CN109870584A - A kind of neutrophil gelatinase-associated lipocalin detection kit - Google Patents
A kind of neutrophil gelatinase-associated lipocalin detection kit Download PDFInfo
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- CN109870584A CN109870584A CN201711257965.9A CN201711257965A CN109870584A CN 109870584 A CN109870584 A CN 109870584A CN 201711257965 A CN201711257965 A CN 201711257965A CN 109870584 A CN109870584 A CN 109870584A
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- associated lipocalin
- gelatinase
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Abstract
The present invention relates to a kind of neutrophil gelatinase-associated lipocalin detection kits, belong to technical field of immune assay.The kit include Sample dilution, cleaning solution, developing solution and terminate liquid, the soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody of horseradish peroxidase-labeled, various concentration soluble neutral granulocyte gelatinase associated lipocalin standard items, soluble neutral granulocyte gelatinase associated lipocalin quality-control product and micro reaction plate, micro reaction plate be coated with soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody.Standard items described in the kit directly use, and realize efficient, quick detection, while the quality-control product can identify whether testing result is accurate, and the detection kit of different size can be obtained in the standard items of various concentration, adapt to different clients demand.
Description
Technical field
The present invention relates to a kind of neutrophil gelatinase-associated lipocalin (NGAL) detection kits, belong to and exempt from
Epidemic disease tests and analyzes technical field.
Background technique
Acute kidney injury (acute kidney injury, AKI) is common in emergency and severe disease patient, and early diagnosis, treatment can be shown
Writing improves prognosis.The main still serum creatinine (Scr) of the lab index of renal dysfunction at present, but Scr is one relative to AKI
Plant marker that is insensitive and extremely lagging.Because only reflecting the filtering function of glomerulus, but due to the compensatory capacity of kidney
Very strong, when glomerular filtration rate is down to normal 30% or less, significant change can just occur for Scr, but be likely to own warp at this time
Miss the critical period for the treatment of.Due to lacking early stage, sending out clinic can not in early days with predictive, Noninvasive marker
Existing injury of kidney, and effective remedy measures are taken in time.
NGAL is the small protein of a 25KD, is made of 178 amino acid, belongs to apolipoprotein family member,
Its function is transhipment hydrophobic small molecules ligand.NGAL results from neutrophil leucocyte, also a variety of normal tissues of the mankind (such as lung,
Tracheae, stomach, colon, kidney, uterus, prostate and salivary gland etc.) epithelial cell in have different degrees of low expression.In various kidneys
NGAL then overexpression in dirty disease.Although its biological function not yet illustrates completely, it is current research shows that: in mouse
Experiment in vitro in, the early stage progenitor cell of kidney mesenchyma generates proliferation effect after NGAL promotes, and promotes original interstitial thin
Born of the same parents generate the shape of kidney and express the surface marker of glomerulus, proximal tubule, distal tubule, to adjust kidney to epithelial differentiation
Dirty generation.In addition, kidney injury can cause free iron in renal epithelial cell to gather, and the accumulation of iron and albuminuria, renal tubule
The lesion degrees such as interstitial lesion, lipid peroxidation are directly related, and the iron of excess load can increase the neurological susceptibility of Ischemic kieney injury.
NGAL reduces the active oxygen that extracellular iron generates by adjusting iron metabolism and mitigates Apoptosis caused by oxidative stress, while thin
Iron intracellular can increase cell repair through a variety of ways, reduce apoptosis.Therefore, NGAL may be played in kidney injury to damage-retardation
The effect of wound.
NGAL is a kind of one of new marker for being possibly used for AKI early diagnosis.Not only there is glomerular filtration when AKI
The sharply decline of function, and usually all with the dysfunction of renal tubule.NGAL not only because glomerular filtration rate decline due to
It is accumulated in blood circulation, renal damage position is even more meeting great expression release, therefore the raising of NGAL refers to compared with other when generation AKI
It marks faster, and higher with the correlation of AKI.Early diagnosis of the NGAL in kidney trouble and the in terms of general to disease prognosis
It plays a significant role.
Therefore, seem to the quick, comprehensive, accurate of neutrophil gelatinase-associated lipocalin, specific detection
It is particularly important.Up to the present, the neutrophil gelatinase-associated lipocalin detection means of commodity all still has one
It settles finally sex-limited.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of detections of neutrophil gelatinase-associated lipocalin
Kit, the kit can carry out neutrophil gelatinase-associated lipocalin quickly, comprehensively, accurately, specifically
Detection.
To achieve the purpose of the present invention, following technical scheme is provided.
A kind of neutrophil gelatinase-associated lipocalin detection kit, the kit include that sample is dilute
Liquid, cleaning solution, developing solution and terminate liquid, enzyme marker are released, the enzyme marker is the solubility of horseradish peroxidase-labeled
Neutrophil gelatinase-associated lipocalin monoclonal antibody；The detection kit further include: multiple and different concentration
Soluble neutral granulocyte gelatinase associated lipocalin standard items, soluble neutral granulocyte gelatinase related lipid
Transporter quality-control product and micro reaction plate, the micro reaction plate are coated with soluble neutral granulocyte gelatinase correlation rouge
Matter transporter monoclonal antibody.
The concentration of the soluble neutral granulocyte gelatinase associated lipocalin standard items of the multiple various concentration
It is followed successively by 0ng/ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml and 200ng/ml.
Preferably, the soluble neutral granulocyte gelatinase associated lipocalin standard items the preparation method comprises the following steps:
Soluble neutral granulocyte gelatinase associated lipocalin antigen is diluted with fetal calf serum processing matrix, with obtained
To soluble neutral granulocyte gelatinase associated lipocalin standard items.
Preferably, the solvable neutrophil gelatinase-associated lipocalin quality-control product includes low value range Quality Control
Product and high level range quality-control product, the low value range quality-control product concentration are 24~36ng/ml, the high level range quality-control product concentration
For 48~72ng/ml.
Preferably, the low value range quality-control product concentration is 30ng/ml, and the high level range quality-control product concentration is 60ng/
ml。
Preferably, the quality-control product the preparation method comprises the following steps: with fetal calf serum processing matrix to solvable neutrophil leucocyte gelatin
Enzyme associated lipocalin antigen is diluted, and preparation obtains soluble neutral granulocyte gelatinase associated lipocalin
Quality-control product.
A kind of preparation method of neutrophil gelatinase-associated lipocalin detection kit of the present invention, packet
Include following steps:
(1) soluble neutral granulocyte gelatinase associated lipocalin standard items and soluble neutral granulocyte are prepared
Gelatinase associated lipocalin quality-control product: using fetal calf serum processing matrix that soluble neutral granulocyte gelatinase is related
Lipocalin protein antigen diluent obtains soluble neutral granulocyte gelatinase associated lipocalin standard items, uses simultaneously
Fetal calf serum handles matrix and soluble neutral granulocyte gelatinase associated lipocalin antigen diluent is obtained quality-control product；
(2) the coated micropore reaction of soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody is prepared
Plate: drawing soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody, and 0.01mol/L, pH=7.2 is added
Phosphate solution in be diluted, 4 DEG C store for future use；Microwell plate is taken, the soluble neutral grain after 100 μ L dilution is added in every hole
Cell gelatinase associated lipocalin monoclonal antibody, overlay film are placed on 4 DEG C and are coated with 18~22 hours；Get rid of liquid in hole
Body, every hole are added 200 μ L and include 1~3% bovine serum albumin(BSA), 3~5% sucrose, 0.05~0.15%Priclin300
The phosphate solution of 0.01mol/L, pH=7.4, overlay film are placed on 4 DEG C and close 18~22 hours；Liquid in hole is got rid of, and is being inhaled
It is patted dry on water paper, vacuum sealed package after drying 2~4 days at room temperature obtains soluble neutral granulocyte gelatinase related lipid
The coated micro reaction plate of transporter monoclonal antibody, the micro reaction plate are stored in 4 DEG C.
(3) the soluble neutral granulocyte gelatinase associated lipocalin Dan Ke of horseradish peroxidase-labeled is prepared
Grand antibody: weighing horseradish peroxidase 2mg, is dissolved in 1mL deionized water, and it is 0.05mol/L's that 1~2mL concentration, which is added,
Sodium periodate solution, 4 DEG C slowly shake 30~60min；It is 0.001mol/L, the sodium acetate solution dialysis of pH=4.4 with concentration
18~22 hours；2mg soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody is added, 4 DEG C are protected from light vibration
It swings 18~22 hours；The NaBH that 400 μ L concentration are 0.2mol/L is added4Solution is 0.02mol/L, the phosphorus of pH=7.4 with concentration
Acid buffer is dialysed 18~22 hours；It is secondarily purified with HPLC, protein peak is collected, isometric glycerol is added, obtains horseradish peroxide
The soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody of compound enzyme label, saves in -20 DEG C.
(4) it prepares Sample dilution: taking sodium dihydrogen phosphate 1.409g, disodium hydrogen phosphate 4.372g, sodium chloride 8.5g, ox blood
Pure albumen 5g, Tween-20 0.5mL, Jia Shui are settled to 1L, with sodium hydroxide tune pH to 7.4 ± 0.1；
(5) it prepares cleaning solution: taking sodium dihydrogen phosphate 28.18g, disodium hydrogen phosphate 87.44g, sodium chloride 170g, tween-
2010mL adds water to be settled to 1L, and when use is diluted with water 20 times；
(6) it prepares developing solution: weighing 100gTMB, dissolved with the mixed liquor that 10mLDMSO and 10mL dehydrated alcohol is made into, 4
It DEG C is kept in dark place spare；Prepare the citric acid solution of 0.1mol/LpH4.0~5.0；Citric acid to preparation described in 1000mL is molten
100~200mg hydrogen peroxide urea is added in liquid；Dissolution sufficiently after, be added PVP, make its final concentration of 0.2~0.6%, 4 DEG C
It places 18~22 hours；The TMB mixed liquor dissolved in advance is added, and 2~4% polyethylene glycol are added, water is added to be settled to 1000mL,
4 DEG C are kept in dark place；
(7) it prepares terminate liquid: measuring the 50mL concentrated sulfuric acid and slowly injected in 870mL water along walls of beaker, mix, keep it dilute
18.4 times are released, 4 DEG C of preservations.
Beneficial effect
The present invention provides a kind of neutrophil gelatinase-associated lipocalin detection kit, in kit
The soluble neutral granulocyte gelatinase associated lipocalin standard items of a variety of various concentrations can without being diluted processing
Directly to use, operating procedure is simplified, shortens detection time, realizes efficient, quick detection, while solvable neutral grain is thin
Born of the same parents' gelatinase associated lipocalin quality-control product can identify whether testing result is accurate, in the solubility of multiple and different concentration
The soluble neutral granulocyte gelatinase related lipid that property granulocyte gelatinase associated lipocalin micro reaction plate uses
Transporter monoclonal antibody has high degree of specificity, and soluble neutral granulocyte gelatinase associated lipocalin standard
Product may be implemented to carry out the soluble neutral granulocyte gelatinase associated lipocalin protein molecular in sample single-minded
Quantitative detection；The soluble neutral granulocyte gelatinase associated lipocalin standard items of the various concentration of offer are available
Standard curve, according to soluble neutral granulocyte gelatinase associated lipocalin in the available sample to be tested of standard curve
Concentration；Kit can be combined with full-automatic enzyme-linked response analysis instrument, can further effectively shorten detection time, reduce inspection
It surveys cost and manpower is spent.
Specific embodiment
A kind of neutrophil gelatinase-associated lipocalin detection kit includes micro reaction plate, enzyme mark
Remember object, Sample dilution, developing solution, terminate liquid, cleaning solution, soluble neutral granulocyte gelatinase associated lipocalin mark
Quasi- product, soluble neutral granulocyte gelatinase associated lipocalin quality-control product, wherein enzyme marker is horseradish peroxidase
The soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody of enzyme label, soluble neutral granulocyte gelatin
Enzyme associated lipocalin standard items are with fetal calf serum processing matrix to soluble neutral granulocyte gelatinase related lipid
Transporter antigen is diluted to obtain, and soluble neutral granulocyte gelatinase associated lipocalin quality-control product is with tire ox
Serum processing matrix is diluted to obtain to soluble neutral granulocyte gelatinase associated lipocalin antigen, micropore reaction
Plate is coating soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody；Various concentration in this kit
Soluble neutral granulocyte gelatinase associated lipocalin standard items can be directly using standard curve be drawn, according to sample
OD value the corresponding concentration of sample, soluble neutral granulocyte gelatinase associated lipocalin Quality Control are obtained by standard curve
Product be used to identify whether testing result accurate, enzyme marker without dilution handle, it is easy to operate, can effectively shorten detection when
Between, and then reduce testing cost and manpower cost.
Above-mentioned fetal calf serum processing matrix the preparation method comprises the following steps: take it is appropriate it is finely ground after ammonium sulfate at room temperature while stirring
It is added in fetal calf serum, makes its final concentration of 100~300g/L, 4 DEG C are placed 18~22 hours；10,000 turns are centrifuged 20~40 points
Clock takes supernatant filter paper to filter；Supernatant fills bag filter after filtering, and dialyses in the physiological saline of 10 times of volumes at 4 DEG C, often
Every the dialyzate of replacement in 3~5 hours, altogether three times；It is transferred in the 0.05mol/L boric acid solution of 10 volumes and dialyses, often
Every the dialyzate of replacement in 3~5 hours, four times altogether；Liquid after dialysis is put into clean container, in 56 DEG C of heat inactivations 1
~2 hours；The gentamicin of 0.05~0.15% Proclin300 and 0.05~0.15% is added, after mixing with 0.22
μm membrane filtration degerming obtains fetal calf serum, saves at -20 DEG C stand-by.
Embodiment 1
A kind of preparation method of neutrophil gelatinase-associated lipocalin detection kit, including walk as follows
It is rapid:
(1) soluble neutral granulocyte gelatinase associated lipocalin standard items and quality-control product are prepared: taking and grinds in right amount
Ammonium sulfate after thin is added in fetal calf serum while stirring at room temperature, makes its final concentration of 100g/L, and 4 DEG C are placed 18 hours；
10,000 turns are centrifuged 20 minutes, and supernatant filter paper is taken to filter；Bag filter will be filled for supernatant after filter, in 10 times of volumes at 4 DEG C
It dialyses in physiological saline, every the dialyzate of replacement in 3 hours, altogether three times；It is transferred to the 0.05mol/L boric acid solution of 10 volumes
In dialyse, every the dialyzate of replacement in 3 hours, four times altogether；Liquid after dialysis is put into clean container, in 56
DEG C heat inactivation 1 hour；The gentamicin of 0.05% Proclin300 and 0.05% is added, after mixing with 0.22 μm of filter membrane
Filtration sterilization obtains fetal calf serum processing matrix, saves at -20 DEG C stand-by；It is handled using the above-mentioned fetal calf serum being prepared
Soluble neutral granulocyte gelatinase associated lipocalin antigen diluent is obtained 6 various concentrations and is followed successively by 0ng/ by matrix
The soluble neutral granulocyte gelatinase related lipid of ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml, 200ng/ml
Transporter standard items, while soluble neutral granulocyte gelatinase related lipid is delivered into egg using fetal calf serum processing matrix
It is 30ng/ml low value range quality-control product that white antigen diluent, which obtains concentration,；
(2) the coated micropore reaction of soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody is prepared
Plate: drawing appropriate soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody, and 0.01mol/L, pH is added
It is diluted in=7.2 phosphate solution, makes its final concentration of 2 μ g/mL, 4 DEG C store for future use；Microwell plate is taken, every hole is added
Soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody after 100 μ L dilution, overlay film are placed on 4 DEG C of packets
By 18 hours；Liquid in hole is got rid of, every hole is added 200 μ L and includes 1% bovine serum albumin(BSA), 3% sucrose, 0.05%
The phosphate solution of the 0.01mol/L of Priclin300, pH=7.4, overlay film are placed on 4 DEG C and close 18 hours；Get rid of liquid in hole
Body, and patted dry on blotting paper, vacuum sealed package after drying 2 days at room temperature obtains soluble neutral granulocyte gelatinase phase
The coated micro reaction plate of lipocalin protein monoclonal antibody is closed, the micro reaction plate is stored in 4 DEG C；
(3) the soluble neutral granulocyte gelatinase associated lipocalin Dan Ke of horseradish peroxidase-labeled is prepared
Grand antibody: weighing horseradish peroxidase 2mg, is dissolved in 1mL deionized water, and 1mL concentration is added as 0.05mol/L and crosses iodine
Acid sodium solution, 4 DEG C slowly shake 30min；It is 0.001mol/L with concentration, the sodium acetate solution of pH=4.4 is dialysed 18 hours；Add
Enter 2mg soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody, 4 DEG C are protected from light oscillation 18 hours；It is added
400 μ L concentration are the NaBH of 0.2mol/L4Solution, is 0.02mol/L with concentration, and the phosphate buffer dialysis 18 of pH=7.4 is small
When；It is secondarily purified with HPLC, protein peak is collected, isometric glycerol is added, the solubility for obtaining horseradish peroxidase-labeled is single
Clonal antibody is saved in -20 DEG C；
(4) it prepares Sample dilution: taking sodium dihydrogen phosphate 1.409g, disodium hydrogen phosphate 4.372g, sodium chloride 8.5g, ox blood
Pure albumen 5g, Tween-20 0.5mL, Jia Shui are settled to 1L, with sodium hydroxide tune pH to 7.4 ± 0.1；
(5) it prepares cleaning solution: taking sodium dihydrogen phosphate 28.18g, disodium hydrogen phosphate 87.44g, sodium chloride 170g, tween-
2010mL adds water to be settled to 1L, and when use is diluted with water 20 times；
(6) it prepares developing solution: weighing 100gTMB, dissolved with the mixed liquor that 10mLDMSO and 10mL dehydrated alcohol is made into, 4
It DEG C is kept in dark place spare；Prepare 0.1mol/L, the citric acid solution of pH=4.0；To the citric acid solution of preparation described in 1000mL
Middle addition 100mg hydrogen peroxide urea；Dissolution sufficiently after, be added PVP, make its final concentration of 0.2%, 4 DEG C place 18 hours；Add
Enter the TMB mixed liquor dissolved in advance, and 2% polyethylene glycol is added, adds water to be settled to 1000mL, 4 DEG C are kept in dark place；
(7) it prepares terminate liquid: measuring the 50mL concentrated sulfuric acid and slowly injected in 870mL water along walls of beaker, mix, keep it dilute
18.4 times are released, 4 DEG C of preservations.
Embodiment 2
A kind of preparation method of neutrophil gelatinase-associated lipocalin detection kit, including walk as follows
It is rapid:
(1) soluble neutral granulocyte gelatinase associated lipocalin standard items and quality-control product are prepared: taking and grinds in right amount
Ammonium sulfate after thin is added in fetal calf serum while stirring at room temperature, makes its final concentration of 200g/L, and 4 DEG C are placed 20 hours；
10,000 turns are centrifuged 30 minutes, and supernatant filter paper is taken to filter；Bag filter will be filled for supernatant after filter, in 10 times of volumes at 4 DEG C
It dialyses in physiological saline, every the dialyzate of replacement in 4 hours, altogether three times；It is transferred to the 0.05mol/L boric acid solution of 10 volumes
In dialyse, every the dialyzate of replacement in 4 hours, four times altogether；Liquid after dialysis is put into clean container, in 56
DEG C heat inactivation 1.5 hours；The gentamicin of 0.1% Proclin300 and 0.1% is added, after mixing with 0.22 μm of filter membrane
Filtration sterilization obtains fetal calf serum processing matrix, saves at -20 DEG C stand-by；It is handled using the above-mentioned fetal calf serum being prepared
Soluble neutral granulocyte gelatinase associated lipocalin antigen diluent is obtained 6 various concentrations and is followed successively by 0ng/ by matrix
The soluble neutral granulocyte gelatinase related lipid of ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml, 200ng/ml
Transporter standard items, while soluble neutral granulocyte gelatinase related lipid is delivered into egg using fetal calf serum processing matrix
White antigen diluent obtains that concentration is 30ng/ml low value range quality-control product and concentration is 60ng/ml high level range quality-control product；
(2) the coated micropore reaction of soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody is prepared
Plate: drawing appropriate soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody, and 0.01mol/L, pH is added
It is diluted in=7.2 phosphate solution, makes its final concentration of 4 μ g/mL, 4 DEG C store for future use；Microwell plate is taken, every hole is added
Soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody after 100 μ L dilution, overlay film are placed on 4 DEG C of packets
By 20 hours；Liquid in hole is got rid of, every hole is added 200 μ L and includes 2% bovine serum albumin(BSA), 4% sucrose, 0.1%
The phosphate solution of the 0.01mol/L of Priclin300, pH=7.4, overlay film are placed on 4 DEG C and close 20 hours；Get rid of liquid in hole
Body, and patted dry on blotting paper, vacuum sealed package after drying 3 days at room temperature obtains soluble neutral granulocyte gelatinase phase
The coated micro reaction plate of lipocalin protein monoclonal antibody is closed, the micro reaction plate is stored in 4 DEG C；
(3) the soluble neutral granulocyte gelatinase associated lipocalin Dan Ke of horseradish peroxidase-labeled is prepared
Grand antibody: weighing horseradish peroxidase 2mg, is dissolved in 1mL deionized water, and the mistake that 1.5mL concentration is 0.05mol/L is added
Sodium iodide solution, 4 DEG C slowly shake 45min；It is 0.001mol/L with concentration, the sodium acetate solution of pH=4.4 is dialysed 20 hours；
2mg soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody is added, 4 DEG C are protected from light oscillation 20 hours；Add
Enter the NaBH that 400 μ L concentration are 0.2mol/L4Solution is 0.02mol/L, the phosphate buffer dialysis 20 of pH=7.4 with concentration
Hour；It is secondarily purified with HPLC, protein peak is collected, isometric glycerol is added, obtains the solubility of horseradish peroxidase-labeled
Monoclonal antibody is saved in -20 DEG C；
(4) it prepares Sample dilution: taking sodium dihydrogen phosphate 1.409g, disodium hydrogen phosphate 4.372g, sodium chloride 8.5g, ox blood
Pure albumen 5g, Tween-20 0.5mL, Jia Shui are settled to 1L, with sodium hydroxide tune pH to 7.4 ± 0.1；
(5) it prepares cleaning solution: taking sodium dihydrogen phosphate 28.18g, disodium hydrogen phosphate 87.44g, sodium chloride 170g, tween-
2010mL adds water to be settled to 1L, and when use is diluted with water 20 times；
(6) it prepares developing solution: weighing 100gTMB, dissolved with the mixed liquor that 10mLDMSO and 10mL dehydrated alcohol is made into, 4
It DEG C is kept in dark place spare；Prepare 0.1mol/L, the citric acid solution of pH=4.5；To the citric acid solution of preparation described in 1000mL
Middle addition 150mg hydrogen peroxide urea；Dissolution sufficiently after, be added PVP, make its final concentration of 0.4%, 4 DEG C place 20 hours；Add
Enter the TMB mixed liquor dissolved in advance, and 3% polyethylene glycol is added, adds water to be settled to 1000mL, 4 DEG C are kept in dark place；
(7) it prepares terminate liquid: measuring the 50mL concentrated sulfuric acid and slowly injected in 870mL water along walls of beaker, mix, keep it dilute
18.4 times are released, 4 DEG C of preservations.
Embodiment 3
A kind of preparation method of neutrophil gelatinase-associated lipocalin kit, includes the following steps:
(1) soluble neutral granulocyte gelatinase associated lipocalin standard items and quality-control product are prepared: taking and grinds in right amount
Ammonium sulfate after thin is added in fetal calf serum while stirring at room temperature, makes its final concentration of 300g/L, and 4 DEG C are placed 22 hours；
10,000 turns are centrifuged 40 minutes, and supernatant filter paper is taken to filter；Bag filter will be filled for supernatant after filter, in 10 times of volumes at 4 DEG C
It dialyses in physiological saline, every the dialyzate of replacement in 5 hours, altogether three times；It is transferred to the 0.05mol/L boric acid solution of 10 volumes
In dialyse, every the dialyzate of replacement in 5 hours, four times altogether；Liquid after dialysis is put into clean container, in 56
DEG C heat inactivation 2 hours；The gentamicin of 0.15% Proclin300 and 0.15% is added, after mixing with 0.22 μm of filter membrane
Filtration sterilization obtains fetal calf serum processing matrix, saves at -20 DEG C stand-by；It is handled using the above-mentioned fetal calf serum being prepared
Soluble neutral granulocyte gelatinase associated lipocalin antigen diluent is obtained 6 various concentrations and is followed successively by 0ng/ by matrix
The soluble neutral granulocyte gelatinase related lipid of ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml, 200ng/ml
Transporter standard items, while soluble neutral granulocyte gelatinase related lipid is delivered into egg using fetal calf serum processing matrix
White antigen diluent obtains that concentration is 30ng/ml low value range quality-control product and concentration is 60ng/ml high level range quality-control product；
(2) the coated micropore reaction of soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody is prepared
Plate: drawing appropriate soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody, and 0.01mol/L, pH is added
It is diluted in=7.2 phosphate solution, makes its final concentration of 6 μ g/mL, 4 DEG C store for future use；Microwell plate is taken, every hole is added
Soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody after 100 μ L dilution, overlay film are placed on 4 DEG C of packets
By 22 hours；Liquid in hole is got rid of, every hole is added 200 μ L and includes 3% bovine serum albumin(BSA), 5% sucrose, 0.15%
The phosphate solution of the 0.01mol/L of Priclin300, pH=7.4, overlay film are placed on 4 DEG C and close 22 hours；Get rid of liquid in hole
Body, and patted dry on blotting paper, vacuum sealed package after drying 4 days at room temperature obtains soluble neutral granulocyte gelatinase phase
The coated micro reaction plate of lipocalin protein monoclonal antibody is closed, the micro reaction plate is stored in 4 DEG C.
(3) the soluble neutral granulocyte gelatinase associated lipocalin Dan Ke of horseradish peroxidase-labeled is prepared
Grand antibody: weighing horseradish peroxidase 2mg, is dissolved in 1mL deionized water, and 2mL concentration is added as 0.05mol/L and crosses iodine
Acid sodium solution, 4 DEG C slowly shake 60min；It is 0.001mol/L with concentration, the sodium acetate solution of pH=4.4 is dialysed 22 hours；Add
Enter 2mg soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody, 4 DEG C are protected from light oscillation 22 hours；It is added
400 μ L concentration are the NaBH of 0.2mol/L4Solution, is 0.02mol/L with concentration, and the phosphate buffer dialysis 22 of pH=7.4 is small
When；It is secondarily purified with HPLC, protein peak is collected, isometric glycerol is added, the solubility for obtaining horseradish peroxidase-labeled is single
Clonal antibody is saved in -20 DEG C.
(4) it prepares Sample dilution: taking sodium dihydrogen phosphate 1.409g, disodium hydrogen phosphate 4.372g, sodium chloride 8.5g, ox blood
Pure albumen 5g, Tween-20 0.5mL, Jia Shui are settled to 1L, with sodium hydroxide tune pH to 7.4 ± 0.1；
(5) it prepares cleaning solution: taking sodium dihydrogen phosphate 28.18g, disodium hydrogen phosphate 87.44g, sodium chloride 170g, tween-
2010mL adds water to be settled to 1L, and when use dilutes 20 times；
(6) it prepares developing solution: weighing 100gTMB, dissolved with the mixed liquor that 10mLDMSO and 10mL dehydrated alcohol is made into, 4
It DEG C is kept in dark place spare；Prepare the citric acid solution of 0.1mol/LpH=5.0；The citric acid (2) prepared to step described in 1000mL
200mg hydrogen peroxide urea is added in solution；After dissolution sufficiently, PVP is added, make its final concentration of 0.6%, 4 DEG C place it is 22 small
When；The TMB mixed liquor dissolved in advance is added, and 4% polyethylene glycol is added, adds water to be settled to 1000mL, 4 DEG C are protected from light guarantor
It deposits；
(7) it prepares terminate liquid: measuring the 50mL concentrated sulfuric acid and slowly injected in 870mL water along walls of beaker, mix, keep it dilute
18.4 times are released, 4 DEG C of preservations.
The application method of neutrophil gelatinase-associated lipocalin detection kit of the present invention are as follows:
(1) reagent needed for testing and sample take out from refrigerator, balance 30 minutes at room temperature；
(2) one piece of micro reaction plate is taken, lath needed for testing is taken out and is placed on grillage, be numbered；
(3) standard items, quality-control product and 20 μ L of experiment sample of each concentration is taken to be separately added into the hole accordingly numbered；
(4) 80 μ L Sample dilutions are added in every hole, vibrate overlay film after mixing 30 seconds and incubate 60 minutes in 37 DEG C；
(5) liquid in hole is removed, the prior 20 times of diluted cleaning solutions of 300 μ L are added in every hole, and oscillation removed washing lotion after 30 seconds,
It is repeated 4 times, pats dry residual liquid；
(6) 100 μ L enzyme markers are added in every hole, vibrate overlay film after mixing 30 seconds and incubate 30 minutes in 37 DEG C；
(7) liquid in hole is removed, the prior 20 times of diluted cleaning solutions of 300 μ L are added in every hole, and oscillation removed washing lotion after 30 seconds,
It is repeated 4 times, pats dry residual liquid；
(8) 100 μ L developing solutions are added in every hole, mix, 37 DEG C Incubation in dark 20 minutes；
(9) 50 μ L terminate liquids are added in every hole, and oscillation is measured respectively after mixing 30 seconds with microplate reader under the conditions of 450 nano wave length
The OD value in hole；
(10) using absorbance OD value as ordinate (Y), corresponding soluble neutral granulocyte gelatinase related lipid delivery
Protein standard substance concentration is abscissa (X), is made corresponding curve by four parametric fit equations, is calculated according to the OD value of sample
From the content for reading the neutrophil gelatinase-associated lipocalin contained in sample on standard curve.
Detection kit technical indicator analysis of the present invention:
Kit detects limit≤2.0ng/mL, with homologous receptoroid protein structure analog without obvious cross reaction, standard
Measurement range needed for curve meets clinical samples is 6.4~200ng/mL.
The data of product effect:
A batch is made a definite diagnosis through hospital respectively with detection kit of the invention acute kidney injury patients serum sample and just
Ordinary person's serum sample is determined test, the results show that kit of the present invention is reachable to the positive rate of acute kidney injury
98%, and be 0.7% to the false positive rate of normal person's detection, therefore kit of the present invention examines the clinical auxiliary of acute kidney injury
It is disconnected that there is highly important reference value.
The concentration of soluble neutral granulocyte gelatinase associated lipocalin standard items of the present invention is not limited to 0ng/
Six concentration of ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml, 200ng/ml；Soluble neutral granulocyte gelatinase
The concentration range of associated lipocalin quality-control product is not limited to 24~36ng/ml, 48~72ng/ml.By adjusting solvable
Property neutrophil gelatinase-associated lipocalin standard items and soluble neutral granulocyte gelatinase related lipid delivery
The concentration of albumen quality-control product, while preparing micro reaction plate according to the method described above, enzyme marker, Sample dilution, cleaning solution, showing
Color liquid and terminate liquid can prepare the soluble neutral granulocyte gelatinase associated lipocalin detection examination of different size
Agent box.
Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example
Property, it is not considered as limiting the invention, those skilled in the art within the scope of the invention can be to above-mentioned
Embodiment is changed, modifies, replacement and variant.
Claims (6)
1. a kind of neutrophil gelatinase-associated lipocalin detection kit, it is characterised in that: the kit packet
Containing Sample dilution, cleaning solution, developing solution and terminate liquid, enzyme marker, the enzyme marker be horseradish peroxidase or
The soluble neutral granulocyte gelatinase associated lipocalin monoclonal antibody of label；The detection kit further include:
Soluble neutral granulocyte gelatinase associated lipocalin standard items, the soluble neutral granulocyte of multiple and different concentration are bright
Glue enzyme associated lipocalin quality-control product and micro reaction plate, the micro reaction plate are coated with soluble neutral granulocyte
Gelatinase associated lipocalin monoclonal antibody；
The concentration of the soluble neutral granulocyte gelatinase associated lipocalin standard items of the multiple various concentration is successively
For 0ng/ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml and 200ng/ml.
2. a kind of neutrophil gelatinase-associated lipocalin detection kit according to claim 1, special
Sign is: the solvable neutrophil gelatinase-associated lipocalin quality-control product includes low value range quality-control product and high level
Range quality-control product, the low value range quality-control product concentration be 24~36ng/ml, the high level range quality-control product concentration be 48~
72ng/ml。
3. a kind of neutrophil gelatinase-associated lipocalin detection kit according to claim 1, special
Sign is: the low value range quality-control product concentration is 30ng/ml, and the high level range quality-control product concentration is 60ng/ml.
4. described in any item a kind of neutrophil gelatinase-associated lipocalin detection examinations according to claim 1~3
Agent box, it is characterised in that: the soluble neutral granulocyte gelatinase associated lipocalin standard items the preparation method comprises the following steps:
Soluble neutral granulocyte gelatinase associated lipocalin antigen is diluted with fetal calf serum processing matrix, with obtained
To soluble neutral granulocyte gelatinase associated lipocalin standard items.
5. special according to a kind of 1~3 described in any item neutrophil gelatinase-associated lipocalin detection kits
Sign is: the quality-control product the preparation method comprises the following steps: with fetal calf serum processing matrix to solvable neutrophil leucocyte gelatinase correlation rouge
Matter transporter antigen is diluted, and preparation obtains soluble neutral granulocyte gelatinase associated lipocalin quality-control product.
6. a kind of neutrophil gelatinase-associated lipocalin detection kit according to claim 4, special
Sign is: solvable neutrophil gelatinase-associated lipocalin antigen is diluted with fetal calf serum processing matrix,
Preparation obtains soluble neutral granulocyte gelatinase associated lipocalin quality-control product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711257965.9A CN109870584A (en) | 2017-12-04 | 2017-12-04 | A kind of neutrophil gelatinase-associated lipocalin detection kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711257965.9A CN109870584A (en) | 2017-12-04 | 2017-12-04 | A kind of neutrophil gelatinase-associated lipocalin detection kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109870584A true CN109870584A (en) | 2019-06-11 |
Family
ID=66914309
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711257965.9A Pending CN109870584A (en) | 2017-12-04 | 2017-12-04 | A kind of neutrophil gelatinase-associated lipocalin detection kit |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109870584A (en) |
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2017
- 2017-12-04 CN CN201711257965.9A patent/CN109870584A/en active Pending
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Application publication date: 20190611 |