CN106443009A - Preparation method of neutrophil gelatinase-associated lipid carrier protein detection kit - Google Patents
Preparation method of neutrophil gelatinase-associated lipid carrier protein detection kit Download PDFInfo
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- CN106443009A CN106443009A CN201610794197.XA CN201610794197A CN106443009A CN 106443009 A CN106443009 A CN 106443009A CN 201610794197 A CN201610794197 A CN 201610794197A CN 106443009 A CN106443009 A CN 106443009A
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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
- G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
Abstract
The invention relates to the field of biochemical technology and discloses a preparation method of a neutrophil gelatinase-associated lipid carrier protein detection kit. The method includes the following steps: activating of hollow polystyrene microspheres, coupling of antibodies, blocking of microspheres, washing and dissolution. According to the preparation method, hollow polystyrene microspheres are used in an neutrophil gelatinase-associated lipid carrier protein (NGAL for short) regent, that is, the polystyrene microspheres are synthesized on the inner core of nano-SiO2, and then the nano-SiO2 is removed with hydrofluoric acid so as to form hollow polystyrene microspheres; the density of the polystyrene microspheres is reduced, and thus the microspheres can be better suspended in a solution; the stability of the reagent improved, the detection sensitivity of detection reagents is thus enhanced, the steady production on a large scale of the NGAL reagent is thus facilitated, and the preparation method has important practical value.
Description
Technical field
The present invention relates to technical field of biochemistry, particularly to a kind of inspection of neutrophil gelatinase-associated lipocalin NGAL
The preparation method of test agent box.
Background technology
Human neutrophil gelatinase-associated lipocalin (neutrophil gelatinase-
Associatedlipocalin, NGAL) molecular weight about 25kDa, it is covalently bonded in neutrophilic granulocyte gelatinase.NGAL physiology shape
It is a kind of somatomedin under state, be primarily involved in generation, the growth of early stage kidney epithelia.Include kidney, lung, stomach in normal structure
And expression is relatively low in the epithelial tissue of colon.After ischemia-reperfusion injury of kidney, proximal convoluted tubule epithelium great expression NGAL, be
It has recently found that the sensitivity of early diagnosiss AKI biological markers.Urine NGAL be one of ischemia-reperfusion injury of kidney quick
The index of sense, its expression and renal ischemic time correlation simultaneously.
During acute kidney injury, NGAL level is higher, and prognosis will develop into acute renal failure.NGAL water
Flat higher generally go out situations below：Operation on vessels of heart patient, the personnel that are critically ill, septic or hemorrhagic shock, renal transplantation, vein X
The reaction of ray-contrast media and nephrotoxicity treatment reflection.Can a certain degree of acute renal and decline in more than 50% patient
Exhaust, the usual serious disease later stage occurs so that mortality rate greatly increases.
The NGAL detection method clinically using at present have enzyme immunoassay (ELISA), radio immunoassay (RIA),
Latex strengthens transmission immunological turbidimetry method, common turbidimetry etc., but ELISA and RIA detection method has complex operation, specimen
Need pretreatment, the shortcoming of detection time length；Common turbidimetry haves the shortcomings that insufficient sensitivity；And latex strengthens transmission
Immunoturbidimetry is usually present agent precipitate and leads to the bad shortcoming of stability, thus leading to the inadequate problem of detection sensitivity.
In sum, the neutrophil gelatinase-associated lipocalin NGAL detection examination that a kind of reagent stability is good is provided
Agent box is the problem of our current urgent need to resolves.
Content of the invention
It is an object of the invention to provide a kind of neutrophil gelatinase-associated lipocalin NGAL detection kit
Preparation method is so that the stability of detectable is more preferable, thus enhancing the detection sensitivity of detectable.
For solving above-mentioned technical problem, embodiments of the present invention provide a kind of neutrophilic granulocyte gelatinase related lipid
The preparation method of transporter detection kit, this preparation method comprises the steps of：The activation of hollow polystyrene microsphere：To
After adding the first buffer to be diluted in hollow polystyrene microsphere, add N-hydroxy-succinamide solution, stirring, mixing
Uniformly, addition 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride solution during stirring, continuation stirring,
After washing, it is dissolved in the first buffer, obtain final product the hollow polystyrene microsphere solution of activation；The coupling of antibody：To activation
Neutrophil gelatinase-associated lipocalin NGAL antibody-solutions, concussion reaction is added in hollow polystyrene microsphere solution
Afterwards, obtain final product the first mixed liquor；The closing of microsphere：Add bovine serum albumin, concussion reaction in the first mixed liquor, obtain final product second
Mixed liquor；Washing, dissolving：Second mixed liquor is dissolved in the second buffer after centrifugation, supersound washing, obtains final product neutral grain
Cell gelatinase associated lipocalin detection kit.
Embodiment of the present invention in terms of existing technologies, by neutrophilic granulocyte gelatinase related lipid deliver egg
In vain using hollow polystyrene microsphere in (NGAL) reagent, that is, pass through in Nano-meter SiO_22Synthetic polystyrene microsphere on kernel, then
Nano-meter SiO_2 is removed by Fluohydric acid.2Kernel, thus defining the polystyrene microsphere of hollow, reduces polystyrene microsphere
Density, such that it is able to make microsphere preferably suspend in the solution, improves the stability of reagent, thus enhancing detectable
Detection sensitivity.Be conducive to the stable mass production of neutrophil gelatinase-associated lipocalin NGAL (NGAL) reagent,
There is important actual application value.
In addition, hollow polystyrene microsphere preparation method comprises the steps of：Manganese sulfate and ferric chloride mixing are added
In solution, stir, add sodium hydroxide, adjustment thermotonuses 1～2h obtains Manganese Ferrite；Add Oleic acid, react 2～3h,
Obtain the Manganese Ferrite of oleic acid modified；
Take silicon dioxide, sodium lauryl sulphate, styrene, potassium peroxydisulfate to be dissolved in deionized water, stir；Add
The Manganese Ferrite of oleic acid modified, adjusts temperature, adds acrylic acid, continue stirring 3～7h after reaction 1～3h；It is subsequently adding Fluohydric acid.,
Maintain stirring 1～3h, obtain final product hollow polystyrene microsphere, by Nano-meter SiO_22Synthetic polystyrene microsphere on kernel, then lead to
Cross Fluohydric acid. and remove Nano-meter SiO_22Kernel, thus defining the polystyrene microsphere of hollow, reduces the close of polystyrene microsphere
Degree is such that it is able to make microsphere preferably suspend in the solution.
In addition, hollow polystyrene microsphere preparation method comprises the steps of：Manganese sulfate and ferric chloride mixing are added
In solution, stir, add sodium hydroxide, adjustment thermotonuses 1～2h obtains Manganese Ferrite；Add Oleic acid, react 2～3h,
Obtain the Manganese Ferrite of oleic acid modified；
Take silicon dioxide, sodium lauryl sulphate, styrene, potassium peroxydisulfate to be dissolved in deionized water, stir；Add
The Manganese Ferrite of oleic acid modified, adjusts temperature, adds Fluohydric acid., maintains stirring 1～3h, adds acrylic acid, continue stirring 3～
7h；Obtain final product hollow polystyrene microsphere, by Nano-meter SiO_22Synthetic polystyrene microsphere on kernel, then removed by Fluohydric acid.
Nano-meter SiO_22Kernel, thus defining the polystyrene microsphere of hollow, reduces the density of polystyrene microsphere such that it is able to make
Microsphere preferably suspends in the solution.
In addition, the mass concentration of silicon dioxide is 0.25～0.1g/ml, the particle diameter of silicon dioxide is 30～80nm.So
, in 30～80nm, the density of hollow polystyrene microsphere is in 1.01～1.03g/ for the diameter that the tiny balloon ultimately forming can be made
cm3Such that it is able to ensure that microsphere can preferably be suspended in water, improve the stability of reagent.
In addition, the mass concentration of sodium lauryl sulphate is 1～20mg/ml.So make the polystyrene thickness of synthesis
Degree is in 30～100nm, thus ensureing that reagent disclosure satisfy that linear in product technology requirement and sensitivity index.
In addition, styrene is 1 with the volume ratio of deionized water:5.So can ensure that cinnamic utilization rate highest, make
More than 95% styrene conversion is polystyrene.
In addition, hollow polystyrene microsphere includes hollow polystyrene microsphere, ferrimanganic layer and Oleic acid layer, ferrimanganic layer passes through
Ferrimanganic layer is uniformly coated on the outside of hollow polystyrene microsphere by Oleic acid layer, wherein, hollow polystyrene microsphere hollow
Section diameter is 30nm～80nm, and the thickness of the polystyrene layer that hollow polystyrene microsphere is formed is 30nm～100nm, ferrimanganic
The thickness of layer is 20nm～50nm, and the thickness of Oleic acid layer is 2nm～10nm, by synthesizing by the Manganese Ferrite cladding of oleic acid modified
Hollow polystyrene microsphere, Manganese Ferrite not only possess very high superparamagnetism as ferroso-ferric oxide, be easy to detached excellent
Point, and have that cost is lower, the advantage that synthesized cladding outer layer can preferably control pattern and size；And it is synthesized
Magnetic hollow polystyrene microsphere preferably can control size so that hollow polystyrene microsphere is more suitable for in-vitro diagnosis
In reagent.
In addition, the first buffer is 2- (N- morpholine) ethanesulfonic acid buffer, the concentration of the first buffer is 25～
100mM, pH are 5.5～6.5.This is the first buffer preferred best ion intensity and pH so that antibody can more wrap
It is layed onto on hollow polystyrene microsphere, thus reaching higher Detection results.
In addition, the concentration of N-hydroxy-succinamide solution is 10～200mg/mL；1- (3- dimethylamino-propyl) -3- second
The concentration of base carbodiimide hydrochloride solution is 5～80mg/mL.So can preferably activate microsphere, improve antibody linked arriving
Efficiency on hollow polystyrene microsphere.
In addition, the second buffer solution is 4- hydroxyethyl piperazine ethanesulfonic acid, the concentration of the second buffer solution is 25～100mM.
Buffer solution is enable to have suitable pH so that hollow polystyrene microsphere is stably dissolved in buffer.
Brief description
Fig. 1 is according to the neutrophil gelatinase-associated lipocalin NGAL detection examination in first embodiment of the invention
The preparation flow figure of agent box；
Fig. 2 is according to the neutrophil gelatinase-associated lipocalin NGAL detection examination in fifth embodiment of the invention
The deviation from linearity detection curve of agent box.
Specific embodiment
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with each reality to the present invention for the accompanying drawing
The mode of applying is explained in detail.However, it will be understood by those skilled in the art that in each embodiment of the present invention,
In order that reader more fully understands that the application proposes many ins and outs.But, even if there is no these ins and outs and base
In following embodiment many variations and modification it is also possible to realize the application technical scheme required for protection.
The first embodiment of the present invention is related to a kind of neutrophil gelatinase-associated lipocalin NGAL detectable
The preparation method of box, this preparation method comprises the steps of：The activation of hollow polystyrene microsphere：To hollow polystyrene microsphere
After middle addition the first buffer is diluted, add N-hydroxy-succinamide solution, stirring, mix homogeneously, in the mistake of stirring
Add 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride solution in journey, continue stirring, after washing, be dissolved into the
In one buffer, obtain final product the hollow polystyrene microsphere solution of activation；The coupling of antibody：Hollow polystyrene microsphere to activation
Add neutrophil gelatinase-associated lipocalin NGAL antibody-solutions in solution, after concussion reaction, obtain final product the first mixed liquor；
The closing of microsphere：Add bovine serum albumin, concussion reaction in the first mixed liquor, obtain final product the second mixed liquor；Washing, dissolving：
Second mixed liquor is dissolved in the second buffer after centrifugation, supersound washing, obtains final product neutrophilic granulocyte gelatinase correlation fat
Matter transporter detection kit.
Specifically, in the present embodiment, hollow polystyrene microsphere preparation method can comprise the steps of：By sulphuric acid
Manganese and ferric chloride mixing add in solution, stir, and add sodium hydroxide, and adjustment thermotonuses 1～2h obtains Manganese Ferrite；
Add Oleic acid, react 2～3h, obtain the Manganese Ferrite of oleic acid modified；Take silicon dioxide, sodium lauryl sulphate, styrene, over cure
Sour potassium is dissolved in deionized water, stirs；Add the Manganese Ferrite of oleic acid modified, adjust temperature, after reaction 1～3h, add propylene
Acid, continues stirring 3～7h；It is subsequently adding Fluohydric acid., maintain stirring 1～3h, obtain final product hollow polystyrene microsphere.But, this reality
Mode of applying should not be as limit, and following manner can also prepare hollow polystyrene microsphere：By manganese sulfate and ferric chloride
Mixing adds in solution, stirs, and adds sodium hydroxide, and adjustment thermotonuses 1～2h obtains Manganese Ferrite；Add Oleic acid, instead
Answer 2～3h, obtain the Manganese Ferrite of oleic acid modified；Silicon dioxide, sodium lauryl sulphate, styrene, potassium peroxydisulfate is taken to be dissolved in
In ionized water, stir；Add the Manganese Ferrite of oleic acid modified, adjust temperature, add Fluohydric acid., maintain stirring 1～3h, then plus
Enter acrylic acid, continue stirring 3～7h；Obtain final product hollow polystyrene microsphere.
Further, can to include hollow polystyrene micro- for the structure of the above-mentioned hollow polystyrene microsphere preparing
Ball, ferrimanganic layer and Oleic acid layer, ferrimanganic layer passes through Oleic acid layer and ferrimanganic layer is uniformly coated on the outer of hollow polystyrene microsphere
Side, wherein, a diameter of 30nm～80nm of hollow parts of hollow polystyrene microsphere, the polyphenyl that hollow polystyrene microsphere is formed
The thickness of pvdf layer is 30nm～100nm, and the thickness of ferrimanganic layer is 20nm～50nm, and the thickness of Oleic acid layer is 2nm～10nm.In
Empty polystyrene microsphere can also be prepared acquisition by other means and can also directly obtain from direct purchase the in market.This area skill
Art personnel can flexibly select the concrete of hollow polystyrene microsphere preparation method or hollow polystyrene microsphere as needed
Structure.
What deserves to be explained is, in the present embodiment, the first buffer can be 2- (N- morpholine) ethanesulfonic acid buffer,
The concentration of the first buffer can be 25～100mM, and pH can be 5.5～6.5.
The concentration of N-hydroxy-succinamide solution can be 10～200mg/mL；1- (3- dimethylamino-propyl) -3- second
The concentration of base carbodiimide hydrochloride solution can be 5～80mg/mL.
Second buffer solution can be 4- hydroxyethyl piperazine ethanesulfonic acid, the concentration of the second buffer solution can for 25～
100mM.
By the above, it is seen that, in the present embodiment, specifically prepare neutrophilic granulocyte gelatinase related lipid
The method of transporter detection kit is as follows：
(1) the preparation of hollow polystyrene microsphere：1.69g manganese sulfate and 5.4g ferric chloride is taken to be separately added into 50ml solution
In, take 1.2g sodium hydroxide to be configured to 10ml solution, be added in mixed solution, adjust reaction temperature to 70 DEG C, react 1h, obtain
To Manganese Ferrite thickness degree a size of 36nm.Add 2ml Oleic acid, continue stirring 2h, obtain acidity of oil Manganese Ferrite a size of 39nm, its
Middle Oleic acid thickness degree 2nm.
Take 1g 30nm silicon dioxide (SiO2), 40mg sodium lauryl sulphate, 10ml styrene, 10mg potassium peroxydisulfate molten
In 40ml deionized water, stir.Obtain the polystyrene microsphere that polystyrene layer thickness is 78nm；Add the above-mentioned oil of 1g
Dissolubility Manganese Ferrite, sets 60 DEG C of temperature of reaction system, adds 0.8ml acrylic acid after reaction 2h, continue stirring 5h, obtain magnetic and gather
Phenylethylene micro ball.It is subsequently adding the Fluohydric acid. 3ml of 40% concentration, maintain stirring 2h, obtain final product hollow polystyrene microsphere.
It should be noted that after setting reaction temperature it is also possible to by add acrylic acid, Fluohydric acid. order exchange under, that is,
It is initially charged Fluohydric acid., maintains stirring 3h, add acrylic acid, continue stirring 7h；Hollow polystyrene microsphere can also be obtained.
Above-mentioned steps synthesis has obtained by the hollow polystyrene microsphere of the Manganese Ferrite cladding of oleic acid modified, and Manganese Ferrite is not only
While possessing very high superparamagnetism as ferroso-ferric oxide, be easy to separate, and have that cost is lower, synthesized bag
Cover the advantage that outer layer can preferably control pattern and size；And synthesized magnetic hollow microsphere can preferably control chi
Very little it is adaptable in external diagnosis reagent.
(2) the activation of polystyrene microsphere：0.2ml hollow polystyrene microsphere 2ml 25mM MES (pH6.5) is taken to buffer
Liquid dilutes, and adds 40mg N-hydroxy-succinamide (NHS) solution, mix homogeneously, adds 20mg 1- (3- bis- in then stirring
Methylaminopropyl) -3- ethyl-carbodiimide hydrochloride (EDC) solution, stirring reaction under room temperature, it is dissolved into 2ml 25mM after washing
In MES (pH 6.5) buffer.
(3) the coupling of antibody：1mg self-control neutrophil cell gelatinase is added in the microspheres solution crossed to first step activated rinse
Associated lipocalin (NGAL) antibody-solutions, concussion reaction 1h at room temperature.
(4) the closing of microsphere：20mg bovine serum albumin, room temperature concussion closing 1h is added in mixed liquor.
(5) washing, dissolving：The mixture that reaction is terminated is centrifuged 10min, ultrasonic 5min under 16000rpm and washs 2 times,
Finally it is dissolved into 50mM HEPES, in 0.5%BSA, 0.05%NaN3 buffer.
In the prior art, in the preparation method of neutrophil gelatinase-associated lipocalin NGAL detection kit
The polymer microsphere of polystyrene microsphere and its synthesis can be added, the polymer microsphere of this polystyrene microsphere and its synthesis refers to
By a kind of polymerization or by existing polymer through either physically or chemically modification obtained from spherical polymer, particle diameter
Typically arrive between hundreds of micron at several nanometers.Because its density is close to water, can be good at being suspended in water, thus logical
Raw material frequently as external diagnosis reagent.But when the particle diameter of polystyrene microsphere constantly increases, it is subject in the solution
Gravity also can constantly increase, and often results in the precipitation of reagent, and then affects the stability of reagent and the sensitivity of detection.
In order to solve this problem, the embodiment in the present invention passes through in neutrophil gelatinase-associated lipocalin NGAL
(NGAL) using hollow polystyrene microsphere in reagent, that is, pass through in Nano-meter SiO_22Synthetic polystyrene microsphere on kernel, then lead to
Cross Fluohydric acid. and remove Nano-meter SiO_22Kernel, thus defining the polystyrene microsphere of hollow, reduces the close of polystyrene microsphere
Degree, such that it is able to make microsphere preferably suspend in the solution, improves the stability of reagent.Thus it is thin that neutral grain is greatly facilitated
The stable mass production of born of the same parents' gelatinase associated lipocalin (NGAL) reagent, has important actual application value.
Second embodiment of the present invention is related to a kind of neutrophil gelatinase-associated lipocalin NGAL detectable
The preparation method of box, this preparation method comprises the steps of：
(1) the preparation of hollow polystyrene microsphere：
Take 1.69g manganese sulfate and 5.4g ferric chloride to be separately added in 50ml solution, take 1.5g sodium hydroxide to be configured to
10ml solution, is added in mixed solution, adjusts reaction temperature to 70 DEG C, reacts 2h, obtain Manganese Ferrite thickness degree a size of
31nm.Add 2ml Oleic acid, continue stirring 3h, obtain acidity of oil Manganese Ferrite a size of 35nm, wherein Oleic acid thickness degree 4nm.
Take 1g 62nm silicon dioxide (SiO2), 60mg sodium lauryl sulphate, 10ml styrene, 10mg potassium peroxydisulfate molten
In 40ml deionized water, stir.Obtain the polystyrene microsphere that polystyrene layer thickness is 56nm, set reaction system
Temperature 60 C, adds 0.8ml acrylic acid after reaction 3h, continues stirring 7h, obtains magnetic polystyrene microsphere.It is subsequently adding 40%
The Fluohydric acid. 3ml of concentration, maintains stirring 3h, obtains final product hollow polystyrene microsphere.
(2) the activation of polystyrene microsphere：0.4ml hollow polystyrene microsphere 2ml 25mM MES (pH 6.5) is taken to delay
Rush liquid dilution, add 40mg N-hydroxy-succinamide (NHS) solution, mix homogeneously, in then stirring, add 30mg 1- (3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) solution, stirring reaction under room temperature, it is dissolved into 2ml after washing
In 25mM MES (pH 6.5) buffer.
(3) the coupling of antibody：Add 0.8mg self-control neutrophilic granulocyte bright in the microspheres solution crossed to first step activated rinse
Glue enzyme associated lipocalin (NGAL) antibody-solutions, concussion reaction 1h at room temperature.
(4) the closing of microsphere：20mg bovine serum albumin, room temperature concussion closing 1h is added in mixed liquor.
(5) washing, dissolving：The mixture that reaction is terminated is centrifuged 10min, ultrasonic 5min under 16000rpm and washs 2 times,
Finally it is dissolved into 50mM HEPES, in 0.5%BSA, 0.05%NaN3 buffer.
Third embodiment of the present invention is related to a kind of neutrophil gelatinase-associated lipocalin NGAL detectable
The preparation method of box, this preparation method comprises the steps of：
(1) the preparation of hollow polystyrene microsphere：
Take 1.69g manganese sulfate and 5.4g ferric chloride to be separately added in 50ml solution, take 1.2g sodium hydroxide to be configured to 8ml
Solution, is added in mixed solution, adjusts reaction temperature to 70 DEG C, reacts 1.5h, obtain Manganese Ferrite thickness degree a size of 42nm.
Add 2ml Oleic acid, continue stirring 2.5h, obtain acidity of oil Manganese Ferrite a size of 48nm, wherein Oleic acid thickness degree 6nm.
Take 1g 35nm silicon dioxide (SiO2), 40mg sodium lauryl sulphate, 10ml styrene, 6mg potassium peroxydisulfate be dissolved in
In 40ml deionized water, stir.Obtain the polystyrene microsphere that polystyrene layer thickness is 85nm, set reaction system temperature
60 DEG C of degree, adds 0.8ml acrylic acid after reaction 1h, continues stirring 3h, obtains magnetic polystyrene microsphere.It is subsequently adding 40% dense
The Fluohydric acid. 3ml of degree, maintains stirring 1h, obtains final product hollow polystyrene microsphere.
It should be noted that after setting reaction temperature it is also possible to by add acrylic acid, Fluohydric acid. order exchange under, that is,
It is initially charged Fluohydric acid., maintains stirring 1h, add acrylic acid, continue stirring 3h；Hollow polystyrene microsphere can also be obtained.
(2) the activation of polystyrene microsphere：0.2ml hollow polystyrene microsphere 2ml 25mM MES (pH 6.5) is taken to delay
Rush liquid dilution, add 60mg N-hydroxy-succinamide (NHS) solution, mix homogeneously, in then stirring, add 20mg 1- (3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) solution, stirring reaction under room temperature, it is dissolved into 2ml after washing
In 25mM MES (pH 6.5) buffer.
(3) the coupling of antibody：Add 1.5mg self-control neutrophilic granulocyte bright in the microspheres solution crossed to first step activated rinse
Glue enzyme associated lipocalin (NGAL) antibody-solutions, concussion reaction 1h at room temperature.
(4) the closing of microsphere：30mg bovine serum albumin, room temperature concussion closing 1h is added in mixed liquor.
(5) washing, dissolving：The mixture that reaction is terminated is centrifuged 10min, ultrasonic 5min under 16000rpm and washs 2 times,
Finally it is dissolved into 50mM HEPES, in 0.5%BSA, 0.05%NaN3 buffer.
4th embodiment of the present invention is related to a kind of neutrophil gelatinase-associated lipocalin NGAL detectable
The preparation method of box, this preparation method comprises the steps of：
Take 1.69g manganese sulfate and 5.4g ferric chloride to be separately added in 50ml solution, take 1.2g sodium hydroxide to be configured to 8ml
Solution, is added in mixed solution, adjusts reaction temperature to 70 DEG C, reacts 1h, obtain Manganese Ferrite thickness degree a size of 42nm.Plus
Enter 2ml Oleic acid, continue stirring 2h, obtain acidity of oil Manganese Ferrite a size of 48nm, wherein Oleic acid thickness degree 6nm.
Take 1g 35nm silicon dioxide (SiO2), 40mg sodium lauryl sulphate, 10ml styrene, 8mg potassium peroxydisulfate be dissolved in
In 40ml deionized water, stir.Obtain the polystyrene microsphere that polystyrene layer thickness is 82nm, set reaction system temperature
60 DEG C of degree, adds 0.8ml acrylic acid after reaction 2h, continues stirring 5h, obtains magnetic polystyrene microsphere.It is subsequently adding 40% dense
The Fluohydric acid. 3ml of degree, maintains stirring 2h, obtains final product hollow polystyrene microsphere.
It should be noted that after setting reaction temperature it is also possible to by add acrylic acid, Fluohydric acid. order exchange under, that is,
It is initially charged Fluohydric acid., maintains stirring 2h, add acrylic acid, continue stirring 5h；Hollow polystyrene microsphere can also be obtained.
(2) the activation of polystyrene microsphere：0.2ml hollow polystyrene microsphere 2ml 25mM MES (pH 6.5) is taken to delay
Rush liquid dilution, add 20mg N-hydroxy-succinamide (NHS) solution, mix homogeneously, in then stirring, add 20mg 1- (3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) solution, stirring reaction under room temperature, it is dissolved into 2ml after washing
In 25mM MES (pH 6.5) buffer.
(3) the coupling of antibody：Add 1.2mg self-control neutrophilic granulocyte bright in the microspheres solution crossed to first step activated rinse
Glue enzyme associated lipocalin (NGAL) antibody-solutions, concussion reaction 1h at room temperature.
(4) the closing of microsphere：30mg bovine serum albumin, room temperature concussion closing 1h is added in mixed liquor.
(5) washing, dissolving：The mixture that reaction is terminated is centrifuged 5min, ultrasonic 8min washing 2 times under 18000rpm,
After be dissolved into 50mM HEPES, in 0.5%BSA, 0.05%NaN3 buffer.
5th embodiment of the present invention is related to a kind of neutrophil gelatinase-associated lipocalin NGAL detectable
The preparation method of box, this preparation method comprises the steps of：
Take 1.69g manganese sulfate and 5.4g ferric chloride to be separately added in 50ml solution, take 1.2g sodium hydroxide to be configured to 8ml
Solution, is added in mixed solution, adjusts reaction temperature to 70 DEG C, reacts 1h, obtain Manganese Ferrite thickness degree a size of 42nm.Plus
Enter 2.5ml Oleic acid, continue stirring 2h, obtain acidity of oil Manganese Ferrite a size of 50nm, wherein Oleic acid thickness degree 6nm.
Take 1g 35nm silicon dioxide (SiO2), 40mg sodium lauryl sulphate, 10ml styrene, 8mg potassium peroxydisulfate be dissolved in
In 40ml deionized water, stir.Obtain the polystyrene microsphere that polystyrene layer thickness is 82nm, set reaction system temperature
60 DEG C of degree, adds 0.8ml acrylic acid after reaction 2h, continues stirring 5h, obtains magnetic polystyrene microsphere.It is subsequently adding 40% dense
The Fluohydric acid. 3ml of degree, maintains stirring 2h, obtains final product hollow polystyrene microsphere.
It should be noted that after setting reaction temperature it is also possible to by add acrylic acid, Fluohydric acid. order exchange under, that is,
It is initially charged Fluohydric acid., maintains stirring 2h, add acrylic acid, continue stirring 5h；Hollow polystyrene microsphere can also be obtained.
(2) the activation of polystyrene microsphere：0.2ml hollow polystyrene microsphere 2ml 25mM MES (pH 6.5) is taken to delay
Rush liquid dilution, add 30mg N-hydroxy-succinamide (NHS) solution, mix homogeneously, in then stirring, add 40mg 1- (3-
Dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride (EDC) solution, stirring reaction under room temperature, it is dissolved into 2ml after washing
In 25mM MES (pH 6.5) buffer.
(3) the coupling of antibody：Add 1.5mg self-control neutrophilic granulocyte bright in the microspheres solution crossed to first step activated rinse
Glue enzyme associated lipocalin (NGAL) antibody-solutions, concussion reaction 1h at room temperature.
(4) the closing of microsphere：40mg bovine serum albumin, room temperature concussion closing 1h is added in mixed liquor.
(5) washing, dissolving：The mixture that reaction is terminated is centrifuged 5min, ultrasonic 8min washing 2 times under 18000rpm,
After be dissolved into 100mM HEPES, in 1%BSA, 0.05%NaN3 buffer.
In the present embodiment, in order to the stability proving the detectable adding hollow polystyrene microsphere can be higher
A bit, respectively two kinds of reagent are tested, a kind of reagent is the NGAL reagent containing hollow polystyrene microsphere, another examination
Agent is the NGAL reagent containing polystyrene microsphere.
Detecting step is：
1st, the coated NGAL R2 reagent of stability assessment batch hollow polystyrene microsphere and polystyrene microsphere is taken to be coated
NGAL R2 reagent be positioned in 4 DEG C of refrigerators place 12 months after take out detection.
2nd, the R1 reagent taking same recipe is divided into two parts respectively as R1 reagent
3rd, detect parameter：3/150/50；Wavelength 800/570；Light-metering point 18～34；
4th, detect sample：
1. it is diluted to linear sample using linear high level to detect 1 time
2. each 10 times of detection 500ug/L and 1000ug/L sample
3. detection quality-control product 1 (220ug/L) and each three times of quality-control product 2 (720ug/L)
Experimental result is as shown in table 1～3：
Table 1：
The linear gradient result of NGAL reagent 12 monthly tests of 4 DEG C of placements
In order to be able to more intuitively see the deviation from linearity between two kinds of reagent, the linear ladder to two kinds of reagent detections in table 1
Shown in degree result corresponding curve Fig. 2,
Table 2
NGAL reagent 12 months CV testing results of 4 DEG C of placements
Table 3
NGAL reagent 12 months accuracy testing results of 4 DEG C of placements
Interpretation：
(1) good stability：Within reagent effect duration, in the range of test linear sample (30-1000) μ g/L, linearly definitely inclined
Difference is less than ± 70 μ g/L；(1001-5000), in the range of μ g/L, linear relative deviation is less than ± 8%.
(2) in the range of reagent effect duration, CV≤3%；Accuracy determination result is to be with the relative deviation of quality-control product target value
Within ± 6%.
It will be understood by those skilled in the art that the respective embodiments described above are to realize the specific embodiment of the present invention,
And in actual applications, can to it, various changes can be made in the form and details, without departing from the spirit and scope of the present invention.
Claims (10)
1. a kind of preparation method of neutrophil gelatinase-associated lipocalin NGAL detection kit is it is characterised in that wrap
Containing following steps：
The activation of hollow polystyrene microsphere：After adding the first buffer to be diluted in hollow polystyrene microsphere, add
N-hydroxy-succinamide solution, stirring, mix homogeneously, described stirring during add 1- (3- dimethylamino-propyl)-
3- ethyl-carbodiimide hydrochloride solution, after continuation stirring, washing, is dissolved in described first buffer, obtains final product in activation
Empty polystyrene microsphere solution；
The coupling of antibody：Neutrophilic granulocyte gelatinase related lipid is added in the hollow polystyrene microsphere solution of described activation
Transporter antibody-solutions, after concussion reaction, obtain final product the first mixed liquor；
The closing of microsphere：Add bovine serum albumin, concussion reaction in described first mixed liquor, obtain final product the second mixed liquor；
Washing, dissolving：Described second mixed liquor is dissolved in the second buffer after centrifugation, supersound washing, obtains final product neutrality
Granulocyte gelatinase associated lipocalin detection kit.
2. preparation method according to claim 1 is it is characterised in that described hollow polystyrene microsphere preparation method comprises
Following steps：
Manganese sulfate and ferric chloride mixing are added in solution, stirs, add sodium hydroxide, adjust thermotonuses 1～2h
Obtain Manganese Ferrite；Add Oleic acid, react 2～3h, obtain the Manganese Ferrite of oleic acid modified；
Take silicon dioxide, sodium lauryl sulphate, styrene, potassium peroxydisulfate to be dissolved in deionized water, stir；Add described
The Manganese Ferrite of oleic acid modified, adjusts temperature, adds acrylic acid, continue stirring 3～7h after reaction 1～3h；It is subsequently adding Fluohydric acid.,
Maintain stirring 1～3h, obtain final product hollow polystyrene microsphere.
3. preparation method according to claim 1 is it is characterised in that described hollow polystyrene microsphere preparation method comprises
Following steps：
Manganese sulfate and ferric chloride mixing are added in solution, stirs, add sodium hydroxide, adjust thermotonuses 1～2h
Obtain Manganese Ferrite；Add Oleic acid, react 2～3h, obtain the Manganese Ferrite of oleic acid modified；Take silicon dioxide, sodium lauryl sulphate,
Styrene, potassium peroxydisulfate are dissolved in deionized water, stir；Add the Manganese Ferrite of described oleic acid modified, adjust temperature, add
Fluohydric acid., maintains stirring 1～3h, adds acrylic acid, continues stirring 3～7h；Obtain final product hollow polystyrene microsphere.
4. the preparation method according to Claims 2 or 3 is it is characterised in that the mass concentration of described silicon dioxide is 0.25
～0.1g/ml, the particle diameter of described silicon dioxide is 30～80nm.
5. the preparation method according to Claims 2 or 3 is it is characterised in that the mass concentration of described sodium lauryl sulphate
For 1～20mg/ml.
6. the preparation method according to Claims 2 or 3 is it is characterised in that the body of described styrene and described deionized water
Long-pending ratio is 1:5.
7. preparation method according to claim 1 is it is characterised in that described hollow polystyrene microsphere includes hollow polyphenyl
Ethylene microsphere, ferrimanganic layer and Oleic acid layer, described ferrimanganic layer is uniformly coated on institute by described Oleic acid layer by described ferrimanganic layer
State the outside of hollow polystyrene microsphere, wherein, a diameter of 30nm～80nm of hollow parts of described hollow polystyrene microsphere,
The thickness of the polystyrene layer that described hollow polystyrene microsphere is formed is 30nm～100nm, and the thickness of described ferrimanganic layer is
20nm～50nm, the thickness of described Oleic acid layer is 2nm～10nm.
8. preparation method according to claim 1 is it is characterised in that described first buffer is 2- (N- morpholine) second sulphur
Acid buffer, the concentration of described first buffer is 25～100mM, and pH is 5.5～6.5.
9. preparation method according to claim 1 is it is characterised in that the concentration of described N-hydroxy-succinamide solution is
10～200mg/mL；The concentration of described 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride solution is 5～80mg/
mL.
10. preparation method according to claim 1 is it is characterised in that described second buffer solution is 4- hydroxyethyl piperazine
Ethyl sulfonic acid, the concentration of described second buffer solution is 25～100mM.
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