CN105334326A - Ceruloplasmin detection kit - Google Patents
Ceruloplasmin detection kit Download PDFInfo
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- CN105334326A CN105334326A CN201510694321.0A CN201510694321A CN105334326A CN 105334326 A CN105334326 A CN 105334326A CN 201510694321 A CN201510694321 A CN 201510694321A CN 105334326 A CN105334326 A CN 105334326A
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- reagent
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- detection kit
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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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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Abstract
The invention discloses a ceruloplasmin detection kit, and belongs to the technical field of clinic in-vitro detection reagents. The kit comprises a reagent R1, a reagent R2 and a calibration sample. By adding a certain amount of a silicon-dioxide-coated magnetic nano-particle into the reagent R1 and adding a certain amount of bovine serum albumin and Kathon-CG into the reagent R2, the kit stability and analysis sensitivity are effectively improved, the linearity scope is relatively good, the reagent accurate is high, and further popularization usage of the kit in the market is facilitated.
Description
Technical field
The present invention relates to clinical vitro detection reagent technique field, particularly a kind of CER detection kit.
Background technology
CER (CP) is also known as CuO-2 layer, and be a kind of α 2 glycoprotein of cupric, molecular weight is about 120,000-16 ten thousand, not easily purifying.Be a single chain polypeptide at present, per molecule, containing 6-7 copper atom, is blue due to cupric, and sugary about 10%, terminal sialic acid is connected with polypeptied chain, has the gene pleomorphism in heredity.It act as the zymoprotein regulating the distribution of copper at each position of body, synthesis cupric, has the effect of antioxidant, and has oxidase active.It is generally acknowledged that CER is synthesized by liver, a part is by biliary excretion, and in urine, content is very micro-.CER measures and has the certain significance to the diagnosis of the diseases such as some liver, courage, kidney.
CP has oxidasic activity, polyphenol and polyamines class substrate is had to the ability of its oxidation of catalysis.Nearest research thinks that CP can catalysis Fe
2+be oxidized to Fe
3+.There are different views in the carrier being whether copper for CP.Though the content of Copper in Serum has 95% to be in CP with non-diffusing state, and have 5% to be that state of can dialysing is absorbed by intestinal tube and is transported to liver, combine through sialic acid again infiltrate CP carrier protein (apoprotein) in liver after, be finally released into blood circulation.What in blood circulation, CP can be considered copper does not have virose metabolic pool.Cell can utilize the copper in CP molecule to synthesize the zymoprotein of cupric, such as monoamine oxidase, ascorbic acid oxidase etc.
Another result of study thinks that CP plays a part antioxidant in recent years.In blood circulation, the anti-oxidant vigor of CP can prevent the generation of Lipid Peroxide In Tissues and free radical, significant when inflammation especially.CER also belongs to a kind of Acute reaction protein.Plasma C P infect, wound and tumour time increase.Its most special effect is the diagnosis of assisting Wilson disease, and namely patients blood plasma CP content obviously declines, and increases with the dialyzable copper content of blood plasma.Major part patient can have hepatic disorder and with neural symptom, and as treated not in time, this disease is Progressive symmetric erythrokeratodermia and fatal, therefore should diagnosis in time, and available copper sequestrant-penicillamine treatment.Plasma C P also often declines when malnutrition, severe liver disease and nephrotic syndrome.When woman gestationperiod, oral contraceptive, its content is significantly increased.
Ceruloplasmin (CP) clinical meaning: (1) raises: 1. severe infection: inflammation, hepatitis, periostitis, pyelonephritis, tuberculosis, pneumoconiosis etc.2. malignant tumour: leukaemia, malignant lymphoma, various cancer.3. the bile stasis of blood is stagnant: primary biliary stasis type cirrhosis, extrahepatic cholestatic jaundice, oxyhepatitis, chronic hepatitis, alcoholic cirrhosis.4. hyperthyroidism, rheumatism, rheumatoid arthritis, alpastic anemia, myocardial infarction, Post operation etc.5. other: acute schizophrenia, delirium tremens, homocystinuria, gestation, oral contraceptive.6. tuberculosis is seen, silicosis, hyperthyroidism, malignant tumour (as leukaemia, Hodgkin's disease, liver cancer etc.) etc.7. gestation under physiological conditions, oral contraceptive, CP also can raise.8. when chronic hepatitis and cirrhosis, the Changing Pattern of CER there is no consistent view, theoretically, when liver cell is undermined, blood level should reduce, but RUSSO etc. measure none example of 54 routine CALD patients serum CERs to be reduced, nearly half case is had to raise on the contrary, with completely different during hepatolenticular degeneration.9. contribute to differentiating cirrhosis and liver cancer, during primary carcinoma of liver, ceruloplasmin is >8.3% higher than normal probability, is 12% higher than normal probability during cirrhosis.(2) reduce: 1. Wilson disease and hepatolenticular degeneration (diagnosis index for most worthy).2. malnutritive: nephrotic syndrome, malabsorption syndrome, spilling property of albumen stomach and intestine disease, nephrotic syndrome, Hypoproteinemia etc.3. primary biliary cirrhosis of liver, primary biliary locking disease etc.4. neonate, premature infant.5. serious Hypoproteinemia is seen, nephrotic syndrome etc.6. diagnose hepatolenticular degeneration: during this disease, CER significantly reduces, excessively high may be deposited on caused by liver and basal nuclei or CER dyssynthesis due to copper.
The antibody response that CP antigen in sample is corresponding to reagent, forms antigen-antibody complex, makes solution form certain turbidity.Detect under 340nm wavelength, the height of this turbidity is directly proportional to the content of antigen when a certain amount of antibody exists.By comparing with the same calibration solution processed, calculate the content of CP in unknown sample.The method is a kind of without the need to pre-service sample, and technology and equipment is less demanding, and precision and the higher analytical approach of specificity.Because the method does not need expensive equipment, can robotization be realized, and a large amount of sample can be measured, therefore be subject to clinical extensive popularization.But common CER detects that reagent stability is bad, sensitivity is not high yet, thus limits its applying clinically.
Summary of the invention
For prior art Problems existing, the invention provides a kind of CER detection kit, this kit is compared with the kit of routine, and stability is better than the detection kit of routine, and sensitivity for analysis is high, is conducive to reagent applying clinically.
The present invention is achieved by the following measures:
A kind of CER detection kit, it is characterized in that, it comprises reagent R1, reagent R2 and calibration object, and wherein reagent R1 consists of: the magnetic nanoparticle of 50mmol/LpH8.0Tris damping fluid, 0.1% Sodium azide, 0.1%-2% coated with silica, 40.0g/L polyglycol; Reagent R2 consists of: 50mmol/LpH8.0Tris damping fluid, 30ml/L goat-anti people CP antibody, 20-40g/L bovine serum albumin(BSA), 0.05%Kathon-CG.
The magnetic nanoparticle of described coated with silica is Fe
3o
4/ SiO
2composite nanoparticle, particle diameter is 20-50nm.
Described reagent R1 and reagent R2 ratio are in use R1:R2=225:75.
The magnetic nanoparticle of coated with silica used in the present invention adopts following methods to prepare:
Polyol process is adopted to prepare Fe
3o
4nano particle.Get certain ferric acetyl acetonade and triethylene glycol joins in reflux heating reaction unit, under the condition of magnetic agitation and Ar gas shielded, device is slowly heated to boiling, and keeps backflow a period of time.Cool in backward reaction solution and add the ferriferrous oxide nano-particle generation flocculation that ethyl acetate makes generation, magnetic resolution black product, cleaning for several times, is distributed in ethanol, namely obtains stable Fe
3o
4nano particle alcohol colloidal solution.Afterwards by gained Fe
3o
4nano particle adopts Stober Hydrolyze method to obtain SiO
2coated Fe
3o
4nano particle.Gained Fe
3o
4/ SiO
2the particle diameter of composite nanoparticle is 20-50nm.
Calibration object used in the present invention is the CP calibration object that Shanghai Yuan Sheng Bioisystech Co., Ltd produces.
Kit of the present invention carries out on the automatic biochemistry analyzer with double reagent function, its concrete using method is: add physiological saline, sample or calibration object 5 μ l, the reagent R2 of 75 μ l is added again after adding R1 reagent 225 μ l preincubate 5min afterwards again, read absorbance A 1 afterwards, after reaction 5min, read absorbance A 2, and calculate Δ A.
Beneficial effect of the present invention:
CER detection kit provided by the invention, by adding the magnetic nanoparticle of coated with silica in reagent R1, this magnetic nanoparticle is combined with goat-anti people CP antibody under the effect of Electrostatic Absorption, thus make antibody form aggegation on magnetic nanoparticle, improve the sensitivity for analysis of reagent greatly.In reagent R2, add bovine serum albumin(BSA) simultaneously, solve antibody this difficult problem unstable in lean solution, it can make antibody stabilization in test, and it is relatively neutral, but the character of antibody can not be affected, and Kathon-CG is a kind of novel high-efficiency environment friendly type wide-spectrum bactericide, antiseptic, in this kit, use Kathon-CG to efficiently solve BSA preserve easily mouldy shortcoming for a long time, therefore BSA and Kathon-CG acting in conjunction effectively enhances the stability of kit, but can not have an impact to the accuracy of reagent, be conducive to this reagent further to promote in the market.
Accompanying drawing explanation
Fig. 1 embodiment 2 accuracy validation laboratory test results and control group testing result correlativity;
Fig. 2 embodiment 3 accuracy validation laboratory test results and control group testing result correlativity;
Fig. 3 embodiment 4 accuracy validation laboratory test results and control group testing result correlativity;
Fig. 4 embodiment 1-4 linear correlation confirmatory experiment testing result;
Fig. 5 stability confirmatory experiment testing result;
Fig. 6 sensitivity for analysis experimental result.
Embodiment
For a better understanding of the present invention, further describe below in conjunction with specific embodiment.
Embodiment 1
A CER detection kit for routine, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH8.0Tris damping fluid 50mmol/L
Sodium azide 0.1%
Polyglycol 40.0g/L
Reagent R2 consists of:
PH8.0Tris damping fluid 50mmol/L
Goat-anti people APOE antibody 30ml/L
The kit that the present embodiment describes, in use, its assay method adopts Toshiba 120 automatic analyzer with double reagent function, operates as follows:
Add physiological saline, sample or calibration object 5 μ l, after adding R1 reagent 225 μ l preincubate 5min afterwards again, add the reagent R2 of 75 μ l again, read absorbance A 1 afterwards, after reaction 5min, read absorbance A 2, and calculate Δ A.
The CP calibration object that the calibration object that the present embodiment uses is produced for Shanghai Yuan Sheng Bioisystech Co., Ltd.
Embodiment 2
A kind of CER detection kit, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH8.0Tris damping fluid 50mmol/L
Sodium azide 0.1%
Polyglycol 40.0g/L
The magnetic nanoparticle 0.1% of coated with silica
Reagent R2 consists of:
PH8.0Tris damping fluid 50mmol/L
Goat-anti people APOE antibody 30ml/L
Bovine serum albumin(BSA) 20g/L
Kathon-CG0.05%
Concrete assay method is with embodiment 1.
Embodiment 3
A kind of CER detection kit, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH8.0Tris damping fluid 50mmol/L
Sodium azide 0.1%
Polyglycol 40.0g/L
The magnetic nanoparticle 1% of coated with silica
Reagent R2 consists of:
PH8.0Tris damping fluid 50mmol/L
Goat-anti people APOE antibody 30ml/L
Bovine serum albumin(BSA) 30g/L
Kathon-CG0.05%
Concrete assay method is with embodiment 1.
Embodiment 4
A kind of CER detection kit, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH8.0Tris damping fluid 50mmol/L
Sodium azide 0.1%
Polyglycol 40.0g/L
The magnetic nanoparticle 2% of coated with silica
Reagent R2 consists of:
PH8.0Tris damping fluid 50mmol/L
Goat-anti people APOE antibody 30ml/L
Bovine serum albumin(BSA) 40g/L
Kathon-CG0.05%
Concrete assay method is with embodiment 1.
Experimental verification is carried out to kit assay performance obtained in above-described embodiment 1-4.
accuracy validation is tested:
Using the kit of embodiment 2,3,4 as experimental group, the CER detection kit (production of Shanghai company) market obtaining a kind of accuracy excellence of accreditation carries out contrast experiment as a control group, detect 40 samples, the result of detection is as Fig. 1-Fig. 3.
Known by the detection data of Fig. 1-Fig. 3, the testing result correlativity of embodiment 2,3,4 detection kit and control test kit is respectively 0.9983,0.9988,0.9985, correlativity is relatively good, show that kit of the present invention and market obtaining the CER detection kit with excellent accuracy approved has high consistency, prove that other various compositions that kit of the present invention adds can not impact its accuracy, kit still keeps good accuracy.
linear dependence confirmatory experiment:
CER high level sample is found to be 80mg/dL, serial dilution is carried out with physiological saline, the sample of preparation 6 variable concentrations, be followed successively by the sample of 80mg/dL, 64mg/dL, 48mg/dL, 32mg/dL, 16mg/dL, 0mg/dL concentration, each concentration level various kinds originally measures three times respectively, gets its mean value respectively.The reagent of embodiment 1,2,3,4 is utilized to detect respectively.Testing result as shown in Figure 4.
Above-mentioned testing result display, embodiment 1-4 testing result correlativity is all greater than 0.999, reach product standard requirement, illustrate that magnetic nanoparticle, bovine serum albumin(BSA) and the Kathon-CG adding coated with silica in reagent can not reduce the linear dependence of reagent detection.
stability confirmatory experiment:
2 DEG C ~ 8 DEG C, store reagents in the light protected environment of non-corrosiveness gas, detect the stability of four kinds of embodiment reagent.Four kinds of reagent are monthly chosen same sample and are measured its absorbance three times, average, and contrast, thus determine the stabilization time of reagent with fresh embodiment 1 reagent testing result.Detect data as Fig. 5.
Experimental result shows, embodiment 1 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 15 months, stablize, and embodiment 2,3,4 reagent 2 DEG C ~ 8 DEG C, in the light protected environment of non-corrosiveness gas storage within 24 months, stablize, illustrate and add bovine serum albumin(BSA) and Kathon-CG in reagent, the two acting in conjunction effectively raises the stability of CER detection kit.
sensitivity for analysis confirmatory experiment:
With the sample of CER kit test concentration known of the present invention at 32.6mg/dL, record absorbance difference.The reagent of embodiment 1,2,3,4 is utilized to detect respectively.Testing result as shown in Figure 6.
Known by detecting data, the absorbance difference of embodiment 2,3,4 detection kit is all than the height of embodiment 1, illustrate that the magnetic nanoparticle adding coated with silica in reagent is combined with goat-anti people CP antibody under the effect of Electrostatic Absorption, thus make antibody form aggegation on magnetic nanoparticle, expand response signal, improve the sensitivity for analysis of reagent greatly.
Comprehensive above analysis, CER detection kit provided by the invention, by adding the magnetic nanoparticle of a certain amount of coated with silica and add stability and the sensitivity for analysis that a certain amount of bovine serum albumin(BSA) and Kathon-CG effectively can improve kit in reagent R2 in reagent R1, the range of linearity is better, and the accuracy of reagent is also better.Therefore, CER detection kit provided by the invention is conducive to further promoting the use of in the market.
Claims (3)
1. a CER detection kit, it is characterized in that, it comprises reagent R1, reagent R2 and calibration object, and wherein reagent R1 consists of: the magnetic nanoparticle of 50mmol/LpH8.0Tris damping fluid, 0.1% Sodium azide, 0.1%-2% coated with silica, 40.0g/L polyglycol; Reagent R2 consists of: 50mmol/LpH8.0Tris damping fluid, 30ml/L goat-anti people CP antibody, 20-40g/L bovine serum albumin(BSA), 0.05%Kathon-CG.
2. kit according to claim 1, is characterized in that, the magnetic nanoparticle of described coated with silica is Fe
3o
4/ SiO
2composite nanoparticle, particle diameter is 20-50nm.
3. kit according to claim 1, is characterized in that, described reagent R1 and reagent R2 ratio are in use R1:R2=225:75.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510694321.0A CN105334326A (en) | 2015-10-24 | 2015-10-24 | Ceruloplasmin detection kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510694321.0A CN105334326A (en) | 2015-10-24 | 2015-10-24 | Ceruloplasmin detection kit |
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| CN105334326A true CN105334326A (en) | 2016-02-17 |
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| CN201510694321.0A Pending CN105334326A (en) | 2015-10-24 | 2015-10-24 | Ceruloplasmin detection kit |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290926A (en) * | 2016-08-13 | 2017-01-04 | 山东博科生物产业有限公司 | A kind of apolipoprotein B immunoturbidimetry detection kit |
| CN106645694A (en) * | 2016-09-24 | 2017-05-10 | 济南博鑫生物技术有限公司 | Fn (fibronectin) detection kit |
| CN112730833A (en) * | 2020-11-23 | 2021-04-30 | 迪瑞医疗科技股份有限公司 | Ceruloplasmin determination kit by using immuno-transmission turbidimetry |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006028321A1 (en) * | 2004-09-07 | 2006-03-16 | Zenovac, Inc. | Method of measuring ceruloplasmin concentration in a blood spot, kit and method of diagnosing wilson's disease |
| CN101718779A (en) * | 2009-10-29 | 2010-06-02 | 广西师范大学 | Kit for detecting immuno-nanogold synchronous scattering spectrum of human serum ceruloplosmin and use method thereof |
| CN104374924A (en) * | 2014-12-15 | 2015-02-25 | 山东博科生物产业有限公司 | Alpha1-AT (antitrypsin) immunoturbidimetry detection kit |
| CN104407159A (en) * | 2014-12-15 | 2015-03-11 | 山东博科生物产业有限公司 | IgM (Immune Globulin M) immune turbidimetry test kit |
| WO2015042593A1 (en) * | 2013-09-23 | 2015-03-26 | Assaypro, Llc | Immunoassays using over-labeled fluorescent probes |
-
2015
- 2015-10-24 CN CN201510694321.0A patent/CN105334326A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006028321A1 (en) * | 2004-09-07 | 2006-03-16 | Zenovac, Inc. | Method of measuring ceruloplasmin concentration in a blood spot, kit and method of diagnosing wilson's disease |
| CN101718779A (en) * | 2009-10-29 | 2010-06-02 | 广西师范大学 | Kit for detecting immuno-nanogold synchronous scattering spectrum of human serum ceruloplosmin and use method thereof |
| WO2015042593A1 (en) * | 2013-09-23 | 2015-03-26 | Assaypro, Llc | Immunoassays using over-labeled fluorescent probes |
| CN104374924A (en) * | 2014-12-15 | 2015-02-25 | 山东博科生物产业有限公司 | Alpha1-AT (antitrypsin) immunoturbidimetry detection kit |
| CN104407159A (en) * | 2014-12-15 | 2015-03-11 | 山东博科生物产业有限公司 | IgM (Immune Globulin M) immune turbidimetry test kit |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106290926A (en) * | 2016-08-13 | 2017-01-04 | 山东博科生物产业有限公司 | A kind of apolipoprotein B immunoturbidimetry detection kit |
| CN106645694A (en) * | 2016-09-24 | 2017-05-10 | 济南博鑫生物技术有限公司 | Fn (fibronectin) detection kit |
| CN112730833A (en) * | 2020-11-23 | 2021-04-30 | 迪瑞医疗科技股份有限公司 | Ceruloplasmin determination kit by using immuno-transmission turbidimetry |
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