CN104330567B - Detection kit of concentration of TNNI3K and its preparation method and application in a kind of blood - Google Patents

Detection kit of concentration of TNNI3K and its preparation method and application in a kind of blood Download PDF

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CN104330567B
CN104330567B CN201410391773.7A CN201410391773A CN104330567B CN 104330567 B CN104330567 B CN 104330567B CN 201410391773 A CN201410391773 A CN 201410391773A CN 104330567 B CN104330567 B CN 104330567B
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concentration
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antibody
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sheep anti
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CN104330567A (en
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刘小珍
王珊珊
刘小舟
陈捷
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Shanghai Institute of Technology
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    • G—PHYSICS
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    • 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
    • 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
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    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure

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Abstract

The present invention discloses detection kit of concentration of TNNI3K and its preparation method and application in a kind of blood, the detection kit of the concentration of TNNI3K in described blood, including concentration be 0.01～0.3 μ g/mL mouse-anti-human T NNI3K monoclonal antibody aqueous solution, concentration be 0.1～2 μ g/mL sheep anti mouse lgG (H+L) antibody aqueous solution, concentration be 1～5mg/mL rare earths salt, the mixed liquor of nanometer gold nano rare earth sheep anti mouse lgG (H+L) antibody, the PBS of glycerol, be respectively the carrier of the ringlet of 0.5mm and 6mm with the degree of depth and diameter.In this blood, the detection kit of the concentration of TNNI3K may be used for detecting the concentration of TNNI3K in blood, can detect 0.008ngTNNI3K/ point, and the shortest, and consumption sample amount is few.

Description

Detection kit of concentration of TNNI3K and its preparation method and application in a kind of blood
Technical field
The present invention relates to detection kit of concentration of TNNI3K and its preparation method and application in a kind of blood.
Background technology
Ischemic heart desease is many by the coronary artery obstruction or narrow caused including that atherosclerotic lesion causes.Due to ischemia Property heart disease have morbidity suddenly, the feature that mortality rate is high, morbidity starts rear a few hours to 24 hours, the time in 3 days to one weeks In early diagnosis and corresponding treatment be the principal element determining its help rate height.In general, although according to electrocardiogram S-T The ischemic of section is raised and CPK, and cardiac muscle troponin I, T (cTnI, cTnT), it is permissible that Myoglobin (MB) rises high index Make a definite diagnosis, but with diagnosis under still having a lot of cases to be difficult to soon.
Particularly measure serum myoglobin (MB) though the early stage that can diagnose as acute myocardial infarction (AMI) is the sensitiveest Index, but poor specificity, the disease such as Skeletal muscle injury, wound, renal failure, all may result in it and raise；Myocardium myo calcium egg White I, T (cTnI, cTnT), have relatively high specificity, but the most also can increase in some skeletal muscle disease blood.CTnI is not But increase in acute myocardial infarction patients blood, also have rising at acute renal failure blood sample.
Due to myocardial ischemia, myocardium cell necrosis in various degree that myocardial infarction causes and leak into and blood vessel cause blood In TNNI3K concentration in various degree increase.In blood, the detection of TNNI3K concentration mainly uses enzyme linked immunosorbent assay to measure at present, but this inspection Survey method is the longest, need the technical problem such as more blood sample and special instrument.
Summary of the invention
The present invention one of in order to solve, the detection of TNNI3K concentration in above-mentioned blood is the longest, need more blood sample And the technical problem such as special instrument, and provide that a kind of detection is the shortest, need blood sample amount few, it is not necessary to the blood of special instrument The detection kit of the concentration of middle TNNI3K.
The two of the purpose of the present invention are to provide the preparation of the detection kit of the concentration of TNNI3K in above-mentioned a kind of blood Method.
The three of the purpose of the present invention are to utilize in above-mentioned blood the detection kit of the concentration of TNNI3K in blood The method that the concentration of TNNI3K carries out detecting.
Technical scheme
The detection kit of the concentration of TNNI3K in a kind of blood, including:
(1), concentration is 0.01～0.3 μ g/mL mouse-anti-human T NNI3K monoclonal antibody aqueous solution；
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 0.1～2 μ g/mL；
(3), concentration is the rare earths salt of 1～5mg/mL；
Its middle rare earth is the one in lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutecium, yttrium, scandium or two Plant above lucium；
(4), the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. receive Meter Jin: nano rare earth: sheep anti mouse lgG (H+L) antibody is 2.5～1.0:1～1.6:0.2～0.24；
(5), the PBS of glycerol
The PBS of described glycerol, will glycerol to join pH value be that in 7.4 disodium hydrogen phosphate aqueous solutions mixing is all Even and obtain, wherein the concentration of volume percent of glycerol is 30%；
(6), with the carrier of ringlet
Described carrier is the polyvinylidene fluoride film of the porous of aperture≤0.25 μm, nitrocellulose filter, polystyrene film Or cellulose acetate membrane；
A diameter of 6mm of described ringlet, the degree of depth are 0.5mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L.
The preparation method of the detection kit of the concentration of TNNI3K in above-mentioned a kind of blood, specifically includes following steps:
(1), mouse-anti-human T NNI3K monoclonal antibody being dissolved with distilled water, being made into concentration is 0.01～0.3 μ g/mL mouse-anti People's TNNI3K monoclonal antibody aqueous solution；
(2), being dissolved by sheep anti mouse lgG (H+L) antibody with distilled water, being made into concentration is 0.1～2 μ g/mL sheep anti mouse lgG (H + L) antibody aqueous solution；
(3), the preparation of rare earths salt
Being dissolved in by rare earth oxide in concentrated nitric acid or aqueous hydrochloric acid solution that mass percent concentration is 37%, obtaining concentration is 1 ～the rare earths salt of 5mg/mL；
Its middle rare earth is lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutecium, yttrium, scandium oxide In a kind of or rare earth oxide of more than two；
(4), the preparation of the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
It is 0.01% gold chloride with the trisodium citrate aqueous solution that mass percent concentration is 1% and mass percent concentration Aqueous solution calculates by volume, i.e. mass percent concentration is the trisodium citrate aqueous solution of 1%: mass percent concentration is 0.01% aqueous solution of chloraurate is the ratio mixing of 1.5mL:100mL, and stirring is claret to solution, is cooled to room temperature, gained Reactant liquor concentration be 0.05～0.1mol/L wet chemical regulation pH value be 6.5～8.0, obtain nano-Au solution；
2., the preparation of nano rare earth solution
Molten with the rare-earth salts that concentration is 1～5mg/mL of (3) gained with the tannic acid solution that mass percent concentration is 1% Liquid calculates by volume, i.e. 1% tannic acid solution: the rare earths salt of 1～5mg/mL is that the ratio of 10mL:1～0.2mL is carried out After mixing, under stirring condition, carry out reduction reaction 30min, the potassium carbonate that reactant liquor concentration is 0.05～0.1mol/L of gained Aqueous solution regulation pH value is 6.5～8.0, obtains nano rare earth solution；
3., the nano rare earth solution of the nano-Au solution of step 1. gained Yu step 2. gained is pressed nanometer gold: Nano Rare The ratio that mass ratio is 2.5～1.0:1～1.6 of soil mixes, and obtains the mixed solution of nanometer gold and nano rare earth；
4. in the nanometer gold of step 3. gained and the mixed solution of nano rare earth, add the sheep anti mouse lgG (H of (2) gained + L) antibody-solutions, obtain the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody；
In the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: receive Rice rare earth: sheep anti mouse lgG (H+L) antibody is 2.5～1.0:1～1.6:0.2～0.24；
(5), the preparation of the PBS of glycerol；
It is the PBS that in 7.4 disodium hydrogen phosphate aqueous solutions, mix homogeneously i.e. obtains glycerol that glycerol joins pH value, its The concentration of volume percent of middle glycerol is 30%；
(6), with the preparation of carrier of ringlet
On carrier, ringlet is imprinted out with card punch；
Described carrier is the polyvinylidene fluoride film of the porous of aperture≤0.25 μm, nitrocellulose filter, polystyrene film Or cellulose acetate membrane；
A diameter of 6mm of described ringlet, the degree of depth are 0.5mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L.
Utilize the side that in above-mentioned blood, the concentration of TNNI3K in blood is detected by the detection kit of the concentration of TNNI3K Method, specifically includes following steps:
(1), the PBS of glycerol is added drop-wise to the ringlet of carrier from the front of carrier；
(2), after the PBS of the glycerol of step (1) penetrates into carrier, then by mouse-anti-human T NNI3K monoclonal antibody Solution is added drop-wise to the ringlet of carrier from the front of carrier；
After mouse-anti-human T NNI3K monoclonal antibody solution penetrates into carrier, blocker is joined load from the front of carrier In the ringlet of body, after blocker penetrates into carrier, carrier is used washing liquid, distilled water wash 3 times successively；
Described blocker be mass percent concentration be the bovine serum albumin of 0.2-1%, gelatin or milk aqueous solution；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
(3), on both sides have above the plastic support of opening and put absorbent material, after then step (2) being processed Carrier faces up and is placed on absorbent material；
Described absorbent material is one or more the mixture in filter paper, absorbent cotton, sponge；
(4), with distilled water by diluting blood sample 60 times to be measured, by the blood sample after dilution from the carrier of step (3) gained Front is added drop-wise in the ringlet of carrier；
After blood penetrates into carrier, by with dilution after the isopyknic nanometer gold-nano rare earth-sheep anti mouse lgG of blood sample (H+L) mixed liquor of antibody joins the ringlet of carrier from the front of carrier；
After the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody penetrates into carrier, carrier is used successively Washing liquid, distilled water wash 3 times, then will join carrier for the silver staining agent of the blood sample volume 5 times after dilution from the front of carrier Ringlet in, lucifuge reaction 1min, then wash away silver staining agent with distilled water；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L；
According to the degree of depth of the grey black spot colors that uniformity occurs in the ringlet of carrier, with colorimetry can obtain hemorrhage in The concentration of TNNI3K.
Beneficial effects of the present invention
The detection kit of the concentration of TNNI3K in a kind of blood of the present invention, owing to have employed mouse-anti-human T NNI3K Dan Ke The carrier of grand antibody high adsorption capacity, gold label silver stain technology, replace static adsorption with dynamic adsorption, we have discovered that Nano Rare Soil can increase substantially the sensitivity of gold label silver stain method, solves and uses enzyme linked immunosorbent assay to measure the inspection of TNNI3K concentration in blood at present Survey method is the longest, need the technical problem such as more blood sample and special instrument.The present invention is the shortest, from fixing mouse-anti People's TNNI3K monoclonal antibody starts, and only needs three minutes to going out result；Need to blood sample less, it is only necessary to 0.033 μ L；Need not inspection The instrument surveyed；0.008ngTNNI3K/ point can be detected.
Accompanying drawing explanation
Fig. 1 a, with the structural representation of carrier of ringlet, wherein 1 is carrier, and 2 is the ringlet on carrier；
The front schematic view of Fig. 2 carrier 1；
The placement relation schematic diagram of Fig. 3 plastic support 4, absorbent material 3 and carrier 1, is followed successively by plastics from bottom to top and props up Support body 4, absorbent material 3 and carrier 1；
There is the structural representation of the plastic support of opening on Fig. 4 both sides.
Detailed description of the invention
Below by specific embodiment and combine accompanying drawing the present invention is expanded on further, but it is not limiting as the present invention.
Embodiment 1
The detection kit of the concentration of TNNI3K in a kind of blood, including:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.01 μ g/mL；
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 0.1 μ g/mL；
(3), la concn is the lanthanum chloride solution of 1mg/mL；
(4), the mixed liquor of nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer Gold: nanometer lanthanum: sheep anti mouse lgG (H+L) antibody is 2.5:1:0.2；
(5), the PBS of glycerol
The PBS of described glycerol, will glycerol to join pH value be that in 7.4 disodium hydrogen phosphate aqueous solutions mixing is all Even and obtain, wherein the concentration of volume percent of glycerol is 30%；
(6), with the carrier of ringlet
Described with ringlet carrier structural representation as shown in Figure 1a, wherein 1 is carrier, and 2 is on carrier Ringlet；Its A-A to schematic diagram as shown in Figure 1 b；
Described carrier 1 is porous polyvinylidene fluoride film that aperture is 0.25 μm；
The degree of depth and the diameter of described ringlet 2 are respectively 0.5mm and 6mm.
In above-mentioned a kind of blood, the preparation method of the detection kit of the concentration of TNNI3K, specifically comprises the following steps that
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, be made into the mouse-anti people that concentration is 0.01 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, add distilled water and make it dissolve, move to In the volumetric flask of 100mL, it is settled to 100ml with distilled water, obtains the mouse-anti-human T NNI3K monoclonal antibody water that concentration is 10 μ g/mL Solution；
Take mouse-anti-human T NNI3K monoclonal antibody aqueous solution that 25 μ L concentration are 10 μ g/mL in 25mL volumetric flask, with steaming Distilled water is settled to 25ml, obtains the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that concentration is 0.01 μ g/mL；
(2), being dissolved by sheep anti mouse lgG (H+L) antibody with distilled water, being made into concentration is 0.1 μ g/mL sheep anti mouse lgG (H+L) Antibody aqueous solution, step is as follows:
By sheep anti mouse lgG (H+L) antibody of 1mg in 50mL small beaker, add distilled water and make it dissolve, move to 100mL's In volumetric flask, it is settled to 100ml with distilled water, obtains sheep anti mouse lgG (H+L) the antibody aqueous solution that concentration is 10 μ g/mL；
Take sheep anti mouse lgG (H+L) antibody aqueous solution that 1.00mL concentration is 10 μ g/mL in 100mL volumetric flask, with distillation Water is settled to 100ml, obtains sheep anti mouse lgG (H+L) the antibody aqueous solution that concentration is 0.1 μ g/mL；
(3), by 0.2932g lanthana it is dissolved in the aqueous hydrochloric acid solution that 2.5mL mass percent concentration is 37%, heating, makes It dissolves, and is settled to 250ml with distilled water, i.e. obtains the lanthanum chloride solution that la concn is 1mg/mL；
(4), the preparation of the mixed liquor of nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
Mass percent concentration with the trisodium citrate aqueous solution that the mass percent concentration of 1.5mL is 1% Yu 100ml Being after 0.01% aqueous solution of chloraurate mixes, stirring is claret to solution, is cooled to room temperature, the reactant liquor concentration of gained Wet chemical regulation pH value for 0.05mol/L is 6.5, is then settled to 100ml with distilled water, obtains nanometer gold molten Liquid；
2., the preparation of nanometer lanthanum solution
It is 1mg/ with the la concn of the tannic acid solution that 10ml mass percent concentration is 1% Yu 1.0ml step (3) gained After the lanthanum chloride solution of mL mixes, stirring carries out reduction reaction 30min, and the reactant liquor concentration of gained is 0.05mol/L Wet chemical regulation pH value be 6.5, be settled to 50ml with distilled water, obtain nanometer lanthanum solution；
3., 62.5 μ L nanometer lanthanum solution of 65.3 μ L nano-Au solutions of step 1. gained with step 2. gained are pressed nanometer Gold: the mass ratio of nanometer lanthanum is that the ratio of 2.5:1 mixes, and obtains nanometer gold and the mixed solution of nanometer lanthanum；
4. in the nanometer gold of step 3. gained and the mixed solution of nanometer lanthanum, add the concentration of 2.5mL step (2) gained It is sheep anti mouse lgG (H+L) the antibody aqueous solution of 0.1 μ g/mL, is then that 0.01 mol/L disodium hydrogen phosphate aqueous solution is fixed by concentration Hold to 25ml, obtain the mixed liquor of nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody；
In the mixed liquor of nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: nanometer Lanthanum: sheep anti mouse lgG (H+L) antibody is 2.5:1:0.2；
(5), the preparation of the PBS of glycerol；
It is the PBS that in 7.4 disodium hydrogen phosphate aqueous solutions, mix homogeneously i.e. obtains glycerol that glycerol joins pH value, its The concentration of volume percent of middle glycerol is 30%；
(6), with the preparation of carrier 1 of ringlet 2
Ringlet 2 is imprinted out on the carrier 1 with card punch；
Wherein carrier 1 is porous polyvinylidene fluoride film that aperture is 0.25 μm；
Described ringlet 2, its degree of depth and diameter are respectively 0.5mm and 6mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L.
Utilize the side that in above-mentioned blood, the concentration of TNNI3K in blood is detected by the detection kit of the concentration of TNNI3K Method, specifically comprises the following steps that
(1), the PBS of 10 μ L glycerol is added drop-wise to the ringlet 2 of carrier 1 from the front of carrier 1, described carrier The front of 1 is as shown in Figure 2；
(2), after the PBS of the glycerol of step (1) penetrates into carrier 1, by 2 μ L mouse-anti-human T NNI3K monoclonal antis Body aqueous solution drips in the ringlet 2 of carrier 1 from the front of carrier 1；
After mouse-anti-human T NNI3K monoclonal antibody aqueous solution penetrates into carrier 1, by 2 μ L blockeres from the front of carrier 1 Join in the ringlet 2 of carrier 1, after blocker penetrates into carrier 1, carrier 1 is used washing liquid, distilled water wash 3 times successively；
Wherein mouse-anti-human T NNI3K monoclonal antibody solution is that mouse-anti-human T NNI3K monoclonal antibody joins in distilled water And the mouse-anti-human T NNI3K monoclonal antibody solution that the concentration obtained is 0.01 μ g/mL；
Described blocker be mass percent concentration be the Bovine Serum Albumin in Aqueous Solution of 0.2%；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
(3), on both sides have above the plastic support 4 of opening and put absorbent material 3, the most again step (2) is processed After the facing up of carrier 1, be placed on absorbent material 3, concrete placement schematic diagram is as it is shown on figure 3, the most successively For plastic support 4, absorbent material 3 and carrier 1；
There is the structural representation of the plastic support 4 of opening on described both sides as shown in Figure 4；
Described absorbent material 3 is filter paper；
(4), with distilled water by diluting blood sample 60 times to be measured, by the blood sample after 2 μ L dilutions from the carrier 1 of step (3) Front is slowly added dropwise in the ringlet 2 of carrier 1；
After blood sample penetrates into carrier 1, by the mixed liquor of 2 μ L nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody from load The front of body 1 is slowly added in the ringlet 2 of carrier 1；
After the mixed liquor of nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody penetrates into carrier 1, carrier 1 is used successively Then 10 μ L silver staining agent are quickly adding into the ringlet 2 of carrier 1, lucifuge by washing liquid, distilled water wash 3 times from the front of carrier 1 Reaction 1min, then washes away silver staining agent with 100 μ L distilled water；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L；
Ringlet 2 according to carrier 1 occurs the degree of depth of the grey black spot colors of uniformity, draws every point by colorimetry The amount of TNNI3K is 0.089ngTNNI3K/ point, concentration 2670ng/mL of TNNI3K in blood.
Embodiment 2
The detection kit of the concentration of TNNI3K in a kind of blood, including:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.3 μ g/mL；
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 2 μ g/mL；
(3), praseodymium concentration is the praseodymium nitrate solution of 5mg/mL；
(4), the mixed liquor of nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer Gold: nanometer praseodymium: sheep anti mouse lgG (H+L) antibody is 1.0:1.6:0.24；
(5), the PBS of glycerol
The PBS of described glycerol, will glycerol to join pH value be that in 7.4 disodium hydrogen phosphate aqueous solutions mixing is all Even and obtain, wherein the concentration of volume percent of glycerol is 30%；
(6), with the carrier of ringlet
Described carrier be aperture be the nitrocellulose filter of the porous of 0.22 μm；
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L.
In above-mentioned a kind of blood, the preparation method of the detection kit of the concentration of TNNI3K, specifically comprises the following steps that
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, be made into the mouse-anti people that concentration is 0.3 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, add distilled water and make it dissolve, move to In the volumetric flask of 100mL, it is settled to 100ml with distilled water, obtains the mouse-anti-human T NNI3K monoclonal antibody water that concentration is 10 μ g/mL Solution；
Take mouse-anti-human T NNI3K monoclonal antibody solution that 0.75mL concentration is 10 μ g/mL in 25mL volumetric flask, with steaming Distilled water is settled to 25ml, obtains the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that concentration is 0.3 μ g/mL；
(2), being dissolved by sheep anti mouse lgG (H+L) antibody with distilled water, being made into concentration is that 2 μ g/mL sheep anti mouse lgG (H+L) resist Body aqueous solution, step is as follows:
By sheep anti mouse lgG (H+L) antibody of 1mg in 50mL small beaker, add distilled water and make it dissolve, move to the appearance of 50mL In measuring bottle, it is settled to 50ml with distilled water, obtains sheep anti mouse lgG (H+L) the antibody aqueous solution that concentration is 20 μ g/mL；
Take sheep anti mouse lgG (H+L) antibody-solutions that concentration is 20 μ g/mL of 10.00mL in 100mL volumetric flask, with steaming Distilled water is settled to 100ml, and obtaining concentration is 2 μ g/mLd sheep anti mouse lgG (H+L) antibody aqueous solutions；
(3), being mixed with 3mL concentrated nitric acid by 0.5852g praseodymium oxide, heating makes it dissolve, and is settled to distilled water after cooling 100mL, obtains the praseodymium nitrate solution that praseodymium concentration is 5mg/mL；
(4), the preparation of the mixed liquor of nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
Mass percent concentration with the trisodium citrate aqueous solution that the mass percent concentration of 1.5mL is 1% Yu 100ml Being after 0.01% aqueous solution of chloraurate mixes, stirring is claret to solution, is cooled to room temperature, the reactant liquor concentration of gained Wet chemical regulation pH value for 0.1mol/L is 8.0, is then settled to 100ml with distilled water, obtains nano-Au solution；
2., the preparation of nanometer praseodymium solution
By the 0.20ml praseodymium concentration of the tannic acid solution that 10ml mass percent concentration is 1% with step (3) gained it is After the praseodymium nitrate solution of 5mg/mL mixes, stirring carries out reduction reaction 30min, and the reactant liquor concentration of gained is The wet chemical regulation pH value of 0.05mol/L is 8, is settled to 50ml with distilled water, obtains nanometer praseodymium solution；
3., 100 μ L nanometer praseodymium solution of 26.2 μ L nano-Au solutions of step 1. gained with step 2. gained are pressed nanometer Gold: the mass ratio of nanometer praseodymium is that the ratio of 1.0:1.6 mixes, and obtains nanometer gold and the mixed solution of nanometer praseodymium；
4., in the nanometer gold of step 3. gained and the mixed solution of nanometer praseodymium, the dense of 0.15mL step (2) gained is added Degree is sheep anti mouse lgG (H+L) the antibody aqueous solution of 2 μ g/mL, is then that 0.01 mol/L disodium hydrogen phosphate aqueous solution is fixed by concentration Hold to 25ml, obtain the mixed liquor of nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody；
In the mixed liquor of nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: nanometer Praseodymium: sheep anti mouse lgG (H+L) antibody is 1.0:1.6:0.24；
(5), the preparation of the PBS of glycerol；
It is the PBS that in 7.4 disodium hydrogen phosphate aqueous solutions, mix homogeneously i.e. obtains glycerol that glycerol joins pH value, its The concentration of volume percent of middle glycerol is 30%；
(6), with the preparation of carrier of ringlet
On carrier, ringlet is imprinted out with card punch；
Described carrier be aperture be the nitrocellulose filter of the porous of 0.22 μm；
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L.
Utilize the side that in above-mentioned blood, the concentration of TNNI3K in blood is detected by the detection kit of the concentration of TNNI3K Method, specifically comprises the following steps that
(1), the PBS of 10 μ L glycerol is added drop-wise to the ringlet of carrier from the front of carrier；
(2), after the PBS of the glycerol of step (1) penetrates into carrier, then by Mus that 2 μ L concentration are 0.3 μ g/mL Anti-human TNNI3K monoclonal antibody aqueous solution is added drop-wise to the ringlet of carrier from the front of carrier；
After the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that concentration is 0.3 μ g/mL penetrates into carrier, 2 μ L are blocked Agent addition is added drop-wise to the ringlet of carrier from the front of carrier；
After blocker penetrates into carrier, carrier is used washing liquid, distilled water wash 3 times successively；
Described blocker be mass percent concentration be the aqueous solution of the milk of 1%；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
(3), on both sides have above the plastic support of opening and put absorbent material, after the most again step (2) being washed The facing up of carrier, be placed on absorbent material；
Described absorbent material is absorbent cotton；
(4), with distilled water by diluting blood sample 60 times to be measured, by the blood sample after 2 μ L dilutions from the carrier of step (3) gained Front be slowly dropped in the ringlet of carrier；
After blood sample penetrates into carrier, by the mixed liquor of 2 μ L nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody from load The front of body is slowly dropped in the ringlet of carrier；
After the mixed liquor of nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody penetrates into carrier, by carrier successively with washing Then 10 μ L silver staining agent are quickly adding into the ringlet of carrier by liquid, distilled water wash 3 times from the front of carrier, and lucifuge is reacted 1min, then washes away silver staining agent with 100 μ L distilled water；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L；
According to the degree of depth of the grey black spot colors that uniformity occurs in the ringlet of carrier, draw every point by colorimetry The amount of TNNI3K is 0.008ngTNNI3K/ point, concentration 240ng/mL of TNNI3K in blood.
Embodiment 3
The detection kit of the concentration of TNNI3K in a kind of blood, including:
(1), concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.15 μ g/mL；
(2), concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1 μ g/mL；
(3), europium concentration is the europium nitrate solution of 2mg/mL and Gadolinium trinitrate solution that gadolinium concentration is 2.5mg/mL；
(4), nanometer gold-nanometer europium and the mixed liquor of gadolinium-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nanometer gold-nanometer europium and gadolinium-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. Nanometer gold: nanometer europium and gadolinium: sheep anti mouse lgG (H+L) antibody is 1.65:1.34:0.22；
Wherein nanometer europium and gadolinium, calculate in mass ratio, europium: gadolinium is 1:1；
(5), the PBS of glycerol
The PBS of described glycerol, will glycerol to join pH value be that in 7.4 disodium hydrogen phosphate aqueous solutions mixing is all Even and obtain, wherein the concentration of volume percent of glycerol is 30%；
(6), with the carrier of ringlet
Described carrier be aperture be the nitrocellulose filter of the porous of 0.22 μm；
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L.
In above-mentioned a kind of blood, the preparation method of the detection kit of the concentration of TNNI3K, specifically comprises the following steps that
(1), with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, be made into the mouse-anti people that concentration is 0.15 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, add distilled water and make it dissolve, move to In the volumetric flask of 100mL, being settled to 100ml with distilled water, obtaining concentration is 10 μ g/mLd mouse-anti-human T NNI3K monoclonal antibody water Solution；
Take the concentration of 375 μ L be 10 μ g/mL mouse-anti-human T NNI3K monoclonal antibody solutions in 25mL volumetric flask, with distillation Water is settled to 25ml, obtains the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that concentration is 0.15 μ g/mL；
(2), being dissolved by sheep anti mouse lgG (H+L) antibody with distilled water, being made into concentration is that 1 μ g/mL sheep anti mouse lgG (H+L) resists Body aqueous solution, step is as follows:
By sheep anti mouse lgG (H+L) antibody of 1mg in 50mL small beaker, add distilled water and make it dissolve, move to 100mL's In volumetric flask, it is settled to 100ml with distilled water, obtains sheep anti mouse lgG (H+L) the antibody aqueous solution that concentration is 10 μ g/mL；
Taking 5.00mL concentration is that 10 μ g/mL sheep anti mouse lgG (H+L) antibody aqueous solutions, in 50mL volumetric flask, use distilled water It is settled to 50ml, obtains sheep anti mouse lgG (H+L) the antibody aqueous solution that concentration is 1 μ g/mL；
(3), the preparation of rare earths salt
Being mixed with 5mL concentrated nitric acid by 0.2316g europium oxide, heating makes it dissolve, and after it cools down, is settled to distilled water 100mL, obtains the europium nitrate solution that europium concentration is 2mg/mL；
Being mixed with 5mL concentrated nitric acid by 0.2886g Gadolinia., heating makes it dissolve, and after it cools down, is settled to distilled water 100mL, obtains the Gadolinium trinitrate solution that gadolinium concentration is 2.5mg/mL；
(4), the preparation of the mixed liquor of nanometer gold-nanometer europium and gadolinium-sheep anti mouse lgG (H+L) antibody
1., the preparation of nano-Au solution
Dense with the mass percent of 100.0mL with the trisodium citrate aqueous solution that the mass percent concentration of 1.5mL is 1% Degree is after 0.01% aqueous solution of chloraurate mixes, and stirring is claret to solution, is cooled to room temperature, and the reactant liquor of gained is with dense The wet chemical regulation pH value that degree is 0.1mol/L is 7.5, is then settled to 100ml with distilled water, obtains nanometer gold molten Liquid；
2., the preparation of the mixed solution of nanometer europium and gadolinium
By the 0.25mL europium concentration of the tannic acid solution that 10mL mass percent concentration is 1% with step (3) gained it is After the europium nitrate solution of 2mg/mL and the Gadolinium trinitrate solution that 0.20mL gadolinium concentration is 2.5mg/mL mix, enter under stirring condition Row reduction reaction 30min, the wet chemical that reactant liquor concentration the is 0.05mol/L regulation pH value of gained is 7.5, then It is settled to 50mL with distilled water, obtains the mixed solution of nanometer europium and gadolinium；
3., by 83.7 μ L nanometer europium and the mixing of gadolinium of 43.1 μ L nano-Au solutions of step 1. gained Yu step 2. gained Solution is in nanometer gold: the europium of nanometer and the ratio that the mass ratio of gadolinium is 1.65:1.34 mix, and obtain nanometer gold and nanometer europium Mixed solution with gadolinium；
4., in the nanometer gold of step 3. gained and the mixed solution of nanometer europium and gadolinium, 0.275mL step (2) gained is added Sheep anti mouse lgG (H+L) the antibody aqueous solution that concentration is 1 μ g/mL, be then that 0.01 mol/L disodium hydrogen phosphate is water-soluble by concentration Liquid is settled to 25ml, obtains nanometer gold-nanometer europium and the mixed liquor of gadolinium-sheep anti mouse lgG (H+L) antibody；
In the mixed liquor of nanometer gold-nanometer europium and gadolinium-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: Nanometer europium and gadolinium: sheep anti mouse lgG (H+L) antibody is 1.65:1.34:0.22；
(5), the preparation of the PBS of glycerol；
It is the PBS that in 7.4 disodium hydrogen phosphate aqueous solutions, mix homogeneously i.e. obtains glycerol that glycerol joins pH value, its The concentration of volume percent of middle glycerol is 30%；
(6), with the preparation of carrier of ringlet
On carrier, ringlet is imprinted out with card punch；
Wherein carrier be aperture be the porous cellulose acetate membrane of 0.20 μm；
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L.
Utilize the side that in above-mentioned blood, the concentration of TNNI3K in blood is detected by the detection kit of the concentration of TNNI3K Method, specifically comprises the following steps that
(1), the PBS of 10 μ L glycerol is added drop-wise to the ringlet of carrier from the front of carrier；
(2), after the PBS of the glycerol of step (1) penetrates into carrier, then by Mus that 2 μ L concentration are 0.15 μ g/mL Anti-human TNNI3K monoclonal antibody aqueous solution is added drop-wise to the ringlet of carrier from the front of carrier；
After the mouse-anti-human T NNI3K monoclonal antibody aqueous solution that concentration is 0.15 μ g/mL penetrates into carrier, then by 2 μ L resistances Disconnected agent joins the ringlet of carrier from the front of carrier；
After blocker penetrates into carrier, carrier is used washing liquid, distilled water wash 3 times successively；
Described blocker be mass percent concentration be the aqueous gelatin solution of 0.5%；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
(3), on both sides have above the plastic support of opening and put absorbent material, after the most again step (2) being washed The facing up of carrier, be placed on absorbent material；
Described absorbent material is sponge；
(4), with distilled water by diluting blood sample 60 times to be measured, by the blood sample after 2 μ L dilutions from the carrier of step (3) gained Front be slowly dropped in the ringlet of carrier；
After blood sample penetrates into carrier, by 2 μ L nanometer gold-nanometer europium and the mixed liquor of gadolinium-sheep anti mouse lgG (H+L) antibody It is slowly added into the ringlet of carrier from the front of carrier；
After the mixed liquor of nanometer gold-nanometer europium and gadolinium-sheep anti mouse lgG (H+L) antibody penetrates into carrier, by carrier successively With washing liquid, distilled water wash 3 times, then 10 μ L silver staining agent are quickly adding into the ringlet of carrier from the front of carrier, lucifuge Reaction 1min, then washes away silver staining agent with 100 μ L distilled water；
Described washing liquid be pH value be that 7.4 disodium hydrogen phosphate aqueous solutions add the solution that obtains of Tween80, NaCl, Wherein the mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is The concentration of 0.015mol/L and hydroquinone is 0.075mol/L；
According to the degree of depth of the grey black spot colors that uniformity occurs in the ringlet of carrier, draw every point by colorimetry The amount of TNNI3K is 0.056ngTNNI3K/ point, concentration 1680ng/mL of TNNI3K in blood.
Embodiment 4
Embodiment 4 is identical with the experiment condition of embodiment 3, simply by molten for the standard that concentration is 1680ng/mL of TNNI3K Liquid substitutes blood sample, does 3 parallel laboratory tests.I.e. use distilled waterWillThe concentration of TNNI3K is the standard solution dilution 60 of 1680ng/mL Times, test with the solution of the TNNI3K after 2 μ L dilutions.According to the grey black spot colors that uniformity occurs in ringlet Degree of depth colorimetry draw the amount of every some TNNI3K be respectively 0.055,0.055,0.055ngTNNI3K/ point, TNNI3K in blood Concentration 1650,1650, the response rate of 1650ng/mL, TNNI3K be respectively 98.21%, 98.21% and 98.21%.
Test result indicate that from above-mentioned, by the detection kit of the concentration of TNNI3K in the blood of the present invention in blood The method that the concentration of TNNI3K carries out detecting is reliable and stable.
Comparative examples 1(enzyme linked immunosorbent assay measures the concentration of TNNI3K in embodiment 3 blood sample)
With Kirkegaard & Perry Laboratories, " the Protein Detector of Inc.TM ELISA Kit " test, specifically comprise the following steps that
(1), by the mouse-anti-human T NNI3K monoclonal antibody of 25mg in 50mL small beaker, add distilled water and make it dissolve, move To the volumetric flask of 25mL, it is settled to 25ml with distilled water, obtains the mouse-anti-human T NNI3K monoclonal antibody water that concentration is 1mg/mL Solution；
(2), taking the concentration of 88 μ L with pipettor is that 1mg/mL mouse-anti-human T NNI3K monoclonal antibody aqueous solution is in 96 orifice plates A hole in, add 100 μ L fixatives, place 120min, remove loose solution, with 150 μ L washing liquids cleanings, wash 3 times；
(3), add 300 μ L blockeres, place 60min, remove unnecessary blocker；
(4), add 2.5 μ L sample of blood, place 120min, clean by 150 μ L washing liquids, wash 4 times；
(5), add the 100 anti-solution of μ L bis-, place 60min, clean by 100 μ L washing liquids, wash 4 times；
(6), add 100 μ L enzyme stains, place 30min；
(7), adding 100 μ L stopping agents, determining the concentration of TNNI3K in this blood sample by MTP-310Lab type microplate reader is 1678ng/mL。
Contrasted by the testing result of above-mentioned comparative examples 1 with embodiment 3, it can be seen that utilize the present invention to try Agent box detects, and compared with the testing result of gained is carried out with traditional enzyme linked immunosorbent assay, its testing result is accurate, and degree of accuracy is high.Enter One step, it can be seen that utilizing test kit of the present invention to detect, it is the shortest, from fixing mouse-anti-human T NNI3K monoclonal antibody Start, only need three minutes to going out result, and enzyme linked immunosorbent assay traditional in comparative examples 1, from fixing mouse-anti-human T NNI3K monoclonal Antibody starts, and needs 390 minutes to going out result.And the test kit utilizing the present invention detects, required blood sample is less, it is only necessary to 0.033 μ L, and traditional enzyme linked immunosorbent assay, need 2.5 μ L.Further, blood sample is detected by the test kit utilizing the present invention, it is not necessary to Special detecting instrument, and traditional enzyme linked immunosorbent assay need to use microplate reader.
The above is only the citing of embodiments of the present invention, it is noted that for the ordinary skill of the art For personnel, on the premise of without departing from the technology of the present invention principle, it is also possible to make some improvement and modification, these improve and become Type also should be regarded as protection scope of the present invention.

Claims (7)

1. the detection kit of the concentration of TNNI3K in a blood, it is characterised in that the detection of the concentration of TNNI3K in described blood Test kit, including:
(1) concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.01～0.3 μ g/mL；
(2) concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 0.1～2 μ g/mL；
(3) concentration is the rare earths salt of 1～5mg/mL；
Its middle rare earth be in lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutecium, yttrium, scandium one or both with On lucium；
(4) mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody
The mixed liquor of described nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody is by the method system comprised the steps For forming:
1. the preparation of nano-Au solution
It is that 0.01% gold chloride is water-soluble with the trisodium citrate aqueous solution that mass percent concentration is 1% and mass percent concentration Liquid calculates by volume, i.e. mass percent concentration is the trisodium citrate aqueous solution of 1%: mass percent concentration is 0.01% Aqueous solution of chloraurate is after the ratio of 1.5mL:100mL mixes, and the reactant liquor of stirring reaction to gained is claret, is cooled to Room temperature, the wet chemical that reactant liquor concentration the is 0.05～0.1mol/L regulation pH value of gained is 6.5～8.0, to obtain final product Nano-Au solution；
2. the preparation of nano rare earth solution
With the rare earths salt that concentration is 1～5mg/mL of the tannic acid solution that mass percent concentration is 1% and (3) gained by Volume ratio calculates, i.e. 1% tannic acid solution: the rare earths salt of 1～5mg/mL is that the ratio of 10mL:1～0.2mL mixes After, carrying out reduction reaction 30min under stirring condition, the potassium carbonate that reactant liquor concentration is 0.05～0.1mol/L of gained is water-soluble Liquid regulation pH value is 6.5～8.0, obtains nano rare earth solution；
3. the nano rare earth solution of the nano-Au solution of step 1. gained Yu step 2. gained is pressed nanometer gold: the matter of nano rare earth Amount mixes than the ratio being 2.5～1.0:1～1.6, obtains nanometer gold and nano rare earth mixed solution；
4. in the nanometer gold and nano rare earth mixed solution of step 3. gained, add sheep anti mouse lgG (H+L) antibody of (2) gained Aqueous solution, obtains the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody；
In the mixed liquor of described nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: Nano rare earth: sheep anti mouse lgG (H+L) antibody is 2.5～1.0:1～1.6:0.2～0.24；
(5) PBS of glycerol
The PBS of described glycerol, will glycerol join pH value be in 7.4 disodium hydrogen phosphate aqueous solutions mix homogeneously and , wherein the concentration of volume percent of glycerol is 30%；
(6) with the carrier of ringlet
Described carrier is the polyvinylidene fluoride film of the porous of aperture≤0.25 μm, nitrocellulose filter, polystyrene film or second Acid cellulose film；
A diameter of 6mm of described ringlet, the degree of depth are 0.5mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, wherein the concentration of silver nitrate be 0.015mol/L and The concentration of hydroquinone is 0.075mol/L.
The detection kit of the concentration of TNNI3K in a kind of blood the most as claimed in claim 1, it is characterised in that in described blood The detection kit of the concentration of TNNI3K, including:
(1) concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.01 μ g/mL；
(2) concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 0.1 μ g/mL；
(3) la concn is the lanthanum chloride solution of 1mg/mL；
(4) mixed liquor of nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nanometer gold-nanometer lanthanum-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: receive Rice lanthanum: sheep anti mouse lgG (H+L) antibody is 2.5:1:0.2；
(5) PBS of glycerol
The PBS of described glycerol, will glycerol join pH value be in 7.4 disodium hydrogen phosphate aqueous solutions mix homogeneously and , wherein the concentration of volume percent of glycerol is 30%；
(6) with the carrier of ringlet
Described carrier be aperture be the porous polyvinylidene fluoride film of 0.25 μm；
The degree of depth and the diameter of described ringlet are respectively 0.5mm and 6mm.
3. the detection kit of the concentration of TNNI3K in a kind of blood described in claim 1, it is characterised in that in described blood The detection kit of the concentration of TNNI3K, including:
(1) concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.3 μ g/mL；
(2) concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 2 μ g/mL；
(3) praseodymium concentration is the praseodymium nitrate solution of 5mg/mL；
(4) mixed liquor of nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nanometer gold-nanometer praseodymium-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: receive Rice praseodymium: sheep anti mouse lgG (H+L) antibody is 1.0:1.6:0.24；
(5) PBS of glycerol
The PBS of described glycerol, will glycerol join pH value be in 7.4 disodium hydrogen phosphate aqueous solutions mix homogeneously and , wherein the concentration of volume percent of glycerol is 30%；
(6) with the carrier of ringlet
Described carrier be aperture be the nitrocellulose filter of the porous of 0.22 μm；
Described ringlet, its degree of depth and diameter are respectively 0.5mm and 6mm.
The detection kit of the concentration of TNNI3K in a kind of blood the most as claimed in claim 1, it is characterised in that in described blood The detection kit of the concentration of TNNI3K, including:
(1) concentration is the mouse-anti-human T NNI3K monoclonal antibody aqueous solution of 0.15 μ g/mL；
(2) concentration is sheep anti mouse lgG (H+L) the antibody aqueous solution of 1 μ g/mL；
(3) europium concentration is the europium nitrate solution of 2mg/mL and Gadolinium trinitrate solution that gadolinium concentration is 2.5mg/mL；
(4) nanometer gold-nanometer europium and the mixed liquor of gadolinium-sheep anti mouse lgG (H+L) antibody
In the mixed liquor of described nanometer gold-nanometer europium and gadolinium-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer Gold: nanometer europium and gadolinium: sheep anti mouse lgG (H+L) antibody is 1.65:1.34:0.22；
Wherein nanometer europium and gadolinium, calculate in mass ratio, europium: gadolinium is 1:1；
(5) PBS of glycerol
The PBS of described glycerol, will glycerol join pH value be in 7.4 disodium hydrogen phosphate aqueous solutions mix homogeneously and , wherein the concentration of volume percent of glycerol is 30%；
(6) with the carrier of ringlet
Described carrier be aperture be the nitrocellulose filter of the porous of 0.22 μm；
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 a kind of blood the most as claimed in claim 1, it is characterised in that Specifically include following steps:
(1) with distilled water, mouse-anti-human T NNI3K monoclonal antibody is dissolved, be made into the mouse-anti people that concentration is 0.01～0.3 μ g/mL TNNI3K monoclonal antibody aqueous solution；
(2) with distilled water, sheep anti mouse lgG (H+L) antibody is dissolved, be made into the sheep anti mouse lgG (H+L) that concentration is 0.1～2 μ g/mL Antibody aqueous solution；
(3) preparation of rare earths salt
Rare earth oxide is dissolved in concentrated nitric acid or aqueous hydrochloric acid solution that mass percent concentration is 37%, obtain concentration be 1～ The rare earths salt of 5mg/mL；
Its middle rare earth is in lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutecium, yttrium, scandium oxide A kind of or the rare earth oxide of more than two；
(4) preparation of the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody
1. the preparation of nano-Au solution
It is that 0.01% gold chloride is water-soluble with the trisodium citrate aqueous solution that mass percent concentration is 1% and mass percent concentration Liquid calculates by volume, i.e. mass percent concentration is the trisodium citrate aqueous solution of 1%: mass percent concentration is 0.01% Aqueous solution of chloraurate is after the ratio of 1.5mL:100mL mixes, and the reactant liquor of stirring reaction to gained is claret, is cooled to Room temperature, the wet chemical that reactant liquor concentration the is 0.05～0.1mol/L regulation pH value of gained is 6.5～8.0, to obtain final product Nano-Au solution；
2. the preparation of nano rare earth solution
Molten with the rare-earth salts that concentration is 1～5mg/mL of step (3) gained with the tannic acid solution that mass percent concentration is 1% Liquid calculates by volume, i.e. 1% tannic acid solution: the rare earths salt of 1～5mg/mL is that the ratio of 10mL:1～0.2mL is carried out After mixing, under stirring condition, carry out reduction reaction 30min, the potassium carbonate that reactant liquor concentration is 0.05～0.1mol/L of gained Aqueous solution regulation pH value is 6.5～8.0, obtains nano rare earth solution；
3. the nano rare earth solution of the nano-Au solution of step 1. gained Yu step 2. gained is pressed nanometer gold: the matter of nano rare earth Amount mixes than the ratio being 2.5～1.0:1～1.6, obtains nanometer gold and nano rare earth mixed solution；
4. in the nanometer gold and nano rare earth mixed solution of step 3. gained, add the sheep anti mouse lgG (H+L) of step (2) gained Antibody aqueous solution, obtains the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody；
In the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody, calculate in mass ratio, i.e. nanometer gold: Nano Rare Soil: sheep anti mouse lgG (H+L) antibody is 2.5～1.0:1～1.6:0.2～0.24；
(5) preparation of the PBS of glycerol
It is the PBS that in 7.4 disodium hydrogen phosphate aqueous solutions, mix homogeneously i.e. obtains glycerol that glycerol joins pH value, the sweetest The concentration of volume percent of oil is 30%；
(6) with the preparation of carrier of ringlet
On carrier, ringlet is imprinted out with card punch；
Described carrier is the polyvinylidene fluoride film of the porous of aperture≤0.25 μm, nitrocellulose filter, polystyrene film or second Acid cellulose film；
A diameter of 6mm of described ringlet, the degree of depth are 0.5mm；
(7) silver staining agent:
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, wherein the concentration of silver nitrate be 0.015mol/L and The concentration of hydroquinone is 0.075mol/L.
6. the detection kit of the concentration of TNNI3K TNNI3K in blood in the blood as described in any one of claim 1-4 Concentration detects.
7. in blood as claimed in claim 6, the detection kit of the concentration of TNNI3K is used for carrying out the concentration of TNNI3K in blood The method of detection, it is characterised in that specifically include following steps:
(1) PBS of glycerol is added drop-wise to the ringlet of carrier from the front of carrier；
(2) after the PBS until the glycerol of step (1) penetrates into carrier, then by mouse-anti-human T NNI3K monoclonal antibody solution It is added drop-wise to the ringlet of carrier from the front of carrier；
After mouse-anti-human T NNI3K monoclonal antibody solution penetrates into carrier, blocker is joined carrier from the front of carrier In ringlet, after blocker penetrates into carrier, carrier is used washing liquid, distilled water wash 3 times successively；
Described blocker be mass percent concentration be in the bovine serum albumin of 0.2-1%, gelatin or milk aqueous solution Kind；
Described washing liquid be pH value be 7.4 disodium hydrogen phosphate aqueous solutions to add the solution that obtains of Tween80, NaCl, wherein The mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
(3) having above the plastic support of opening on both sides and put absorbent material, the carrier after then step (2) being processed is just Face up and be placed on absorbent material；
Described absorbent material is one or more the mixture in filter paper, absorbent cotton, sponge；
(4) with distilled water by diluting blood sample 60 times to be measured, the blood sample after dilution is dripped from the front of the carrier of step (3) gained It is added in the ringlet of carrier；
After blood penetrates into carrier, by with dilution after the isopyknic nanometer gold-nano rare earth of blood sample-sheep anti mouse lgG (H+L) The mixed liquor of antibody joins the ringlet of carrier from the front of carrier；
After the mixed liquor of nanometer gold-nano rare earth-sheep anti mouse lgG (H+L) antibody penetrates into carrier, by carrier successively with washing Liquid, distilled water wash 3 times, then will join carrier for the silver staining agent of the blood sample volume 5 times after dilution from the front of carrier In ringlet, lucifuge reaction 1min, then wash away silver staining agent with distilled water；
Described washing liquid be pH value be 7.4 disodium hydrogen phosphate aqueous solutions to add the solution that obtains of Tween80, NaCl, wherein The mass percent concentration of Tween80 is 0.5%, and the mass percent concentration of NaCl is 10%；
Described silver staining agent is the aqueous solution containing silver nitrate and hydroquinone, and wherein the concentration of silver nitrate is 0.015mol/L and right The concentration of Benzodiazepines is 0.075mol/L；
According to the degree of depth of the grey black spot colors that uniformity occurs in the ringlet of carrier, with colorimetry can obtain hemorrhage in The concentration of TNNI3K.
CN201410391773.7A 2014-08-11 2014-08-11 Detection kit of concentration of TNNI3K and its preparation method and application in a kind of blood Expired - Fee Related CN104330567B (en)

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