CN102818903A - Double-particle compounded Lp-a detection kit - Google Patents
Double-particle compounded Lp-a detection kit Download PDFInfo
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- CN102818903A CN102818903A CN201210302800XA CN201210302800A CN102818903A CN 102818903 A CN102818903 A CN 102818903A CN 201210302800X A CN201210302800X A CN 201210302800XA CN 201210302800 A CN201210302800 A CN 201210302800A CN 102818903 A CN102818903 A CN 102818903A
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Abstract
Relating to the field of in vitro diagnosis of medical immunology, the invention provides a double-particle compounded Lp-a detection kit, which is composed of an R1 reagent, an R2 reagent and a calibrator. The R1 reagent consists of: a phosphate buffer solution with PH of 7.0-7.6 and a concentration of 30-80mmol/l, polyethylene glycol 6000-10000 with a concentration of 60-120mm/l, and disodium ethylenediamine tetraacetate with a concentration of 10-20mmol/l. The R2 reagent consists of: Goat anti-human Lp-a polyclonal antibody, a polystyrene latex coating, the phosphate buffer solution with PH of 7.0-7.6 and disodium ethylenediamine tetraacetate, etc. And the calibrator consists of: calibrator for: a bovine serum matrix with a concentration of 0-10mg/l, 0.2-2.2% of sodium azide, 1-10% of tween-20, and 1-3% of bovine serum albumin. The kit has the advantages of simple operation and good specificity, greatly improves the antibody purity, maximumly avoids non-specific reactions, and greatly enhances the result accuracy.
Description
Technical field:
The present invention relates to medical immunology in-vitro diagnosis field, be specifically related to a kind of pair of Lp-a detection kit that particle is compound.
Background technology:
Berg in 1963 finds that when the plasma lipoprotein electrophoresis beta lipoprotein partly has a kind of new antigenic component, and combines with LDL, with this antigenic component called after lipoprotein (a).Confirm that the Lp-a core is made up of lipid such as triglyceride, phosphatide, cholesterol, cholesterol ester and apolipoprotein B thereafter, similar LDL, but also contain the Apo (a) of a uniqueness, the latter does not exist in other any lipoprotein.Research shows that liver is the main place of synthetic Apo (a).Lp-a and LDL are different, are not to be transformed by VLDL, can not be converted into other lipoprotein, are one type of independently lipoprotein.The prominent feature of human Lp-a metabolism is, the Lp-a level can differ 100 times between individuality, but the variation of same individual blood plasma Lp-a level is then less relatively.The ldl receptor defective can influence Lp-a concentration between individuality, maybe be relevant with the synthetic increase of Lp-a in the body.
Lp-a is an independent hazard factor of coronary heart disease, and all irrelevant with smoking, hypertension, LDL-C and HDL-C and ApoA-1 and ApoB etc.Compare with other lipoprotein or apolipoprotein, the relation of Lp-a and males with coronary disease is particularly close.The dangerous critical level of blood plasma Lp-a is generally at 0.2～0.3g/L, as surpass＞0.30g/L then the danger of AS rise 2 times, as rising with LDL-C simultaneously, the relative risk of CHD rises 5 times.And the Lp-a level is high more, CHD takes place then more early.Lp-a has the multiple-factor inheritance characteristic, and CHD family history person is arranged, and the Lp-a positive rate is apparently higher than no family history person.
It is occur in recent years a kind of comparatively stable, body fluid albumen homogeneous phase immunoturbidimetry detection method accurately that latex particle strengthens turbidimetry.The PETIA method is divided into two kinds substantially.A kind of is the scattering turbidimetry detection method; Another kind is the turbid detection method of transmittance.The ultimate principle of these two kinds of methods is closely similar; It all is surface-crosslinked monoclonal antibody at the polymer latex microballoon; When the crosslinked microballoon that antibody arranged with after antigen combines, can flock together rapidly at short notice, changed the astigmatic performance or the light transmission of reactant liquor.And the reactant liquor astigmatism performance or the change of light transmission (being absorbance) and the concentration of tested antigen have stronger correlativity, can reflect the concentration of tested antigen within the specific limits.The PETIA detection method is the mensuration of in homogeneous reaction system, carrying out antigen, antibody response and result.Behind antigen, the antibody response, directly the absorbance of assaying reaction liquid has been save the ELISA method and has been hatched and washed loaded down with trivial details operation stepss such as plate repeatedly, and a few minutes just can obtain the result, and are time saving and energy saving.In addition, the interference of many manual operation factors and extraneous factors such as reagent, environment has also correspondingly been avoided in the simplification of nano immune turbidimetry operation steps, and stability and repeatability are all better, can reflect the content of measured matter more truly.Though the sensitivity of immunoturbidimetry is more weaker than ELISA method, the lower limit of foot many marker proteins in detecting human normal plasma can satisfy the Clinical detection requirement fully.
Because the content of serum Lp-a is low, its assay method needs higher sensitivity and specificity.By the qualitative detection of initial immunoelectrophoresis, to radio-immunity, fluorescence immunoassay and immunoenzymatic detection by quantitative, and the robotization detection of arriving immunoturbidimetry, development is very rapidly.Unidirectional immunodiffusion (SRID), radioimmunoassay (RIA), FIA are sent out (FIA), time-resolved fluorescent immunoassay (TRFIA), enzymoimmunoassay (ELISA), latex particle method, particle enhancing transmission immunoturbidimetry (PETIA), particle enhancing scattering immunoturbidimetry (PENIA).At present, immunoturbidimetry is the most general method in common, but immunoturbidimetry highest detection limit can only reach 30mg/l, and linearity can only reach 800mg/l, in clinical practice, exists limitation.
Summary of the invention:
The purpose of this invention is to provide a kind of pair of LP-a detection kit that particle is compound, it is easy and simple to handle, quick, highly sensitive, specificity is good, has improved the purity of antibody greatly, has avoided non-specific responding to greatest extent, and making as a result, accuracy is greatly improved.
In order to solve the existing problem of background technology; The present invention adopts following technical scheme: it is made up of R1 reagent, R2 reagent and calibration object three parts, and the mass ratio of R1 reagent consists of: the phosphate buffer 30-80mmol/l of PH=7.0-7.6, Macrogol 6000-1000060-120mm/l and disodium ethylene diamine tetraacetate 10-20mmol/L; Consisting of of R2 reagent: goat-anti people Lp-a polyclonal antibody, polystyrene latex encapsulate, the phosphate buffer of PH=7.0-7.6 and disodium ethylene diamine tetraacetate etc.; Consisting of of calibration object: 0-10mg/l cow's serum matrix, Sodium azide 0.2-2.2%, Tween-20 1-10%, bovine serum albumin(BSA) 1-3%, above-mentioned number percent are the number percent of cow's serum matrix volumetric usage.
To encapsulate mass ratio be 1/10-5/10 for goat-anti people Lp-a polyclonal antibody and polystyrene latex in the described R2 reagent.
In the described R2 reagent in the antibody sandwich process; The confining liquid component comprises the glycocoll of 0.5%-1.5% skimmed milk power, 10-60mm; Latex dilution component comprises PH7.0-7.6 phosphate buffer 30-80mmol/l, 0.5%-1.5% skimmed milk power, 0.9%NaN3.
The preparation process of described R2 reagent is: with the polystyrene latex particles of the polystyrene latex particles of 190nm and the 45nm mixed according to 1: 4, goat-anti people Lp-a polyclonal antibody and mixed polystyrene latex mix with 30: 100 mass ratio, and damping fluid adopts the phosphate buffer of 0.02M; With 37 ℃ of absorption behind both mixings 8 hours, the antibody on connecting was removed not in dialysis afterwards, adds the glycocoll of confining liquid 0.1% skimmed milk power and 0.2mm; Sealed 2 hours; The centrifugal supernatant that goes is with PH7.4 phosphate buffer 45mmol/l, disodium ethylene diamine tetraacetate 10.2mmol/l; 0.05% skimmed milk power, 0.9%NaN3 is diluted to 0.18%;
Described calibration object prepares process: with treated cow's serum, add BAS 0.1%, NaN3 0.9% obtains the calibration object dilution;
The present invention has following beneficial effect: it is easy and simple to handle, quick, highly sensitive, specificity is good, has improved the purity of antibody greatly, has avoided non-specific responding to greatest extent, and making as a result, accuracy is greatly improved.
Description of drawings:
Fig. 1 is the structural representation of embodiment 1 among the present invention.
Embodiment:
This embodiment is taked following technical scheme: it is made up of R1 reagent, R2 reagent and calibration object three parts, and the mass ratio of R1 reagent consists of: the phosphate buffer 30-80mmol/l of PH=7.0-7.6, Macrogol 6000-10000 60-120mm/l and disodium ethylene diamine tetraacetate 10-20mmol/L; Consisting of of R2 reagent: goat-anti people Lp-a polyclonal antibody, polystyrene latex encapsulate, the phosphate buffer of PH=7.0-7.6 and disodium ethylene diamine tetraacetate etc.; Consisting of of calibration object: 0-10mg/l cow's serum matrix, Sodium azide 0.2-2.2%, Tween-20 1-10%, bovine serum albumin(BSA) 1-3%, above-mentioned number percent are the number percent of cow's serum matrix volumetric usage.
To encapsulate mass ratio be 1/10-5/10 for goat-anti people Lp-a polyclonal antibody and polystyrene latex in the described R2 reagent.
In the described R2 reagent in the antibody sandwich process; The confining liquid component comprises the glycocoll of 0.5%-1.5% skimmed milk power, 10-60mm; Latex dilution component comprises PH7.0-7.6 phosphate buffer 30-80mmol/l, 0.5%-1.5% skimmed milk power, 0.9%NaN3.
The preparation process of described R2 reagent is: with the polystyrene latex particles of the polystyrene latex particles of 190nm and the 45nm mixed according to 1: 4, goat-anti people Lp-a polyclonal antibody and mixed polystyrene latex mix with 30: 100 mass ratio, and damping fluid adopts the phosphate buffer of 0.02M; With 37 ℃ of absorption behind both mixings 8 hours, the antibody on connecting was removed not in dialysis afterwards, adds the glycocoll of confining liquid 0.1% skimmed milk power and 0.2mm; Sealed 2 hours; The centrifugal supernatant that goes is with PH7.4 phosphate buffer 45mmol/l, disodium ethylene diamine tetraacetate 10.2mmol/l; 0.05% skimmed milk power, 0.9%NaN3 is diluted to 0.18%;
Described calibration object prepares process: with treated cow's serum, add BAS 0.1%, NaN3 0.9% obtains the calibration object dilution;
This embodiment has following beneficial effect: it is easy and simple to handle, quick, highly sensitive, specificity is good, has improved the purity of antibody greatly, has avoided non-specific responding to greatest extent, and making as a result, accuracy is greatly improved.
Embodiment 1: correlation test; Use the Lp-a latex enhancement mode reagent of this law invention reagent and contrast agents A company; Adopt automatic 7170 automatic clinical chemistry analyzers that 50 parts of human serums are measured by each autoregressive parameter simultaneously, measured value carried out correlation analysis, according to above-mentioned " the Lp-a assay method " in parameter measure the result and see Fig. 1; X, Y axle are measured value (the content mg/L of Lp-a).
Result by Fig. 1 finds out that the facies relationship of two kinds of reagent is R2=0.985, and regression equation is y=0.978x+5.896.It is good that the result shows that this reagent and import reagent are measured patients serum's correlativity, has excellent specificity and accuracy.
Claims (5)
1. two compound Lp (a) detection kit of particle; It is characterized in that it is made up of R1 reagent, R2 reagent and calibration object three parts, the consisting of of R1 reagent quality ratio: the phosphate buffer 30-80mmol/l of PH=7.0-7.6, Macrogol 6000-1000060-120mm/l and disodium ethylene diamine tetraacetate 10-20mmol/L; Consisting of of R2 reagent: goat-anti people Lp-a polyclonal antibody, polystyrene latex encapsulate, the phosphate buffer of PH=7.0-7.6 and disodium ethylene diamine tetraacetate etc.; Consisting of of calibration object: 0-10mg/l cow's serum matrix, Sodium azide 0.2-2.2%, Tween-20 1-10%, bovine serum albumin(BSA) 1-3%, above-mentioned number percent are the number percent of cow's serum matrix volumetric usage.
2. Lp (a) detection kit that a kind of pair of particle according to claim 1 is compound is characterized in that in the described R2 reagent that it is 1/10-5/10 that goat-anti people Lp-a polyclonal antibody and polystyrene latex encapsulate mass ratio.
3. Lp (a) detection kit that a kind of pair of particle according to claim 1 is compound; It is characterized in that in the described R2 reagent in the antibody sandwich process; The confining liquid component comprises the glycocoll of 0.5%-1.5% skimmed milk power, 10-60mm; Latex dilution component comprises PH7.0-7.6 phosphate buffer 30-80mmol/l, 0.5%-1.5% skimmed milk power, 0.9%NaN3.
4. Lp (a) detection kit that a kind of pair of particle according to claim 1 is compound is characterized in that the preparation process of described R2 reagent is: with the polystyrene latex particles of the polystyrene latex particles of 190nm and the 45nm mixed according to 1: 4, goat-anti people Lp-a polyclonal antibody and mixed polystyrene latex mix with 30: 100 mass ratio; Damping fluid adopts the phosphate buffer of 0.02M; With 37 ℃ of absorption behind both mixings 8 hours, the antibody on connecting was removed not in dialysis afterwards, adds the glycocoll of confining liquid 0.1% skimmed milk power and 0.2mm; Sealed 2 hours; The centrifugal supernatant that goes is with PH7.4 phosphate buffer 45mmol/l, disodium ethylene diamine tetraacetate 10.2mmol/l; 0.05% skimmed milk power, 0.9%NaN3 is diluted to 0.18%.
5. Lp (a) detection kit that a kind of pair of particle according to claim 1 is compound is characterized in that described calibration object prepares process and is: with treated cow's serum, add BAS 0.1%, NaN3 0.9% obtains the calibration object dilution.
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| CN201210302800XA CN102818903A (en) | 2012-08-23 | 2012-08-23 | Double-particle compounded Lp-a detection kit |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103399160A (en) * | 2013-08-07 | 2013-11-20 | 上海睿康生物科技有限公司 | Immunoturbidimetric assay apolipoprotein E detection kit |
| CN103604930A (en) * | 2013-11-07 | 2014-02-26 | 北京利德曼生化股份有限公司 | Lipoprotein (a) detection kit |
| CN104215754A (en) * | 2014-08-14 | 2014-12-17 | 上海睿康生物科技有限公司 | SLO protection eukaryotic expression, and kit containing eukaryotically expressed SLO protein |
| CN105675872A (en) * | 2014-11-21 | 2016-06-15 | 上海科华生物工程股份有限公司 | Lipoprotein (a) quantitative assay kit, use method and application thereof |
| CN106501505A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | A kind of latex orientation coupling technology detection lipoprotein（a）Method |
| CN106501536A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | A kind of latex orientation coupling technology detection lipoprotein（a）Test kit |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1065729A (en) * | 1991-03-25 | 1992-10-28 | 第一化学药品株式会社 | Monoclonal antibody |
| CN102636653A (en) * | 2012-04-19 | 2012-08-15 | 上海蓝怡科技有限公司 | Compounded latex particle-enveloped cystatin C detection kit |
-
2012
- 2012-08-23 CN CN201210302800XA patent/CN102818903A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1065729A (en) * | 1991-03-25 | 1992-10-28 | 第一化学药品株式会社 | Monoclonal antibody |
| CN102636653A (en) * | 2012-04-19 | 2012-08-15 | 上海蓝怡科技有限公司 | Compounded latex particle-enveloped cystatin C detection kit |
Non-Patent Citations (1)
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| ABE ET AL: "Fully mechanized latex immunoassay for serum lipoprotein(a)", 《CLINICA CHIMICA ACTA》 * |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103399160A (en) * | 2013-08-07 | 2013-11-20 | 上海睿康生物科技有限公司 | Immunoturbidimetric assay apolipoprotein E detection kit |
| CN103604930A (en) * | 2013-11-07 | 2014-02-26 | 北京利德曼生化股份有限公司 | Lipoprotein (a) detection kit |
| CN103604930B (en) * | 2013-11-07 | 2015-11-18 | 北京利德曼生化股份有限公司 | A kind of lipoprotein (a) detection kit |
| CN104215754A (en) * | 2014-08-14 | 2014-12-17 | 上海睿康生物科技有限公司 | SLO protection eukaryotic expression, and kit containing eukaryotically expressed SLO protein |
| CN104215754B (en) * | 2014-08-14 | 2016-05-11 | 上海睿康生物科技有限公司 | The eukaryotic expression of SLO albumen and containing the kit of eukaryotic expression SLO albumen |
| CN105675872A (en) * | 2014-11-21 | 2016-06-15 | 上海科华生物工程股份有限公司 | Lipoprotein (a) quantitative assay kit, use method and application thereof |
| CN106501505A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | A kind of latex orientation coupling technology detection lipoprotein（a）Method |
| CN106501536A (en) * | 2016-10-03 | 2017-03-15 | 王贤俊 | A kind of latex orientation coupling technology detection lipoprotein（a）Test kit |
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