CN103076456A - Kit for detecting alpha 1-acidoglycoprotein by using immunity transmission turbidity method - Google Patents
Kit for detecting alpha 1-acidoglycoprotein by using immunity transmission turbidity method Download PDFInfo
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Abstract
The invention discloses a kit for detecting alpha 1-acidoglycoprotein by using an immunity transmission turbidity method. A reagent R1 comprises 10-500 mM buffer liquid, a 0.5-50 g/L surfactant, a 5-50 g/L electrolyte, a 2-100 g/L high molecular accelerant, a 5-50 g/L stabilizing agent and a 1-10 g/L preservative; a reagent R2 comprises 10-500 mM buffer liquid, a 150-300 g/L anti-human alpha 1-acidoglycoprotein antibody, a 5-50 g/L electrolyte and a 1-10 g/L preservative; and a standard product comprises 10-500 mM buffer liquid, a 5-50 g/L alpha 1-acidoglycoprotein antigen, a 5-50 g/L stabilizing agent, a 1-10 g/L preservative and a 0.01-10 g/L antioxidant. The kit has the advantages of simplicity and fastness in use, remarkably improved detection efficiency and stable reagent, can satisfy the requirement of clinical and quick high throughout sample detection, and is suitable for clinical promotion.
Description
Technical field
The present invention relates to medical immunology in-vitro diagnosis field, be specifically related to the kit that the immune turbidimetry of a kind of usefulness detects α 1-acidoglycoprotein.
Background technology
α 1-acidoglycoprotein, molecular weight approximately 40000 is a kind of non-specific acute phase reactive proteins, also is the maximum glycoprotein of (sugary approximately 45%), acidity the strongest (PI is 2.7～3.5) of sugar content in the serum human.α 1-acidoglycoprotein is mainly produced by liver macrophage and granulocyte, and cancer cell also can synthesize.
α 1-acidoglycoprotein is the present comparatively marker of inflammation of responsive acute phase response, is one of acute phase reactant.It reacts the variation early than body temperature and leukocyte count to inflammation, infection, therefore be widely used in clinical: (1) α 1-acidoglycoprotein content raises: under the pathologic condition, interleukin-11 stimulates phagocyte to discharge the fat polysaccharide, what can promote α 1-acidoglycoprotein syntheticly makes that level raises in the blood, therefore α 1-acidoglycoprotein is the most stable a kind of acute phase reactant that is positive in early days.α 1-acidoglycoprotein content raises during such as infection (inflammation), wound, burn, operation, acute myocardial infarction AMI.Rheumatoid arthritis, systemic loupus erythematosus, clone's disease, malignant tumour also increase in addition, raise when metastasis of cancer more obvious.(2) α 1-acidoglycoprotein concentration reduces: the albumen quality that filters in the urine due to liver cell lesion late period, nephrotic syndrome or the other reasons is lost, inherent cause and the disease such as malnutritive.
α 1-acidoglycoprotein detection method commonly used in the prior art is radio immunoassay, euzymelinked immunosorbent assay (ELISA).There are many deficiencies in these technology, the equipment special such as needs, and sample needs pre-service, can not go up automatic clinical chemistry analyzer and carry out batch detection analysis etc.There are the problems such as radiation and pollution in radiommunoassay.It is commonplace that euzymelinked immunosorbent assay (ELISA) is used, but the method is heterogeneous immune detection system, and the mensuration process is comparatively loaded down with trivial details, and length consuming time lacks unit testing working time; Automaticity is not high, and difference between batch and repeatability are relatively large; Need to be equipped with multiple Special Equipment, increased to a certain extent cost.
Summary of the invention
Technical matters to be solved by this invention provides the kit that the immune turbidimetry of a kind of usefulness detects α 1-acidoglycoprotein, and it is simple to operate, good reproducibility, can carry out that batch sample is full-automatic to be detected, thus defective in the elimination above-mentioned background technology.
For solving the problems of the technologies described above, technical scheme of the present invention is:
The immune turbidimetry of a kind of usefulness detects the kit of α 1-acidoglycoprotein, and this kit comprises reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 comprises damping fluid 10～500mM, surfactant 0.5～50g/L, electrolyte 5～50g/L, macromolecule promoter 2～100g/L, stabilizing agent 5～50g/L, antiseptic 1～10g/L;
Reagent R2 comprises damping fluid 10～500mM, anti-human α 1-acidoglycoprotein antibody 15%～30%(w/v), electrolyte 5～50g/L, antiseptic 1～10g/L;
Calibration object comprises damping fluid 10～500mM, α 1-acidoglycoprotein antigen, stabilizing agent 5～50g/L, antiseptic 1～10g/L, antioxidant 0.01～10g/L.
Above-mentioned damping fluid 10～500mM, namely concentration is 10～500mmo l/L.
Anti-human α 1-acidoglycoprotein antibody 15%～30%(w/v), namely concentration is 150～300g/L.
Wherein, described damping fluid is selected from one or more in phosphate buffer, glycine buffer, TRIS damping fluid, CAPSO, MOPS, borate buffer solution, the Hepes damping fluid;
The pH scope of damping fluid is 5.5～9.0;
Described surfactant be selected from Triton series, Tween series, bay ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene nonylplenyl ether, polyoxyethylene octyl group phenylate one or more;
Described electrolyte be selected from sodium chloride, potassium chloride, magnesium chloride, magnesium sulphate one or more;
Described macromolecule promoter is selected from one or more in AEO, polyvinylpyrrolidone, ethylene polyethenoxy ether, hydroxypropyl methylcellulose, sodium cellulose glycolate, the sodium polyacrylate;
Described stabilizing agent is selected from one or more in mannose, glucose, shitosan, sorbierite, bovine serum albumin, disodium ethylene diamine tetraacetate, the fructose;
Described antiseptic is selected from one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate, the ethyl mercury sodium thiosulfate;
Described anti-human α 1-acidoglycoprotein antibody is selected from that mouse-anti people, rabbit are anti-human, goat-anti people, chicken are anti-human, in the anti-human α 1-of the duck acidoglycoprotein antibody one or both;
Described anti-human α 1-acidoglycoprotein antibody type comprises monoclonal antibody and polyclonal antibody;
Described antioxidant is selected from one or more in butylated hydroxy anisole, dibutyl hydroxy toluene, benzene polyphenol, Licorice root antioxidant, dilauryl thiodipropionate, n-propyl gallate, ditert-butylhydro quinone, the vitamin series.
For the buffer system of guarantee reagent R1, reagent R2 is consistent, as preferred technical scheme, the damping fluid kind of described reagent R1 and reagent R2 is identical.
As a kind of preferred technical scheme, in the described kit,
It is 7.0～8.0 TRIS damping fluid 10～50mM, Tween200.5～5g/L, sodium chloride 5～15g/L, ethylene polyethenoxy ether 50～100g/L, disodium ethylene diamine tetraacetate 5～10g/L, Sodium azide 1～5g/L that reagent R1 comprises pH;
Reagent R2 comprises that comprising pH is 7.0～8.0 TRIS damping fluid 10～50mM, goat-anti people α 1-acidoglycoprotein polyclonal antibody 15%～30%(w/v), electrolyte 5～50g/L, antiseptic 1～10g/L;
Calibration object comprises TRIS damping fluid 10～500mM, α 1-acidoglycoprotein antigen 0.05～5.0g/L, disodium ethylene diamine tetraacetate 5～50g/L, Sodium azide 1～10g/L, butylated hydroxy anisole 0.01～10g/L.
Above-mentioned goat-anti people α 1-acidoglycoprotein polyclonal antibody 15%～30%(w/v), namely the concentration of goat-anti people α 1-acidoglycoprotein polyclonal antibody is 150～300g/L.
Wherein, in the described calibration object, the preferred 0.1～3.0g/L of α 1-acidoglycoprotein antigen concentration.
Described calibration object can be prepared into high concentration single-point calibration product, becomes in use the reference calibrations product of 6 variable concentrations with normal saline dilution, also can directly be prepared into the reference calibrations product of 6 variable concentrations.
Immunoturbidimetry of the present invention is that antigen-antibody is in conjunction with the dynamic measurement method, its ultimate principle is: corresponding antigen-antibody reaction occurs in the α 1-acidoglycoprotein antigen in the sample and the α 1-acidoglycoprotein antibody among the reagent R2, form insoluble antigen-antibody complex, make reactant liquor produce certain turbidity.During α 1-acidoglycoprotein antibody excess, the amount of the immune complex of formation increases along with the increase of α 1-acidoglycoprotein antigen amount in the sample in the reactant liquor, and the turbidity of reactant liquor is corresponding increase also.Measure the light absorption value of this compound under specific wavelength, the calibration curve of demarcating with calibration object compares, and can calculate the content of the α 1-acidoglycoprotein in the sample.
Owing to having adopted technique scheme, the invention has the beneficial effects as follows:
The present invention detects reagent, uses simple to operately, can be applied to automatic clinical chemistry analyzer and the semi-automatic biochemical analyzer of different types of machines, can satisfy clinical fast, high flux detects the requirement of sample; Repeated experiment shows that kit has high precision, the high characteristics of accuracy; Show that with the import reagent contrast experiment kit correlativity of the present invention is good, good linearity, repeatability is high; Kit has been owing to having used the optimization agent combination of screening, so that the stability of reagent improves greatly, 2-8 ° of C can stablize and deposit more than 1 year.Therefore kit of the present invention has very high clinical value.
Description of drawings
Fig. 1 is the method flow schematic diagram that the embodiment of the invention 4 detects α 1-acidoglycoprotein content;
Fig. 2 is the canonical plotting of the α 1-acidoglycoprotein calibration object of the embodiment of the invention 4;
Fig. 3 is the reagent of the present invention of the embodiment of the invention 6 and the correlativity curve map of import contrast agent.
Embodiment
For technological means, creation characteristic that the present invention is realized, reach purpose and effect is easy to understand, below in conjunction with specific embodiment, further set forth the present invention.
Embodiment 1
1. reagent preparation:
Reagent R2:
Calibration object:
Calibration object concentration is as required added the α 1-acidoglycoprotein antigen of respective amount in the mentioned solution to, prepares α 1-acidoglycoprotein calibration object.This calibration object can be high concentration single-point calibration product, becomes in use the reference calibrations product of 6 variable concentrations with normal saline dilution, also can directly be prepared into the reference calibrations product of 6 variable concentrations.The reference calibrations product of 6 variable concentrations of the present embodiment preparation are respectively 0g/L, 0.15g/L, 0.31g/L, 0.62g/L, 1.24g/L, 2.50g/L.Then use the membrane filtration degerming of 0.22 μ m, place 2～8 ℃ of preservations.
Embodiment 2
1. reagent preparation:
Reagent R1:
Reagent R2:
Calibration object:
Calibration object concentration is as required added the α 1-acidoglycoprotein antigen of respective amount in the mentioned solution to, prepares α 1-acidoglycoprotein calibration object.What the present embodiment was selected is high concentration single-point reference calibrations product, and α 1-acidoglycoprotein antigen concentration is 2.7g/L, then uses the membrane filtration degerming of 0.22 μ m, places 2～8 ℃ of preservations.Become the reference calibrations product of 5 variable concentrations during use with normal saline dilution, one of them does not add α 1-acidoglycoprotein antigen, as blank reagent, α 1-acidoglycoprotein antigen concentration is respectively 0g/L, 0.17g/L, 0.34g/L, 0.68g/L, 1.35g/L, 2.70g/L.
Embodiment 3
1. reagent preparation:
Reagent R1:
Reagent R2:
Calibration object:
Calibration object concentration is as required added the α 1-acidoglycoprotein antigen of respective amount in the mentioned solution to, prepares α 1-acidoglycoprotein calibration object.The reference calibrations product of 6 variable concentrations of the present embodiment preparation are respectively 0g/L, 0.15g/L, 0.31g/L, 0.62g/L, 1.24g/L, 2.50g/L.Then use the membrane filtration degerming of 0.22 μ m, place 2～8 ℃ of preservations.
Embodiment 4
1. reagent preparation:
Reagent R1:
Reagent R2:
Calibration object:
Calibration object concentration is as required added the α 1-acidoglycoprotein antigen of respective amount in the mentioned solution to, prepares α 1-acidoglycoprotein calibration object.This calibration object can be high concentration single-point calibration product, becomes in use the reference calibrations product of 6 variable concentrations with normal saline dilution, also can directly be prepared into the reference calibrations product of 6 variable concentrations.What the present embodiment was selected is high concentration single-point reference calibrations product, and α 1-acidoglycoprotein antigen concentration is 2.7g/L, then uses the membrane filtration degerming of 0.22 μ m, places 2～8 ℃ of preservations.Become the reference calibrations product of 5 variable concentrations during use with normal saline dilution, one of them does not add α 1-acidoglycoprotein antigen, as blank reagent, α 1-acidoglycoprotein antigen concentration is respectively 0g/L, 0.17g/L, 0.34g/L, 0.68g/L, 1.35g/L, 2.70g/L.
2. detect:
α 1-acidoglycoprotein detection method as shown in Figure 1, analytical approach is Two point end assay, specifically: the sample application amount is 12 μ l, reagent R1 and R2 application of sample amount are respectively 225 μ l and 75 μ l; 225 μ l reagent R1 add 12 μ l samples 37 ℃ of insulations 5 minutes, behind the record absorbance OD1, add 75 μ l reagent R2, and 37 ℃ are continued insulation and record absorbance OD2 after 5 minutes; The detection wavelength is respectively: predominant wavelength 340nm, commplementary wave length 660nm.
3. Specification Curve of Increasing:
Adopt the present embodiment reagent and said determination method, the typical curve of 6 variable concentrations α 1-acidoglycoprotein standard items that use Hitachi 7080 automatic clinical chemistry analyzers record, as shown in Figure 2.X-axis represents α 1-acidoglycoprotein content, and Y-axis represents absorbance.This typical curve can be for detection of sample α 1-acidoglycoprotein content.
Embodiment 5
Adopt detection method and the typical curve of embodiment 4, get at random a clinical serum sample, use Hitachi's 7080 automatic clinical chemistry analyzers to same sample duplicate detection 10 times, testing result is as shown in table 1.
Table 1 detects sample α 1-acidoglycoprotein concentration (10 times) and the coefficient of variation
Result's demonstration, the coefficient of variation is 1.26%, simultaneously, adopts the coefficient of variation of the described kit testing result of embodiment 1-3 less than 5%, shows that kit of the present invention has than high precision.
Embodiment 6
Use respectively reagent of the present invention and import contrast agent (Luo Shi reagent), use Hitachi's 7080 automatic clinical chemistry analyzers that 50 parts of clinical samples are measured simultaneously by each autoregressive parameter, measured value is carried out linear regression.Reagent parameter of the present invention is seen embodiment 4.The regression equation that linear regression obtains is Y=1.0106X-0.05118, coefficient R
2=0.9924, as shown in Figure 3.Wherein X is reagent of the present invention, and Y is contrast agent.The result shows that reagent of the present invention and contrast agent correlativity are fine, illustrates that reagent of the present invention has good specificity and accuracy.Reagent of the present invention goes for the full-automatic and semi-automatic biochemical analyzer of the series such as Hitachi, Olympus, Beckman, Abbott Laboratories, and analytical approach and parameter can suitably be adjusted according to different type of machines.
The present invention is not limited to above-mentioned embodiment, and all are based on technical conceive of the present invention, and the structural improvement of having done all falls among protection scope of the present invention.
Claims (7)
1. a kit that detects α 1-acidoglycoprotein with immune turbidimetry is characterized in that: described examination
The agent box comprises reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 comprises damping fluid 10～500mM, surfactant 0.5～50g/L, electrolyte 5～50g/L, macromolecule promoter 2～100g/L, stabilizing agent 5～50g/L, antiseptic 1～10g/L;
Reagent R2 comprises damping fluid 10～500mM, anti-human α 1-acidoglycoprotein antibody 150～300g/L, electrolyte 5～50g/L, antiseptic 1～10g/L;
Calibration object comprises damping fluid 10～500mM, α 1-acidoglycoprotein antigen, stabilizing agent 5～50g/L, antiseptic 1～10g/L, antioxidant 0.01～10g/L.
2. the immune turbidimetry of a kind of usefulness as claimed in claim 1 detects the kit of α 1-acidoglycoprotein, and it is characterized in that: described damping fluid is selected from one or more in phosphate buffer, glycine buffer, TRIS damping fluid, CAPSO, MOPS, borate buffer solution, the Hepes damping fluid;
Described surfactant is selected from that Tr iton series, Tween are serial, bay ether, polyoxyethylene alkyl phenyl ether, polyoxyethylene nonylplenyl ether, polyoxyethylene octyl group phenylate one or more;
Described electrolyte be selected from sodium chloride, potassium chloride, magnesium chloride, magnesium sulphate one or more;
Described macromolecule promoter is selected from one or more in AEO, polyvinylpyrrolidone, ethylene polyethenoxy ether, hydroxypropyl methylcellulose, sodium cellulose glycolate, the sodium polyacrylate;
Described stabilizing agent is selected from one or more in mannose, glucose, shitosan, sorbierite, bovine serum albumin, disodium ethylene diamine tetraacetate, the fructose;
Described antiseptic is selected from one or more in Sodium azide, phenol, P-hydroxybenzoic acid, ethyl-para-hydroxybenzoate, the ethyl mercury sodium thiosulfate;
Described anti-human α 1-acidoglycoprotein antibody is selected from that mouse-anti people, rabbit are anti-human, goat-anti people, chicken are anti-human, in the anti-human α 1-of the duck acidoglycoprotein antibody one or both;
Described antioxidant is selected from one or more in butylated hydroxy anisole, dibutyl hydroxy toluene, benzene polyphenol, Licorice root antioxidant, dilauryl thiodipropionate, n-propyl gallate, ditert-butylhydro quinone, the vitamin series.
3. the immune turbidimetry of a kind of usefulness as claimed in claim 2 detects the kit of α 1-acidoglycoprotein, and it is characterized in that: described anti-human α 1-acidoglycoprotein antibody type comprises monoclonal antibody and polyclonal antibody.
4. the immune turbidimetry of a kind of usefulness as claimed in claim 2 detects the kit of α 1-acidoglycoprotein, and it is characterized in that: the pH scope of described damping fluid is 5.5～9.0.
5. detect the kit of α 1-acidoglycoprotein such as the immune turbidimetry of each described a kind of usefulness of claim 1～4, it is characterized in that: the damping fluid kind of described reagent R1 and reagent R2 is identical.
6. the immune turbidimetry of a kind of usefulness as claimed in claim 5 detects the kit of α 1-acidoglycoprotein, it is characterized in that: in the described kit,
It is 7.0～8.0 TRIS damping fluid 10～50mM, Tween200.5～5g/L, sodium chloride 5～15g/L, ethylene polyethenoxy ether 50～100g/L, disodium ethylene diamine tetraacetate 5～10g/L, Sodium azide 1～5g/L that reagent R1 comprises pH;
Reagent R2 comprises that comprising pH is 7.0～8.0 TRIS damping fluid 10～50mM, goat-anti people α 1-acidoglycoprotein polyclonal antibody 150～300g/L, electrolyte 5～50g/L, antiseptic 1～10g/L; Calibration object comprises TRI S damping fluid 10～500mM, α 1-acidoglycoprotein antigen 0.05～5.0g/L, disodium ethylene diamine tetraacetate 5～50g/L, Sodium azide 1～10g/L, butylated hydroxy anisole 0.01～10g/L.
7. the immune turbidimetry of a kind of usefulness as claimed in claim 6 detects the kit of α 1-acidoglycoprotein, and it is characterized in that: in the described calibration object, α 1-acidoglycoprotein antigen concentration is 0.1～3.0g/L.
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Cited By (11)
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| CN103529221A (en) * | 2013-10-22 | 2014-01-22 | 青岛金洋生物技术有限公司 | Kit for detecting acid glycoprotein content in serum |
| CN105403712A (en) * | 2016-01-12 | 2016-03-16 | 柏荣诊断产品（上海）有限公司 | High performance detection kit for human urine alpha 1 acidoglycoprotein |
| CN105628942A (en) * | 2015-12-21 | 2016-06-01 | 北京九强生物技术股份有限公司 | Human urine alpha 1-acid glycoprotein detection kit |
| CN105785057A (en) * | 2015-12-22 | 2016-07-20 | 山东博科生物产业有限公司 | Alpha 1-acid glycoprotein detection kit |
| CN107589080A (en) * | 2016-07-08 | 2018-01-16 | 华仁药业股份有限公司 | Phosphatic detection method in a kind of parenteral solution |
| CN108120839A (en) * | 2017-12-27 | 2018-06-05 | 山东博科生物产业有限公司 | A kind of Immunoglobulin IgG detection kit and its preparation, application method |
| CN110501501A (en) * | 2019-07-23 | 2019-11-26 | 武汉大学 | Lung cancer early diagnoses application and the kit of tumor markers |
| CN111888470A (en) * | 2020-08-12 | 2020-11-06 | 赵静 | Flushing fluid with rabies virus neutralizing effect and activity detection method thereof |
| CN112285340A (en) * | 2019-07-25 | 2021-01-29 | 苏州普瑞斯生物科技有限公司 | Latex immunoturbidimetry method of alpha 1-acid glycoprotein detection kit and preparation method thereof |
| 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 |
| CN114544734A (en) * | 2021-12-02 | 2022-05-27 | 浙江鑫科医疗科技有限公司 | Reagent matched with electrolyte module of Beckmann AU series analyzer |
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| CN103529221A (en) * | 2013-10-22 | 2014-01-22 | 青岛金洋生物技术有限公司 | Kit for detecting acid glycoprotein content in serum |
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| CN105403712A (en) * | 2016-01-12 | 2016-03-16 | 柏荣诊断产品（上海）有限公司 | High performance detection kit for human urine alpha 1 acidoglycoprotein |
| CN107589080A (en) * | 2016-07-08 | 2018-01-16 | 华仁药业股份有限公司 | Phosphatic detection method in a kind of parenteral solution |
| CN108120839A (en) * | 2017-12-27 | 2018-06-05 | 山东博科生物产业有限公司 | A kind of Immunoglobulin IgG detection kit and its preparation, application method |
| CN110501501A (en) * | 2019-07-23 | 2019-11-26 | 武汉大学 | Lung cancer early diagnoses application and the kit of tumor markers |
| CN112285340A (en) * | 2019-07-25 | 2021-01-29 | 苏州普瑞斯生物科技有限公司 | Latex immunoturbidimetry method of alpha 1-acid glycoprotein detection kit and preparation method thereof |
| CN111888470A (en) * | 2020-08-12 | 2020-11-06 | 赵静 | Flushing fluid with rabies virus neutralizing effect and activity detection method thereof |
| 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 |
| CN114544734A (en) * | 2021-12-02 | 2022-05-27 | 浙江鑫科医疗科技有限公司 | Reagent matched with electrolyte module of Beckmann AU series analyzer |
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