 1 from the front of the carrier 1 of step (3) gained;
After blood sample penetrates into carrier 1, again the mixed liquor of 2 μ L nm of gold-sheep anti mouse lgG (H+L) antibody is slowly added to the ringlet 2 of carrier 1 from the front of carrier 1, mixed liquor until nm of gold-sheep anti mouse lgG (H+L) antibody penetrates into after carrier 1, carrier 1 is washed 3 times with washing lotion, distilled water successively, then rapidly 10 μ L silver stains are joined the ringlet 2 of carrier 1 from the front of carrier 1, lucifuge reaction 1min, then washes away silver-colored stain with 100 μ L distilled water;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
Described silver-colored stain is the aqueous solution that contains silver nitrate and p-dihydroxy-benzene, and wherein the concentration of silver nitrate is that the concentration of 0.015mol/L and p-dihydroxy-benzene is 0.075mol/L;
According to there is the degree of depth of the grey black spot colors of uniformity in the ringlet 2 of carrier 1, by colourimetry, show that the amount of every some TNNI3K is 0.67ngTNNI3K/ point, the concentration 2010ng/mL of TNNI3K in blood.
embodiment 2
A detection kit for the concentration of TNNI3K in blood, comprising:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 3 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 20 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.24;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with the carrier of ringlet
Described carrier is that aperture is the nitrocellulose filter of the porous of 0.22 μ m;
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm.
The preparation method of the detection kit of the concentration of TNNI3K in above-mentioned a kind of blood, concrete steps are as follows:
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, being made into concentration is the mouse-anti people of 3 μ g/mL
TNNI3K monoclonal antibody aqueous solution, step is as follows:
By the mouse-anti-human T NNI3K monoclonal antibody of 1mg, in 50mL small beaker, adding distil water dissolves it, moves in the volumetric flask of 100mL, with distilled water, is settled to 100mL, and obtaining concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 10 μ g/mL;
Get 7.5mL concentration and be the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 10 μ g/mL in 25mL volumetric flask, with distilled water, be settled to 25mL, obtaining concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 3 μ g/mL;
(2), with distilled water, sheep anti mouse lgG (H+L) antibody is dissolved, being made into concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 20 μ g/mL, and step is as follows:
By sheep anti mouse lgG (H+L) antibody of 1mg, in 50mL small beaker, adding distil water dissolves it, moves in the volumetric flask of 50mL, with distilled water, is settled to 50mL, and obtaining concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 20 μ g/mL;
(3), the preparation of the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
With the trisodium citrate aqueous solution that the mass percent concentration of 1.5mL is 1%, be after 0.01% aqueous solution of chloraurate mixes with the mass percent concentration of 100ml, stirring reaction to the reactant liquor of gained is claret, be chilled to room temperature, then it is 8.0 that the wet chemical that is 0.1mol/L by concentration regulates pH value, then with distilled water, be settled to 100ml, obtain nano-Au solution;
2., in 1.31mL step, 1. adding the concentration of step (2) gained of 0.15mL in the nano-Au solution of gained is sheep anti mouse lgG (H+L) the antibody aqueous solution of 20 μ g/mL, then by the sodium hydrogen phosphate aqueous solution that concentration is 0.01 mol/L, be settled to 25ml, obtain the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody;
In the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.24;
(4), the preparation of the PBS damping fluid of glycerine
It is in 7.4 sodium hydrogen phosphate aqueous solution, to mix and obtain the PBS damping fluid of glycerine that glycerine is joined to pH value, and wherein the concentration of volume percent of glycerine is 30%;
(5), with the preparation of carrier of ringlet
With card punch, on carrier, impress out ringlet;
Described carrier is that aperture is the nitrocellulose filter of the porous of 0.22 μ m;
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm.
The method of utilizing the detection kit of the concentration of TNNI3K in above-mentioned blood to detect the concentration of TNNI3K in blood, concrete steps are as follows:
(1), first, the PBS damping fluid of 10 μ L glycerine is added drop-wise to the ringlet of carrier from the front of carrier;
(2), until the PBS damping fluid of the glycerine of step (1), penetrate into after carrier, then by 2 μ L concentration, be that the mouse-anti-human T NNI3K monoclonal antibody solution of 3 μ g/mL is added drop-wise to the ringlet of carrier from the front of carrier;
After the mouse-anti-human T NNI3K monoclonal antibody solution that is 3 μ g/mL until concentration penetrates in carrier, then 2 μ L blocking agents are added drop-wise to the ringlet of carrier from the front of carrier, after blocking agent penetrates into carrier, carrier are washed 3 times with washing lotion, distilled water successively;
Described blocking agent is that mass percent concentration is 10% milk aqueous solution;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
(3), on there is the plastic support of opening on both sides, put absorbent material, and then the facing up of the carrier after step (2) is processed, be placed on absorbent material;
Described absorbent material is absorbent cotton;
(4), then, with distilled water, by 6 times of diluting blood samples to be measured, the blood sample after 2 μ L dilutions is slowly dripped in the ringlet of carrier from the front of the carrier of step (3) gained;
After blood sample penetrates into carrier, again the mixed liquor of 2 μ L nm of gold-sheep anti mouse lgG (H+L) antibody is slowly added to the ringlet of carrier from the front of carrier, mixed liquor until nm of gold-sheep anti mouse lgG (H+L) antibody penetrates into after carrier, carrier is washed 3 times with washing lotion, distilled water successively, then rapidly 10 μ L silver stains are joined the ringlet of carrier from the front of carrier, lucifuge reaction 1min, then washes away silver-colored stain with 100 μ L distilled water;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
Described silver-colored stain is the aqueous solution that contains silver nitrate and p-dihydroxy-benzene, and wherein the concentration of silver nitrate is that the concentration of 0.015mol/L and p-dihydroxy-benzene is 0.075mol/L;
According to the degree of depth that occurs the grey black spot colors of uniformity in the ringlet of carrier, by colourimetry, show that the amount of every some TNNI3K is 0.08ngTNNI3K/ point, the concentration 240ng/mL of TNNI3K in blood.
embodiment 3
A detection kit for the concentration of TNNI3K in blood, comprising:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 1.5 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 10 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.22;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with the carrier of ringlet
Described carrier is that aperture is the porous cellulose acetate membrane of 0.20 μ m;
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm.
The preparation method of the detection kit of the concentration of TNNI3K in above-mentioned a kind of blood, concrete steps are as follows:
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, being made into concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 1.5 μ g/mL, and step is as follows:
By the mouse-anti-human T NNI3K monoclonal antibody of 1mg, in 50mL small beaker, adding distil water dissolves it, moves in the volumetric flask of 100mL, with distilled water, is settled to 100mL, and obtaining concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 10 μ g/mL;
Get 3.75mL concentration and be the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 10 μ g/mL in 25mL volumetric flask, with distilled water, be settled to 25mL, obtaining concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 1.5 μ g/mL;
(2), with distilled water, sheep anti mouse lgG (H+L) antibody is dissolved, being made into concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 10 μ g/mL, and step is as follows:
By sheep anti mouse lgG (H+L) antibody of 1mg, in 50mL small beaker, adding distil water dissolves it, moves in the volumetric flask of 100mL, with distilled water, is settled to 100mL, and obtaining concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 10 μ g/mL;
(3), the preparation of the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
With the trisodium citrate aqueous solution that the mass percent concentration of 1.5mL is 1%, be after 0.01% aqueous solution of chloraurate mixes with the mass percent concentration of 100.0mL, stirring reaction to the reactant liquor of gained is claret, be chilled to room temperature, then it is 7.5 that the wet chemical that is 0.1mol/L by concentration regulates pH value, then with distilled water, be settled to 100ml, obtain nano-Au solution;
2., in 2.61mL step, 1. adding the concentration of 0.55mL step (2) gained in the nano-Au solution of gained is sheep anti mouse lgG (H+L) the antibody aqueous solution of 10 μ g/mL, then the sodium hydrogen phosphate aqueous solution that is 0.01mol/L by concentration is settled to 25ml, obtains the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody;
In the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.22;
(4), the preparation of the PBS damping fluid of glycerine
It is in 7.4 sodium hydrogen phosphate aqueous solution, to mix and obtain the PBS damping fluid of glycerine that glycerine is joined to pH value, and wherein the concentration of volume percent of glycerine is 30%;
(5), with the preparation of carrier of ringlet
With card punch, on carrier, impress out ringlet;
Wherein carrier is that aperture is the porous cellulose acetate membrane of 0.20 μ m;
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm.
The method of utilizing the detection kit of the concentration of TNNI3K in above-mentioned blood to detect the concentration of TNNI3K in blood, concrete steps are as follows:
(1), first, the PBS damping fluid of 10 μ L glycerine is added drop-wise to the ringlet of carrier from the front of carrier;
(2), until the PBS damping fluid of the glycerine of step (1), penetrate into after carrier, then the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that is 1.5 μ g/mL by 2 μ L concentration is added drop-wise to the ringlet of carrier from the front of carrier;
The mouse-anti-human T NNI3K monoclonal antibody aqueous solution that is 1.5 μ g/mL until concentration penetrates into after carrier, then 2 μ L blocking agents are joined the ringlet of carrier from the front of carrier, after blocking agent penetrates into carrier, carrier is washed 3 times with washing lotion, distilled water successively;
Described blocking agent is that mass percent concentration is the aqueous solution of 5% gelatin;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
(3), on there is the plastic support of opening on both sides, put absorbent material, and then by the facing up of the carrier after step (2) washing, be placed on absorbent material;
Described absorbent material is sponge;
(4), then, with distilled water, by 6 times of diluting blood samples to be measured, the blood sample after 2 μ L dilutions is slowly dripped in the ringlet of carrier from the front of the carrier of step (3) gained;
After blood sample penetrates into carrier, again the mixed liquor of 2 μ L nm of gold-sheep anti mouse lgG (H+L) antibody is slowly added to the ringlet of carrier from the front of carrier, mixed liquor until nm of gold-sheep anti mouse lgG (H+L) antibody penetrates into after carrier, carrier is washed 3 times with washing lotion, distilled water successively, then rapidly 10 μ L silver stains are joined the ringlet of carrier from the front of carrier, lucifuge reaction 1min, then washes away silver-colored stain with 100 μ L distilled water;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
Described silver-colored stain is the aqueous solution that contains silver nitrate and p-dihydroxy-benzene, and wherein the concentration of silver nitrate is that the concentration of 0.015mol/L and p-dihydroxy-benzene is 0.075mol/L;
According to the degree of depth that occurs the grey black spot colors of uniformity in the ringlet of carrier, by colourimetry, show that the amount of every some TNNI3K is 0.57ngTNNI3K/ point, the concentration 1710ng/mL of TNNI3K in blood.
embodiment 4
Embodiment 4 is identical with the experiment condition of embodiment 3, and the standard solution that is just 1710ng/mL by the concentration of TNNI3K substitutes blood sample, does 3 parallel laboratory tests.6 times of the standard solution that is 1710ng/mL with distilled water by the concentration of TNNI3K dilutions
,with the solution of the TNNI3K after 2 μ L dilutions, test.
According to the degree of depth that occurs the grey black spot colors of uniformity in the ringlet of carrier, by colourimetry, show that the amount of every some TNNI3K is respectively 0.56,0.56,0.56ngTNNI3K/ point, concentration 1680,1680, the 1680ng/mL of TNNI3K in blood, the recovery of TNNI3K is respectively 98.25%, 98.25% and 98.25%.
From above-mentioned experimental result, showing, is reliable and stable by the method that the detection kit of the concentration of TNNI3K in blood of the present invention detects the concentration of TNNI3K in blood.
control Example 1(measures the concentration of TNNI3K in embodiment 3 blood samples with enzyme linked immunosorbent assay)
With Kirkegaard & Perry Laboratories, " the Protein Detector of Inc.
tMeLISA Kit " to test, concrete steps are as follows:
(1), by the mouse-anti-human T NNI3K monoclonal antibody of 25mg in 50mL small beaker, adding distil water dissolves it, moves in the volumetric flask of 25mL, with distilled water, is settled to scale, obtaining concentration is 1mg/mL mouse-anti-human T NNI3K monoclonal antibody solution;
(2), with pipettor get 88 μ L concentration be 1mg/mL mouse-anti-human T NNI3K monoclonal antibody aqueous solution in a hole of 96 orifice plates, add 100 μ L immobile liquids, place 120min, remove loose solution, with 150 μ L washing lotions cleanings, wash 3 times;
(3), add 300 μ L blocking agents, place 60min, remove unnecessary blocking agent;
(4), add 2.5 μ L blood samples, place 120min, with 150 μ L washing lotions cleanings, wash 4 times;
(5), add the anti-solution of 100 μ L bis-, place 60min, with 100 μ L washing lotions cleanings, wash 4 times;
(6), add 100 μ L enzyme stains, placement 30min;
(7), add 100 μ L and stop agent, the concentration that determines TNNI3K in this blood sample by MTP-310Lab type microplate reader is 1706ng/mL.
By above-mentioned control Example 1 and the testing result of embodiment 3, contrast, can find out, utilize kit of the present invention to detect, the testing result of gained is compared with traditional enzyme linked immunosorbent assay, and its testing result is accurate, and degree of accuracy is high.Further, can find out and utilize kit of the present invention to detect, it is consuming time shorter, from fixing mouse-anti-human T NNI3K monoclonal antibody, to going out result, only need three minutes, and traditional enzyme linked immunosorbent assay in control Example 1, from fixing mouse-anti-human T NNI3K monoclonal antibody, needs 390 minutes to going out result.And utilize kit of the present invention to detect, required blood sample is less, only need 0.33 μ L, and traditional enzyme linked immunosorbent assay needs 2.5 μ L.Further, can find out and utilize kit of the present invention to detect blood sample, do not need special detecting instrument, and traditional enzyme linked immunosorbent assay need be used microplate reader.
The above is only giving an example of embodiments of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the technology of the present invention principle; can also make some improvement and modification, these improve and modification also should be considered as protection scope of the present invention.
Claims (7)
1. a detection kit for the concentration of TNNI3K in blood, is characterized in that comprising the detection kit of the concentration of TNNI3K in described blood:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1～3 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1～20 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
The mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody is prepared by a method comprising the following steps and forms:
1., the preparation of nano-Au solution
The trisodium citrate aqueous solution that is 1% with mass percent concentration and mass percent concentration are that 0.01% aqueous solution of chloraurate calculates by volume, be that mass percent concentration is 1% trisodium citrate aqueous solution: mass percent concentration is that 0.01% aqueous solution of chloraurate is after the ratio of 1.5mL:100mL is mixed, stirring reaction to the reactant liquor of gained is claret, be chilled to room temperature, then it is 6.5～8.0 that the wet chemical that is 0.05～0.1mol/L by concentration regulates pH value, obtains nano-Au solution;
2., in step, 1. to add the concentration of step (2) in the nano-Au solution of gained be sheep anti mouse lgG (H+L) the antibody aqueous solution of 1～20 μ g/mL, obtains the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody;
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.2～0.24;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with diameter, be the carrier of 6mm, the degree of depth ringlet that is 0.5mm
Described carrier is polyvinylidene fluoride film, nitrocellulose filter, polystyrene film or the cellulose acetate membrane of the porous of aperture≤0.25 μ m.
2. the detection kit of the concentration of TNNI3K in a kind of blood as claimed in claim 1, is characterized in that comprising the detection kit of the concentration of TNNI3K in described blood:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.2;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with the carrier of ringlet
Described carrier is that aperture is the porous polyvinylidene fluoride film of 0.25 μ m;
The degree of depth of described ringlet and diameter are respectively 0.5mm and 6mm.
3. the detection kit of the concentration of TNNI3K in a kind of blood as claimed in claim 1, is characterized in that comprising the detection kit of the concentration of TNNI3K in described blood:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 3 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 20 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.24;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with the carrier of ringlet
Described carrier is that aperture is the nitrocellulose filter of the porous of 0.22 μ m;
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm.
4. the detection kit of the concentration of TNNI3K in a kind of blood as claimed in claim 1, is characterized in that comprising the detection kit of the concentration of TNNI3K in described blood:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 1.5 μ g/mL;
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 10 μ g/mL;
(3), the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.22;
(4), the PBS damping fluid of glycerine
The PBS damping fluid of described glycerine, being about to glycerine, to join pH value be to mix and obtain in 7.4 sodium hydrogen phosphate aqueous solution, wherein the concentration of volume percent of glycerine is 30%;
(5), with the carrier of ringlet
Described carrier is that aperture is the porous cellulose acetate membrane of 0.20 μ m;
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm.
5. the preparation method of the detection kit of the concentration of TNNI3K in a kind of blood as claimed in claim 1, is characterized in that specifically comprising the steps:
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, being made into concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.1～3 μ g/mL;
(2), with distilled water, sheep anti mouse lgG (H+L) antibody is dissolved, being made into concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1～20 μ g/mL;
(3), the preparation of the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
The trisodium citrate aqueous solution that is 1% with mass percent concentration and mass percent concentration are that 0.01% aqueous solution of chloraurate calculates by volume, be that mass percent concentration is 1% trisodium citrate aqueous solution: mass percent concentration is that 0.01% aqueous solution of chloraurate is after the ratio of 1.5mL:100mL is mixed, stirring reaction to the reactant liquor of gained is claret, be chilled to room temperature, then it is 6.5～8.0 that the wet chemical that is 0.05～0.1mol/L by concentration regulates pH value, obtains nano-Au solution;
2., in step, 1. to add the concentration of step (2) gained in the nano-Au solution of gained be sheep anti mouse lgG (H+L) the antibody aqueous solution of 1～20 μ g/mL, obtains the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody;
In the mixed liquor of nm of gold-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nm of gold: sheep anti mouse lgG (H+L) antibody is 5:0.2～0.24;
(4), the preparation of the PBS damping fluid of glycerine
It is in 7.4 sodium hydrogen phosphate aqueous solution, to mix and obtain the PBS damping fluid of glycerine that glycerine is joined to pH value, and wherein the concentration of volume percent of glycerine is 30%;
(5), with the preparation of carrier of ringlet
With card punch, on carrier, impress ringlet;
Described carrier is polyvinylidene fluoride film, nitrocellulose filter, polystyrene film or the cellulose acetate membrane of the porous of aperture≤0.25 μ m;
The diameter of described ringlet is that 6mm, the degree of depth are 0.5mm.
6. in the blood as described in as arbitrary in claim 1-4, the detection kit of the concentration of TNNI3K is for the detection of the concentration of blood TNNI3K.
7. the method that the detection kit of utilizing the concentration of TNNI3K in blood as claimed in claim 6 detects the concentration of TNNI3K in blood, is characterized in that specifically comprising the steps:
(1), the PBS damping fluid of glycerine is added drop-wise to the ringlet of carrier from the front of carrier;
(2), until the PBS damping fluid of the glycerine of step (1), penetrate into after carrier, mouse-anti-human T NNI3K monoclonal antibody aqueous solution is added drop-wise to the ringlet of carrier from the front of carrier;
Until mouse-anti-human T NNI3K monoclonal antibody aqueous solution, penetrate into after carrier, blocking agent is added drop-wise to the ringlet of carrier from the front of carrier;
After blocking agent penetrates into carrier, carrier is washed 3 times with washing lotion, distilled water successively;
Described blocking agent is that mass percent concentration is the aqueous solution of bovine serum albumin(BSA), gelatin or the milk of 2-10%;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
(3), on there is the plastic support of opening on both sides, put absorbent material, and then by the facing up of the carrier after step (2) washing, be placed on absorbent material;
Described absorbent material is one or more the potpourri in filter paper, absorbent cotton, sponge, water-absorption fiber;
(4), with distilled water by 6 times of diluting blood samples to be measured, by dilution after blood sample from the front of the carrier of step (3) gained, be added drop-wise to the ringlet of carrier, after blood sample penetrates into carrier;
By with dilution after the mixed liquor of the isopyknic nm of gold-sheep anti mouse of blood sample lgG (H+L) antibody from the front of carrier, join the ringlet of carrier, mixed liquor until nm of gold-sheep anti mouse lgG (H+L) antibody penetrates into after carrier, carrier is washed 3 times with washing lotion, distilled water successively, then the silver-colored stain of 5 times of the blood sample volume for after dilution is joined the ringlet of carrier from the front of carrier, lucifuge reaction 1min, then washes away silver-colored stain with distilled water;
Described washing lotion is that pH value is the solution that adds Tween80, NaCl to obtain in 7.4 sodium hydrogen phosphate aqueous solution, and the mass percent concentration that wherein mass percent concentration of Tween80 is 0.5%, NaCl is 10%;
Described silver-colored stain is the aqueous solution that contains silver nitrate and p-dihydroxy-benzene, and wherein the concentration of silver nitrate is that the concentration of 0.015mol/L and p-dihydroxy-benzene is 0.075mol/L;
According to the degree of depth that occurs the grey black spot colors of uniformity in the ringlet of carrier, by colourimetry, obtain the concentration of hemorrhage middle TNNI3K.
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| CN104330567A (en) * | 2014-08-11 | 2015-02-04 | 上海应用技术学院 | Detection kit of concentration of TNNI3K in blood, preparation method and application thereof |
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| WO2006057278A1 (en) * | 2004-11-24 | 2006-06-01 | Kumamoto University | Remedy for heart disease using map kinase tnni3k |
| WO2010062365A2 (en) * | 2008-10-31 | 2010-06-03 | Duke University | A method of protecting against heart failure |
| CN102965428A (en) * | 2011-09-30 | 2013-03-13 | 康旭基因技术（北京）有限公司 | Kit for testing and identifying genetic cardiac hypertrophy related gene mutation |
| CN103267855A (en) * | 2013-05-06 | 2013-08-28 | 南京凯基生物科技发展有限公司 | CTnI, Myo and CK-MB three-in-one colloidal gold test strip, kit thereof, and making methods of test strip and kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2006057278A1 (en) * | 2004-11-24 | 2006-06-01 | Kumamoto University | Remedy for heart disease using map kinase tnni3k |
| EP1832299A1 (en) * | 2004-11-24 | 2007-09-12 | Kumamoto University | Remedy for heart disease using map kinase tnni3k |
| WO2010062365A2 (en) * | 2008-10-31 | 2010-06-03 | Duke University | A method of protecting against heart failure |
| CN102965428A (en) * | 2011-09-30 | 2013-03-13 | 康旭基因技术（北京）有限公司 | Kit for testing and identifying genetic cardiac hypertrophy related gene mutation |
| CN103267855A (en) * | 2013-05-06 | 2013-08-28 | 南京凯基生物科技发展有限公司 | CTnI, Myo and CK-MB three-in-one colloidal gold test strip, kit thereof, and making methods of test strip and kit |
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| CN104330567A (en) * | 2014-08-11 | 2015-02-04 | 上海应用技术学院 | Detection kit of concentration of TNNI3K in blood, preparation method and application thereof |
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