CN109870578A - A kind of cystatin C detection kit - Google Patents
A kind of cystatin C detection kit Download PDFInfo
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- CN109870578A CN109870578A CN201711259264.9A CN201711259264A CN109870578A CN 109870578 A CN109870578 A CN 109870578A CN 201711259264 A CN201711259264 A CN 201711259264A CN 109870578 A CN109870578 A CN 109870578A
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Abstract
The present invention relates to a kind of cystatin C detection kits, belong to technical field of immune assay.The kit includes Sample dilution, cleaning solution, developing solution and terminate liquid, enzyme marker, the enzyme marker is the soluble cystatin C monoclonal antibody of horseradish peroxidase-labeled, the detection kit further include: the soluble cystatin C standard items of multiple and different concentration, soluble cystatin C quality-control product and micro reaction plate, the micro reaction plate are coated with soluble cystatin C monoclonal antibody.The soluble cystatin C standard items of a variety of various concentrations in the kit can be used directly without being diluted processing, simplify operating procedure, shorten detection time, realize efficient, quick detection, soluble cystatin C quality-control product can identify whether testing result is accurate simultaneously, the detection kit of the available different size of cystatin C standard items of various concentration, adapts to the use demand of different clients.
Description
Technical field
The present invention relates to a kind of cystatin C (CysC) detection kits, belong to technical field of immune assay.
Background technique
Kidney trouble is a kind of clinically common disease, so far, early diagnosis, early treatment and the kidney for reversing damage
Function is the approach that can uniquely prevent kidney trouble from deteriorating.Clinical evaluation kidney trouble progress and severity are generally with renal function
To refer to, using glomerular filtration rate (GFR) as the reflection most important index of renal function.
Cystatin C, is a kind of cystatin, also referred to as γ-trace of albumin or γ-postglobulin,
And be a kind of low molecular weight, alkaline non-glycated protein, molecular weight 13.3KD is made of 122 amino acid residues, can be by
All karyocytes of body generate, and generation rate is constant.Cystatin C in circulation is only removed through glomerular filtration, for reflection
The endogenous marker of glomerular filtration rate variation, and in proximal convoluted tubule reabsorption, but by complete metabolic breakdown after reabsorption, no
Return to blood.Therefore, the filtering rate of glomerulus can be reflected by the concentration of cystatin C in blood.Moreover, cystatin C
Serum-concentration is not influenced by quick disease process, gender, age and muscle weight, is to reflect that the ideal of glomerular filtration rate variation is homologous
Property marker, and gradually development is good for a sensibility of assessment renal function, the high index of specificity.
Therefore, the quick, comprehensive, accurate of cystatin C, specific detection are particularly important.Up to the present, commodity
Cystatin C detection means all still has certain limitation.
Summary of the invention
In view of this, the kit can cystatin the purpose of the present invention is to provide a kind of cystatin C detection kit
C carries out quick, comprehensive, accurate, special detection.
To achieve the purpose of the present invention, following technical scheme is provided.
A kind of cystatin C detection kit, the kit include Sample dilution, cleaning solution, developing solution and termination
Liquid, enzyme marker, the enzyme marker are the soluble cystatin C monoclonal antibody of horseradish peroxidase-labeled；The inspection
Test agent box further include: the soluble cystatin C standard items of multiple and different concentration, soluble cystatin C quality-control product and micropore
Reaction plate, the micro reaction plate are coated with soluble cystatin C monoclonal antibody.
The concentration of the soluble cystatin C standard items of the multiple various concentration be followed successively by 0ng/ml, 6.4ng/ml,
16ng/ml, 40ng/ml, 100ng/ml and 200ng/ml.
Preferably, the soluble cystatin C standard items the preparation method comprises the following steps: with fetal calf serum processing matrix to solubility
Cystatin C antigen is diluted, and preparation obtains soluble cystatin C standard items.
Preferably, the solvable cystatin C quality-control product includes low value range quality-control product and high level range quality-control product, described low
Value range quality-control product concentration is 24~36ng/ml, and the high level range quality-control product concentration is 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 solvable cystatin C antigen is carried out
Dilution, preparation obtain soluble cystatin C quality-control product.
A kind of preparation method of cystatin C detection kit of the present invention, includes the following steps:
(1) soluble cystatin C standard items and soluble cystatin C quality-control product are prepared: handling matrix using fetal calf serum
Soluble cystatin C antigen diluent is obtained into solvable cystatin C standard items, while will be soluble using fetal calf serum processing matrix
Cystatin C dilutes to obtain quality-control product；
(2) it prepares the solvable coated micro reaction plate of cystatin C monoclonal antibody: drawing soluble cystatin C monoclonal
Antibody is added 0.01mol/L, is diluted in the phosphate solution of pH=7.2,4 DEG C store for future use；Microwell plate is taken, every hole adds
Soluble cystatin C monoclonal antibody after entering 100 μ L dilution, 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, it is coated micro- to obtain soluble cystatin C monoclonal antibody for vacuum sealed package after drying 2~4 days at room temperature
Hole reaction plate, the micro reaction plate are stored in 4 DEG C.
(3) it prepares the soluble cystatin C monoclonal antibody of horseradish peroxidase-labeled: weighing horseradish peroxidase
2mg is dissolved in 1mL deionized water, the sodium periodate solution that 1~2mL concentration is 0.05mol/L is added, 4 DEG C slowly shake 30
~60min；It is 0.001mol/L with concentration, the sodium acetate solution of pH=4.4 is dialysed 18~22 hours；2mg solubility Guang is added
Chalone C monoclonal antibody, 4 DEG C are protected from light oscillation 18~22 hours；The NaBH that 400 μ L concentration are 0.2mol/L is added4Solution, use are dense
Degree is 0.02mol/L, and the phosphate buffer of pH=7.4 is dialysed 18~22 hours；It is secondarily purified with HPLC, protein peak is collected, is added
Enter isometric glycerol, obtain the soluble cystatin C monoclonal antibody of horseradish peroxidase-labeled, 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 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 cystatin C detection kit, the soluble Guang of a variety of various concentrations in kit presses down
Plain C standard items can be used directly without being diluted processing, simplified operating procedure, shortened detection time, realize height
Effect, quickly detection, while solvable cystatin C quality-control product can identify whether testing result accurate, multiple and different concentration it is solvable
Property the soluble cystatin C monoclonal antibody that uses of cystatin C micro reaction plate there is high degree of specificity, and soluble cystatin
C standard items may be implemented to carry out single-minded quantitative detection to the soluble cystatin C protein molecular in sample；The difference of offer is dense
The soluble available standard curve of cystatin C standard items of degree, according to soluble in the available sample to be tested of standard curve
The concentration of cystatin C；Kit can be combined with full-automatic enzyme-linked response analysis instrument, can further effectively shorten detection when
Between, it reduces testing cost and manpower is spent.
Specific embodiment
A kind of cystatin C detection kit includes micro reaction plate, enzyme marker, Sample dilution, developing solution, end
Only liquid, cleaning solution, soluble cystatin C standard items, soluble cystatin C quality-control product, wherein enzyme marker is horseradish peroxidating
The soluble cystatin C monoclonal antibody of object enzyme label, soluble cystatin C standard items are with fetal calf serum processing matrix to can
Dissolubility cystatin C antigen is diluted to obtain, and soluble cystatin C quality-control product is with fetal calf serum processing matrix to soluble Guang
Chalone C antigen is diluted to obtain, and micro reaction plate is to be coated with soluble cystatin C monoclonal antibody；In this kit not
It can be obtained according to the OD value of sample by standard curve directly using standard curve is drawn with concentration solubility cystatin C standard items
To the corresponding concentration of sample, soluble cystatin C quality-control product is used to identify whether testing result is accurate, and enzyme marker is without dilute
Processing is released, it is easy to operate, detection time can be effectively shortened, and then reduce testing cost and manpower is spent.
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 cystatin C detection kit, includes the following steps:
(1) soluble cystatin C standard items and quality-control product are prepared: take it is appropriate it is finely ground after ammonium sulfate side is stirred at room temperature
Side is added in fetal calf serum, makes its final concentration of 100g/L, and 4 DEG C are placed 18 hours；10,000 turns are centrifuged 20 minutes, take supernatant
Filter paper filtering；Supernatant fills bag filter after filtering, and dialyses in the physiological saline of 10 times of volumes at 4 DEG C, replaces every 3 hours
Dialyzate, altogether three times；It is transferred in the 0.05mol/L boric acid solution of 10 volumes and dialyses, it is primary every replacement in 3 hours
Dialyzate, four times altogether；Liquid after dialysis is put into clean container, is inactivated 1 hour in 56 DEG C of heat；It is added 0.05%
The gentamicin of Proclin300 and 0.05% obtains fetal calf serum processing base with 0.22 μm of membrane filtration degerming after mixing
Matter saves stand-by at -20 DEG C；It is using the above-mentioned fetal calf serum processing matrix being prepared that soluble cystatin C antigen is dilute
Release to obtain 6 various concentrations are followed successively by 0ng/ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml, 200ng/ml can
Dissolubility cystatin C standard items, while soluble cystatin C antigen diluent is obtained into concentration using fetal calf serum processing matrix and is
30ng/ml low value range quality-control product；
(2) it prepares the coated micro reaction plate of soluble cystatin C monoclonal antibody: drawing appropriate soluble cystatin C
Monoclonal antibody, is added 0.01mol/L, is diluted in the phosphate solution of pH=7.2, makes its final concentration of 2 μ g/mL, and 4 DEG C
It stores for future use；Microwell plate is taken, the soluble cystatin C monoclonal antibody after 100 μ L dilution is added in every hole, and overlay film is placed on 4 DEG C
Coating 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 cystatin C monoclonal antibody packet
The micro reaction plate of quilt, the micro reaction plate are stored in 4 DEG C；
(3) it prepares the soluble cystatin C monoclonal antibody of horseradish peroxidase-labeled: weighing horseradish peroxidase
2mg is dissolved in 1mL deionized water, the sodium periodate solution that 1mL concentration is 0.05mol/L is added, 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；2mg solubility cystatin C list is added
Clonal antibody, 4 DEG C are protected from light oscillation 18 hours；The NaBH that 400 μ L concentration are 0.2mol/L is added4Solution is with concentration
The phosphate buffer of 0.02mol/L, pH=7.4 are dialysed 18 hours；It is secondarily purified with HPLC, protein peak is collected, is added isometric
Glycerol obtains the soluble monoclonal antibody of horseradish peroxidase-labeled, 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 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 cystatin C detection kit, includes the following steps:
(1) soluble cystatin C standard items and quality-control product are prepared: take it is appropriate it is finely ground after ammonium sulfate side is stirred at room temperature
Side is added in fetal calf serum, makes its final concentration of 200g/L, and 4 DEG C are placed 20 hours；10,000 turns are centrifuged 30 minutes, take supernatant
Filter paper filtering；Supernatant fills bag filter after filtering, and dialyses in the physiological saline of 10 times of volumes at 4 DEG C, replaces every 4 hours
Dialyzate, altogether three times；It is transferred in the 0.05mol/L boric acid solution of 10 volumes and dialyses, it is primary every replacement in 4 hours
Dialyzate, four times altogether；Liquid after dialysis is put into clean container, is inactivated 1.5 hours in 56 DEG C of heat；It is added 0.1%
The gentamicin of Proclin300 and 0.1% obtains fetal calf serum processing base with 0.22 μm of membrane filtration degerming after mixing
Matter saves stand-by at -20 DEG C；It is using the above-mentioned fetal calf serum processing matrix being prepared that soluble cystatin C antigen is dilute
Release to obtain 6 various concentrations are followed successively by 0ng/ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml, 200ng/ml can
Dissolubility cystatin C standard items, while soluble cystatin C antigen diluent is obtained into concentration using fetal calf serum processing matrix and is
30ng/ml low value range quality-control product and concentration are 60ng/ml high level range quality-control product；
(2) it prepares the coated micro reaction plate of soluble cystatin C monoclonal antibody: drawing appropriate soluble cystatin C
Monoclonal antibody, is added 0.01mol/L, is diluted in the phosphate solution of pH=7.2, makes its final concentration of 4 μ g/mL, and 4 DEG C
It stores for future use；Microwell plate is taken, the soluble cystatin C monoclonal antibody after 100 μ L dilution is added in every hole, and overlay film is placed on 4 DEG C
Coating 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 cystatin C monoclonal antibody packet
The micro reaction plate of quilt, the micro reaction plate are stored in 4 DEG C；
(3) it prepares the soluble cystatin C monoclonal antibody of horseradish peroxidase-labeled: weighing horseradish peroxidase
2mg is dissolved in 1mL deionized water, the sodium periodate solution that 1.5mL concentration is 0.05mol/L is added, 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 solubility cystatin C list is added
Clonal antibody, 4 DEG C are protected from light oscillation 20 hours；The NaBH that 400 μ L concentration are 0.2mol/L is added4Solution is with concentration
The phosphate buffer of 0.02mol/L, pH=7.4 are dialysed 20 hours；It is secondarily purified with HPLC, protein peak is collected, is added isometric
Glycerol obtains the soluble monoclonal antibody of horseradish peroxidase-labeled, 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 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 (1) TMB mixed liquor that the step dissolves in advance, and 3% 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.
Embodiment 3
A kind of preparation method of cystatin C kit, includes the following steps:
(1) soluble cystatin C standard items and quality-control product are prepared: take it is appropriate it is finely ground after ammonium sulfate side is stirred at room temperature
Side is added in fetal calf serum, makes its final concentration of 300g/L, and 4 DEG C are placed 22 hours；10,000 turns are centrifuged 40 minutes, take supernatant
Filter paper filtering；Supernatant fills bag filter after filtering, and dialyses in the physiological saline of 10 times of volumes at 4 DEG C, replaces every 5 hours
Dialyzate, altogether three times；It is transferred in the 0.05mol/L boric acid solution of 10 volumes and dialyses, it is primary every replacement in 5 hours
Dialyzate, four times altogether；Liquid after dialysis is put into clean container, is inactivated 2 hours in 56 DEG C of heat；It is added 0.15%
The gentamicin of Proclin300 and 0.15% obtains fetal calf serum processing base with 0.22 μm of membrane filtration degerming after mixing
Matter saves stand-by at -20 DEG C；It is using the above-mentioned fetal calf serum processing matrix being prepared that soluble cystatin C antigen is dilute
Release to obtain 6 various concentrations are followed successively by 0ng/ml, 6.4ng/ml, 16ng/ml, 40ng/ml, 100ng/ml, 200ng/ml can
Dissolubility cystatin C standard items, while soluble cystatin C antigen diluent is obtained into concentration using fetal calf serum processing matrix and is
30ng/ml low value range quality-control product and concentration are 60ng/ml high level range quality-control product；
(2) it prepares the coated micro reaction plate of soluble cystatin C monoclonal antibody: drawing appropriate soluble cystatin C
Monoclonal antibody, is added 0.01mol/L, is diluted in the phosphate solution of pH=7.2, makes its final concentration of 6 μ g/mL, and 4 DEG C
It stores for future use；Microwell plate is taken, the soluble cystatin C monoclonal antibody after 100 μ L dilution is added in every hole, and overlay film is placed on 4 DEG C
Coating 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 cystatin C monoclonal antibody packet
The micro reaction plate of quilt, the micro reaction plate are stored in 4 DEG C.
(3) it prepares the soluble cystatin C monoclonal antibody of horseradish peroxidase-labeled: weighing horseradish peroxidase
2mg is dissolved in 1mL deionized water, the sodium periodate solution that 2mL concentration is 0.05mol/L is added, 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；2mg solubility cystatin C list is added
Clonal antibody, 4 DEG C are protected from light oscillation 22 hours；The NaBH that 400 μ L concentration are 0.2mol/L is added4Solution is with concentration
The phosphate buffer of 0.02mol/L, pH=7.4 are dialysed 22 hours；It is secondarily purified with HPLC, protein peak is collected, is added isometric
Glycerol obtains the soluble monoclonal antibody of horseradish peroxidase-labeled, 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 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 cystatin C 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 solubility cystatin C standard concentration is abscissa (X),
Be made corresponding curve by four parametric fit equations, according to the OD value of sample calculate from standard curve read sample in contain
Cystatin C content.
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:
Renal disease patient caused by the diabetes B made a definite diagnosis through hospital a batch respectively with detection kit of the invention
Serum sample and normal human serum sample are determined test, the results show that the positive of the kit of the present invention to kidney and side
Recall rate is 0.7% to the false positive rate of normal person's detection, therefore kit of the present invention is to 2 types sugar respectively up to 99.0%
The clinical assistant diagnosis of kidney trouble caused by urine disease has highly important reference value.
The concentration of solubility cystatin C standard items of the invention is not limited to 0ng/ml, 6.4ng/ml, 16ng/ml, 40ng/
Six concentration of ml, 100ng/ml, 200ng/ml；The concentration range of soluble cystatin C quality-control product is not limited to 24~36ng/
Ml, 48~72ng/ml.By adjusting the concentration of soluble cystatin C standard items and soluble cystatin C quality-control product, press simultaneously
Micro reaction plate, enzyme marker, Sample dilution, cleaning solution, developing solution and terminate liquid are prepared according to the above method, can be prepared
The soluble cystatin C detection kit of different size.
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 cystatin C detection kit, it is characterised in that: the kit includes Sample dilution, cleaning solution, colour developing
Liquid and terminate liquid, enzyme marker, the enzyme marker are anti-for the soluble cystatin C monoclonal of horseradish peroxidase or label
Body；The detection kit further include: the soluble cystatin C standard items of multiple and different concentration, soluble cystatin C Quality Control
Product and micro reaction plate, the micro reaction plate are coated with soluble cystatin C monoclonal antibody；
The concentration of the soluble cystatin C standard items of the multiple various concentration be followed successively by 0ng/ml, 6.4ng/ml, 16ng/ml,
40ng/ml, 100ng/ml and 200ng/ml.
2. a kind of cystatin C detection kit according to claim 1, it is characterised in that: the solvable cystatin C Quality Control
Product include low value range quality-control product and high level range quality-control product, and the low value range quality-control product concentration is 24~36ng/ml, described
High level range quality-control product concentration is 48~72ng/ml.
3. a kind of cystatin C detection kit according to claim 1, it is characterised in that: 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 cystatin C detection kits according to claim 1~3, it is characterised in that: the solubility
Being diluted the preparation method comprises the following steps: handling matrix with fetal calf serum to soluble cystatin C antigen for cystatin C standard items, is prepared
Obtain soluble cystatin C standard items.
5. according to a kind of 1~3 described in any item cystatin C detection kits, it is characterised in that: the preparation side of the quality-control product
Method are as follows: solvable cystatin C antigen is diluted with fetal calf serum processing matrix, preparation obtains soluble cystatin C quality-control product.
6. a kind of cystatin C detection kit according to claim 4, it is characterised in that: handle matrix with fetal calf serum
Solvable cystatin C antigen is diluted, preparation obtains soluble cystatin C quality-control product.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711259264.9A CN109870578A (en) | 2017-12-04 | 2017-12-04 | A kind of cystatin C detection kit |
Applications Claiming Priority (1)
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| CN201711259264.9A CN109870578A (en) | 2017-12-04 | 2017-12-04 | A kind of cystatin C detection kit |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112903647A (en) * | 2021-01-26 | 2021-06-04 | 中山大学 | Cystatin C detection kit |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112903647A (en) * | 2021-01-26 | 2021-06-04 | 中山大学 | Cystatin C detection kit |
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