CN105548562A - An immunoturbidimetric detection kit for fibrinogen - Google Patents

An immunoturbidimetric detection kit for fibrinogen Download PDF

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Publication number
CN105548562A
CN105548562A CN201510973243.8A CN201510973243A CN105548562A CN 105548562 A CN105548562 A CN 105548562A CN 201510973243 A CN201510973243 A CN 201510973243A CN 105548562 A CN105548562 A CN 105548562A
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China
Prior art keywords
reagent
fibrinogen
kit
testing result
detection kit
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CN201510973243.8A
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Chinese (zh)
Inventor
甘宜梧
董雯
李静
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Biobase Biodustry Shandong Co Ltd
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Biobase Biodustry Shandong Co Ltd
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Priority to CN201510973243.8A priority Critical patent/CN105548562A/en
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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
    • 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
    • G01N33/54326—Magnetic particles
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/745—Assays involving non-enzymic blood coagulation factors
    • G01N2333/75—Fibrin; Fibrinogen

Abstract

The invention discloses an immunoturbidimetric detection kit for fibrinogen, and belongs to the technical field of clinical in-vitro detection reagents. The kit comprises a reagent R1, a reagent R2 and a calibrating product. By adding a certain amount of silica-covered magnetic nanometer particles into the reagent R1 and adding a certain amount of bovine serum albumin and a certain amount of Kathon-CG into the reagent R2, stability and analysis sensitivity of the kit are effectively improved, and the kit is good in linear range and high in accuracy, and is beneficial to further popularization and application in the market.

Description

A kind of fibrinogen immunoturbidimetry detection kit
Technical field
The present invention relates to clinical vitro detection reagent technique field, particularly a kind of fibrinogen immunoturbidimetry detection kit.
Background technology
Fibrinogen is a kind of protein with coagulation function synthesized by liver, is fibrinous precursor.Molecular weight 340000,4 ~ 6 days half life period.Reference value 2 ~ 4 grams per liter in blood plasma.Fibrin reason α, β, γ tri-formed different polypeptied chain, are connected between polypeptied chain with disulfide bond.Under thrombin action, α chain and β chain discharge A peptide and B peptide respectively, generate fibrin monomer.In the process, owing to releasing Acid polypeptide, electronegativity reduces, and monomer is easy to aggregate into fibrin polymer.But now borrow hydrogen bond to be connected with hydrophobic bond between monomer, still dissolve in diluted acid and urea liquid.Further under Ⅹ III factor effects of Ca+2 and activation, be connected with covalent bond between monomer, then become stable insoluble fibrin grumeleuse, complete coagulation process.Liver function serious hindrance or congenital deficiency, all can make plasma fibrinogen concentration decline, can have hemorrhagic tendency time serious.
Fibrinogen and liver diseases: fibrinogen system liver synthesizes, and is mainly distributed in blood plasma, is also present in blood platelet and megacaryocyte.Normal plasma concentration is 1.5 ~ 3.5g/L, therefore when liver is badly damaged, makes liver synthon proteinogen function generation obstacle, then in blood plasma, fibrinogen concentration reduces.Fibrinogen is a kind of plasma glycoprotein of liver synthesis.Can participate in thrombus and formation and development coronarius, being reflection thrombus state index, is also one of independent predictor of Acute coronary event.Fibrinogen raises prompting body fibrinolytic to be reduced, short thrombosis.
Fibrinogen and nephrotic syndrome (NS): the clotting factor of NS patient changes, increase the most obvious with fibrinogen level.Fibrinogen level increases and can reach 10g/L, and this is the result because synthesis increases, and thisly increases with its amount of losing from urinate proportional, but fibrinogenic kalabolism rate is then normal.Fibrinogen and the cholesterol levels of NS patient have significant correlation, and both and serum albumin levels are negative correlation.
Fibrinogen and atherosis: the relation of fibrinogen and cellulose and atherosclerosis plaque forming is very close.Known fiber protein dissolution mechanism is subject to various factors, such as smoking, diabetes, and especially high serum triglyceride can cause PF activation of zymogen thing inhibitor to raise, thus reduces the synthesis of plasminogen.Blood viscosity is higher, and these are all conducive to cellulosic formation.Fibrinogen is a kind of acute phase protein, enter in arterial wall as factor I by blood, under thrombin action, change fibrin monomer secondary into is cross-linked as fibrin, can directly destroy endothelial cell and be adsorbed on erythrocyte surface, arterial thrombus incidence is increased, and promotes that congee sample spot is in progress soon.Plasma fibrinogen can be deposited on vascular wall in addition, and accelerate atherosclerotic, people have found that the amount group disease fibrinogen content of fibrin condensation product in atherosclerotic patch all increases, and all have blood viscosity and increase.The feature that atherosclerotic is notably blocked.
Fibrinogen and cardiovascular and cerebrovascular disease: the research of thrombus in acute ischemia syndrome is shown, plasma fibrinogen level is independently hazards, have fibrinogen level in the patients blood plasma of coronary occlusion disease higher, it is closely related that the scope of myocardial infarction also increases degree with fibrinogen.There is the patient of unstable angina, before myocardial infarction occurs for it, often have plasma fibrinogen level to raise phenomenon.In the myocardial infarction course of disease, then infarct mostly occurs the patient of fibrinogen level more than 7g/L.
Fibrinogen and Hemorheology: find fibrinogen and between whole blood viscosity, plasma viscosity, erythrocyte sedimentation rate and platelet aggregation in remarkable positive correlation, prompting plasma fibrinogen content raises, and blood viscosity can be made to increase.Red gathering of carefully roaring is increased, and platelet aggregation increases, thus makes blood be in hypercoagulative state promotion thrombosis.Plasma fibrinogen content raises because its molecular weight is large.Concentration is high, has polymerization again, is the key factor making blood viscosity increase except red blood cell; Therefore, plasma fibrinogen content is judging that on the developing of disease, tool is of great significance.
Fibrinogen and other factors: the other factors affecting fibrinogen level is more, as genetic predisposition, age growth, hyperlipidemia, smoking, essential hypertension, obesity, oral contraceptive and the gestational period etc., are all hazards that plasma fibrinogen raises.
Fibrinogen immunoturbidimetry detection kit mixes with reagent based on when sample, Fb in sample forms insoluble antigen-antibody complex with antibody response specifically, turbidity is caused to increase, under 340nm wavelength, detect the change of its turbidity, intensity of variation is directly proportional to the concentration of Fb in 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 fibrinogen immunoturbidimetry detects that reagent stability is bad, sensitivity is not high yet, thus limits its applying clinically.
Summary of the invention
Be directed to prior art Problems existing, the invention provides a kind of fibrinogen immunoturbidimetry detection kit, this kit is compared with the kit of routine, and stability is better than the detection kit of routine, sensitivity for analysis is high, is conducive to reagent applying clinically.
The present invention is achieved by the following measures:
A kind of fibrinogen immunoturbidimetry 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 100mmol/LpH6.8Tris damping fluid, 0.1% Sodium azide, 0.1%-2% coated with silica; Reagent R2 consists of: 100mmol/LpH6.8Tris damping fluid, 30ml/L goat-anti people Fb 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=3:1.
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 Fb calibration object that Jiu Fengrunda 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 shown in Fig. 4, add physiological saline, sample or calibration object 5 μ l, absorbance A 1 is read after adding R1 reagent 225 μ l preincubate 5min afterwards again, after adding the reagent R2 reaction 5min of 75 μ l afterwards again, read absorbance A 2, and calculate Δ A.
Beneficial effect of the present invention:
Fibrinogen immunoturbidimetry 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 Fb 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;
The using method of Fig. 4 kit.
Embodiment
For a better understanding of the present invention, further describe below in conjunction with specific embodiment.
Embodiment 1
A fibrinogen immunoturbidimetry detection kit for routine, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH6.8Tris damping fluid 100mmol/L
Sodium azide 0.1%
Reagent R2 consists of:
PH6.8Tris damping fluid 100mmol/L
Goat-anti people Fb 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, read absorbance A 1, after the reagent R2 adding 75 μ l afterwards again reacts 5min, read absorbance A 2, and calculate Δ A.
The Fb calibration object that the calibration object that the present embodiment uses is produced for Jiu Fengrunda Bioisystech Co., Ltd.
Embodiment 2
A kind of fibrinogen immunoturbidimetry detection kit, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH6.8Tris damping fluid 100mmol/L
Sodium azide 0.1%
The magnetic nanoparticle 0.1% of coated with silica
Reagent R2 consists of:
PH6.8Tris damping fluid 100mmol/L
Goat-anti people Fb antibody 30ml/L
Bovine serum albumin(BSA) 20g/L
Kathon-CG0.05%
Concrete assay method is with embodiment 1.
Embodiment 3
A kind of fibrinogen immunoturbidimetry detection kit, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH6.8Tris damping fluid 100mmol/L
Sodium azide 0.1%
The magnetic nanoparticle 1% of coated with silica
Reagent R2 consists of:
PH6.8Tris damping fluid 100mmol/L
Goat-anti people Fb antibody 30ml/L
Bovine serum albumin(BSA) 30g/L
Kathon-CG0.05%
Concrete assay method is with embodiment 1.
Embodiment 4
A kind of fibrinogen immunoturbidimetry detection kit, it comprises reagent R1, reagent R2, calibration object.
Wherein reagent R1 consists of:
PH6.8Tris damping fluid 100mmol/L
Sodium azide 0.1%
The magnetic nanoparticle 2% of coated with silica
Reagent R2 consists of:
PH6.8Tris damping fluid 100mmol/L
Goat-anti people Fb 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 fibrinogen kit (production of Changchun 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 0.9989,0.9990,0.9988, correlativity is relatively good, show that kit of the present invention and market obtaining the fibrinogen test 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:
Fibrinogen high level sample is found to be 8g/L, serial dilution is carried out with physiological saline, the sample of preparation 6 variable concentrations, be followed successively by the sample of 8g/L, 6.4g/L, 4.8g/L, 3.2g/L, 1.6g/L, 0g/L 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 is as shown in table 1.
Table 1 embodiment 1-4 linear correlation confirmatory experiment testing result
Theoretical concentration (g/L) Embodiment 1 testing result (g/L) Embodiment 2 testing result (g/L) Embodiment 3 testing result (g/L) Embodiment 4 testing result (g/L)
0.00 0.14 0.17 0.17 0.11
1.60 1.57 1.60 1.61 1.57
3.20 3.21 3.19 3.14 3.15
4.80 4.67 4.62 4.64 4.76
6.40 6.60 6.44 6.38 6.24
8.00 7.95 7.96 8.05 8.09
Correlation coefficient r 0.9989 0.9994 0.9995 0.9994
Above-mentioned testing result display, embodiment 1-4 testing result correlativity is all greater than 0.990, but the testing result of embodiment 2,3,4 is greater than 0.999, has better linear dependence compared with embodiment 1, and this illustrates that reagent of the present invention has better linear dependence.
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 table 2.
Table 2 stability confirmatory experiment testing result
Time Embodiment 1 reagent testing result Embodiment 2 reagent testing result Embodiment 3 reagent testing result Embodiment 4 reagent testing result Fresh embodiment 1 reagent testing result
12 months 0.38657 0.38606 0.38687 0.38698 0.38781
13 months 0.28568 0.28587 0.28646 0.28632 0.28682
14 months 0.29656 0.29668 0.29654 0.29674 0.29664
15 months 0.38326 0.38399 0.38387 0.38355 0.38406
16 months 0.26975 0.37966 0.37967 0.38008 0.37987
17 months 0.12621 0.38734 0.38686 0.38691 0.38729
18 months 0.00663 0.29826 0.29757 0.29811 0.29843
19 months 0.00032 0.38453 0.38432 0.38463 0.38457
20 months 0.00001 0.39685 0.39596 0.39623 0.39635
21 months 0.00000 0.37374 0.37421 0.37417 0.37461
22 months 0.00000 0.39284 0.39314 0.39321 0.39358
23 months 0.00000 0.38263 0.38198 0.38167 0.38265
24 months 0.00000 0.28178 0.28213 0.28121 0.28235
25 months 0.00000 0.26357 0.26443 0.26500 0.37232
26 months 0.00000 0.09365 0.08932 0.09397 0.37328
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 fibrinogen test kit.
Sensitivity for analysis confirmatory experiment:
With the sample of fibrinogen kit test concentration known of the present invention at 3.10g/L, record absorbance difference.The reagent of embodiment 1,2,3,4 is utilized to detect respectively.Testing result is as shown in table 3.
Table 3 sensitivity for analysis experimental result
Theoretical concentration (g/L) Embodiment 1 testing result Δ A Embodiment 2 testing result Δ A Embodiment 3 testing result Δ A Embodiment 4 testing result Δ A
3.10 0.2512 0.3258 0.3259 0.3257
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 Fb 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, fibrinogen immunoturbidimetry 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, fibrinogen immunoturbidimetry detection kit provided by the invention is conducive to further promoting the use of in the market.

Claims (3)

1. a fibrinogen immunoturbidimetry 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 100mmol/LpH6.8Tris damping fluid, 0.1% Sodium azide, 0.1%-2% coated with silica; Reagent R2 consists of: 100mmol/LpH6.8Tris damping fluid, 30ml/L goat-anti people Fb 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=3:1.
CN201510973243.8A 2015-12-22 2015-12-22 An immunoturbidimetric detection kit for fibrinogen Pending CN105548562A (en)

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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
CN107328933A (en) * 2017-09-06 2017-11-07 深圳蓝韵生物技术有限公司 Magnetic particle enhancing immunoturbidimetry detection box and its application
CN107402308A (en) * 2017-06-21 2017-11-28 北京科跃中楷生物技术有限公司 Immue quantitative detection reagent boxes of IL 6 and preparation method thereof
CN107422129A (en) * 2017-01-15 2017-12-01 北京科跃中楷生物技术有限公司 A kind of super quick cardiac muscle troponin I magnetic microsphere immunoturbidimetry detection method and detection kit
CN109557301A (en) * 2018-11-29 2019-04-02 山东博科生物产业有限公司 A kind of glycocholic acid immunoturbidimetry detection kit

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* Cited by examiner, † Cited by third party
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
CN107422129A (en) * 2017-01-15 2017-12-01 北京科跃中楷生物技术有限公司 A kind of super quick cardiac muscle troponin I magnetic microsphere immunoturbidimetry detection method and detection kit
CN107402308A (en) * 2017-06-21 2017-11-28 北京科跃中楷生物技术有限公司 Immue quantitative detection reagent boxes of IL 6 and preparation method thereof
CN107328933A (en) * 2017-09-06 2017-11-07 深圳蓝韵生物技术有限公司 Magnetic particle enhancing immunoturbidimetry detection box and its application
CN109557301A (en) * 2018-11-29 2019-04-02 山东博科生物产业有限公司 A kind of glycocholic acid immunoturbidimetry detection kit

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