CN102636653A - Compounded latex particle-enveloped cystatin C detection kit - Google Patents
Compounded latex particle-enveloped cystatin C detection kit Download PDFInfo
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- CN102636653A CN102636653A CN2012101175610A CN201210117561A CN102636653A CN 102636653 A CN102636653 A CN 102636653A CN 2012101175610 A CN2012101175610 A CN 2012101175610A CN 201210117561 A CN201210117561 A CN 201210117561A CN 102636653 A CN102636653 A CN 102636653A
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Abstract
The invention relates to the field of medical immunity in-vitro diagnosis, and in particular relates to a determination kit for detecting cystatin C in blood serum. The kit comprises a reagent R1, a reagent R2 and a calibrator, wherein the reagent R1 is a phosphate buffer system which comprises 30-60mmol/l phosphate buffer solution with PH value of 7.2-7.6, 60-95mmol/l polyethylene glycol 6000-8000 and 6-13mmol/l ethylene diamine tetraacetic acid. The reagent R2 comprises two kinds of polystyrene latex particle sensitization particles with different sizes and enveloped by goat anti-human cystatin C polyclonal antibody; and the reagent R2 also comprises latex diluent and confining liquid. The calibrator is 0-10mg/l bovine serum matrix and also comprises 0.2-2.2% preservative and 1-10% stabilizer. Compared with the prior art, the kit provided by the invention has the characteristics of high sensitivity, good specificity, high speed and accurate and reliable determination result.
Description
[technical field]
The present invention relates to medical immunology in-vitro diagnosis field, particularly a kind of bladder chalone C determining reagent kit that detects in the serum.
[background technology]
Kidney trouble is a kind of clinically common disease, and the kidney function damage that a variety of causes causes is the hazards of progress for whole end property ephrosis and angiocardiopathy.Unique method that can stop kidney trouble to worsen is exactly that diagnosis early, early treatment are to reverse the renal function of damage.The concentration of bladder chalone C receives the influence of the preceding factor of kidney hardly, can freely pass through glomerular filtration, does not have the heavily absorption and the drainage of renal tubule, does not also have the outer excretion pathway of kidney.The more bladder chalone C of discovering is the sensitive indicator of reflection kidney function damage.
Latex particle strengthens turbidimetry, and (particle-enhanced turbidimetric immunoassay PETIA) is occur in recent years a kind of comparatively stable, body fluid albumen homogeneous phase immunoturbidimetry detection method accurately.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 bladder chalone C is very 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, it is the most general method in common that latex particle strengthens turbidimetry, and the highest highest detection limit can only reach 0.1mg/l but latex particle strengthens turbidimetry, and linearity can only reach 8mg/l, in clinical practice, exists limitation.
[summary of the invention]
The present invention is in order to overcome the deficiency of prior art; Invention provides a kind of test kit of composite-grain diameter latex particle coated antibody to detect the bladder chalone C content in the serum; With reach easy and simple to handle, highly sensitive, specificity is good, fast, measure result's purpose accurately and reliably.
To achieve these goals, the present invention has designed a kind of compounded latex particle and has encapsulated the bladder chalone C detection kit, comprises reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 is a phosphate buffer, and its component is that pH value is the phosphate buffer 30-60mmol/l of 7.2-7.6, Macrogol 6000-8000 60-95mmol/l and disodium ethylene diamine tetraacetate 6-13mmol/l.
Reagent R2 comprises two kinds of polystyrene latex particle sensitization particles that vary in size that goat-anti human cystatin C polyclonal antibody encapsulates; Also comprise the latex dilution, and confining liquid.
Calibration object is a 0-10mg/l cow's serum matrix, also comprises the antiseptic of 0.2-2.2% and the stabilizing agent of 1-10%.
Among the said reagent R2, said confining liquid comprises the 0.01%-0.1% skimmed milk power, and the glycocoll of 0.1-0.6mm.
Among the said reagent R2, the latex dilution comprises that pH value is the phosphate buffer 35-60mmol/l of 7.2-7.6, disodium ethylene diamine tetraacetate 10.2mmol/l, the skimmed milk power of 0.01%-0.1%, 0.9% NaN3.
Among the said reagent R2, the diameter of two kinds of polystyrene latex particles is respectively 40-70nm and 150-190nm.
Among the said reagent R2, large grain size latex and small grain size latex are with the mixed of 1/3-1/5.
Among the said reagent R2, goat-anti human cystatin C polyclonal antibody is 1/10-3/10 with the mass ratio of the polystyrene latex particle that encapsulates.
In the described calibration object, antiseptic is a Sodium azide.
In the described calibration object, stabilizing agent is one or more potpourris in glycerine, sucrose, BSA, sweet mellow wine or the sorbierite.
The present invention compares with prior art, have highly sensitive, specificity good, fast, measure result's characteristics accurately and reliably.
[description of drawings]
Fig. 1 is the typical curve of the bladder chalone C normative reference of five kinds of variable concentrations of the present invention.
Fig. 2 is clinical correlation tracing analysis figure of the present invention.
[embodiment]
Below in conjunction with specific embodiment, further set forth the present invention.Should understand these embodiment and only be used to explain that the present invention is not used in restriction scope of the present invention.
Embodiment 1:
Dilution of the present invention is formed by following material configuration.
Reagent R1 prepares as follows:
PH7.4 phosphate buffer 45mmol/l;
Macrogol 6000 85mmol/l;
Disodium ethylene diamine tetraacetate 8.5mmol/l;
NaN3 accounts for 0.9% of total reagent R1.
Various compositions add under can room temperature successively, perhaps add simultaneously, or respectively separately packing and with detect before in instant preparation.
Reagent R2 prepares as follows:
Cut-off is that the polystyrene latex particles of 45nm was according to 1: 4 mixed for the polystyrene latex particles of 186nm and diameter directly.Goat-anti human cystatin C polyclonal antibody and mixed polystyrene latex particles are mixed with 30: 100 mass ratio; With behind both mixings 37 ℃ absorption 8 hours; The antibody that does not connect is removed in dialysis afterwards; Add the confining liquid of the glycocoll composition of 0.1% skimmed milk power and 0.2mm, sealed 2 hours.
The use pH value is 7.4 phosphate buffer 45mmol/l, disodium ethylene diamine tetraacetate 10.2mmol/l, 0.05% skimmed milk power, the latex dilution that 0.9% NaN3 forms, the centrifugal supernatant that goes.
The preparation of cow's serum matrix calibration object:
With treated cow's serum, add BSA 0.1%, NaN3 0.9% obtains the calibration object dilution.Use reorganization bladder chalone C albumen to be dissolved in the solution of the similar human serum matrix of preparation, its concentration of standard items of preparation variable concentrations is respectively 0; 0.5mg/l; 1.5mg/l; 3.0mg/l; 6.0mg/l; 10.0mg/l.
The bladder chalone C detection kit that present embodiment is described is applicable to various types of full automatic biochemical apparatus, is example with Hitachi's 7170 full automatic biochemical apparatus, and it is operated like table 1.Analytical approach: 2 end-point methods, i.e. reagent R1; The consumption of R2 is respectively 230ul and 50ul, sample size 3ul; 230ul reagent R1 adds the 3ul sample and behind 37C5min, adds 50ulR2, promptly begins to read a little, reads another point behind the reaction 5min; Detect wavelength predominant wavelength 546nm commplementary wave length 800nm respectively.
Adopt this reagent and said determination method; The curve of the bladder chalone C standard items (self-control) of 5 kinds of different contents that employing Hitachi 7170 Biochemical Analyzers record; As shown in Figure 1, each point is represented the normative reference article of a content, and wherein the X axle is represented bladder chalone C content (mg/L); The Y axle is represented absorbance.
Table 1:
Experiment one, the correlation test of detectable:
Correlation test:
Use this law invention reagent; Concrete prescription is with embodiment 1 and the existing bladder chalone C latex enhancement mode reagent of contrast; Adopt automatic 7170 automatic clinical chemistry analyzers that 40 parts of human serums (comprising normal and monstrosity) are measured by each autoregressive parameter simultaneously, measured value is carried out correlation analysis.According to above-mentioned " bladder chalone C determining method " in parameter measure, measure the result and see Fig. 2, X, Y axle are measured value (the content mg/L of bladder chalone C),
Result by Fig. 2 finds out that the facies relationship of two kinds of reagent is R2=0.984, and regression equation is y=1.004x.It is good that the result shows that this reagent and import reagent are measured patients serum's correlativity, has excellent specificity and accuracy.
In addition, more than experiment is that 7170 full automatic biochemical apparatus that adopt Hitachi, Ltd to make carry out, but reagent of the present invention is not limited to above-mentioned instrument, also is applicable to other full-automatic or semi-automatic biochemical analyzers.
Test Example two, the LDL test:
This experiment purpose is that the minimum of detectable when the test clinical sample detects sensitivity.
Adopt experimental example 1 reagent, contrast agents, standard items, blank solution (generally being normal saline solution and Purified Water), normal human serum sample, low value sample (sample of numerical value in reagent range of linearity lower limit ± 1/3).
Machine: Hitachi's 7170 automatic biochemistry analyzers.
Operation steps: use normal saline solution or deionized water dissolving low value sample, 50% be diluted to 5 points and zero point together then, each test sample 5 times, calculating mean value is tried to achieve SD numerical value.
The result resolves: according to detecting data, calculates SD numerical value and CV numerical value, calculates 1SD respectively, and 2SD, from the beginning of minimum, the numerical value of its mean value-2SD is exactly that the minimum of reagent detects sensitivity more than zero point mean value+2SD.
Table 2,3 results show that when reagent of the present invention was measured dilution 1/16,1/8,1/4,1/2 serum, the numerical value of measuring mean value-2SD showed that all greater than mean value+2SD at zero point reagent LDL of the present invention can reach 0.038mg/ at least.And the contrast available reagent is measured dilution 1/16,1/8,1/4,1/2 serum; And relatively serum mean value-2SD with zero point, mean value+2SD was big or small; Wherein the numerical value of 1/16,1/8 dilution mean value-2SD is less than mean value+2SD at zero point; 1/4, the numerical value of 1/2 dilution mean value-2SD shows that greater than mean value+2SD at zero point the available reagent lowest detection is limited to about 0.124mg/l.
Table 2:
Table 3:
Test Example three, sensitivity experiment:
This experiment purpose is the absorbance changing value of detectable when test physiological saline and certain density management serum.
Adopt the reagent, contrast agents, standard items, blank solution of experimental example 1,0.9% normal saline solution, the absorbance changing value during the management serum of concentration.
Machine: Hitachi's 7170 automatic biochemistry analyzers.
Operation steps: use normal saline solution, low value sample, each test sample 5 times calculates absorbance.
Table 4,5 shows; Absorbance was changed to-2.8 (1/10000A) when reagent of the present invention was measured physiological saline; Absorbance was changed to 323.6 (1/10000A) when theoretical concentration was the 0.35mg/l serum sample; And A company reagent when measuring physiological saline absorbance be changed to-21.75 (10000A), absorbance was changed to 31.5 (1/10000A) when theoretical concentration was the 0.35mg/l serum sample, showed that reagent sensitivity of the present invention will be significantly higher than available reagent.
Table 4:
Table 5:
Claims (8)
1. a compounded latex particle encapsulates the bladder chalone C detection kit, comprises reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 is a phosphate buffer, and its component is that pH value is the phosphate buffer 30-60mmol/l of 7.2-7.6, Macrogol 6000-8000 60-95mmol/l and disodium ethylene diamine tetraacetate 6-13mmol/l;
Reagent R2 comprises two kinds of polystyrene latex particle sensitization particles that vary in size that goat-anti human cystatin C polyclonal antibody encapsulates; Also comprise the latex dilution, and confining liquid;
Calibration object is a 0-10mg/l cow's serum matrix, also comprises the antiseptic of 0.2-2.2% and the stabilizing agent of 1-10%.
2. compounded latex particle according to claim 1 encapsulates the bladder chalone C detection kit, it is characterized in that: among the said reagent R2, said confining liquid comprises the 0.01%-0.1% skimmed milk power, and the glycocoll of 0.1-0.6mm.
3. compounded latex particle according to claim 1 encapsulates the bladder chalone C detection kit; It is characterized in that: among the said reagent R2; The latex dilution comprises that pH value is the phosphate buffer 35-60mmol/l of 7.2-7.6; Disodium ethylene diamine tetraacetate 10.2mmol/l, the skimmed milk power of 0.01%-0.1%, 0.9% NaN3.
4. compounded latex particle according to claim 1 encapsulates the bladder chalone C detection kit, it is characterized in that: among the said reagent R2, the diameter of two kinds of polystyrene latex particles is respectively 40-70nm and 150-190nm.
5. compounded latex particle according to claim 1 encapsulates the bladder chalone C detection kit, it is characterized in that: among the said reagent R2, large grain size latex and small grain size latex are with the mixed of 1/3-1/5.
6. compounded latex particle according to claim 1 encapsulates the bladder chalone C detection kit, it is characterized in that: among the said reagent R2, goat-anti human cystatin C polyclonal antibody is 1/10-3/10 with the mass ratio of the polystyrene latex particle that encapsulates.
7. compounded latex particle according to claim 1 encapsulates the bladder chalone C detection kit, it is characterized in that: in the described calibration object, antiseptic is a Sodium azide.
8. compounded latex particle according to claim 1 encapsulates the bladder chalone C detection kit, it is characterized in that: in the described calibration object, stabilizing agent is one or more potpourris in glycerine, sucrose, BSA, sweet mellow wine or the sorbierite.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102809654A (en) * | 2012-08-23 | 2012-12-05 | 上海睿康生物科技有限公司 | Double-particle compounded C-reactive protein detection kit |
| CN102818903A (en) * | 2012-08-23 | 2012-12-12 | 上海睿康生物科技有限公司 | Double-particle compounded Lp-a detection kit |
| CN103018465A (en) * | 2012-12-16 | 2013-04-03 | 北京利德曼生化股份有限公司 | Human cystatin C chemiluminescence quantitative detection method |
| CN103134934A (en) * | 2013-02-27 | 2013-06-05 | 宁波美康生物科技股份有限公司 | Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample |
| CN103389385A (en) * | 2013-08-07 | 2013-11-13 | 上海睿康生物科技有限公司 | Latex-coated troponin detection kit |
| CN103389383A (en) * | 2013-08-07 | 2013-11-13 | 上海睿康生物科技有限公司 | Detection kit for measuring content of myohemoglobin in serum |
| CN103645323A (en) * | 2013-08-02 | 2014-03-19 | 浙江夸克生物科技有限公司 | Cystatin C detection kit and preparation method therefor |
| CN104049084A (en) * | 2013-03-11 | 2014-09-17 | 南京澳林生物科技有限公司 | Cystine protease inhibitor C detection kit |
| CN105061599A (en) * | 2015-08-05 | 2015-11-18 | 海奥斯生物科技镇江有限公司 | Method for producing goat-anti-human cystatin C protein antiserum |
| CN105738617A (en) * | 2016-04-01 | 2016-07-06 | 武汉生之源生物科技股份有限公司 | Cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof |
| CN106932594A (en) * | 2017-03-14 | 2017-07-07 | 吉林省富生医疗器械有限公司 | A kind of preferred cystatin C detection kit and preparation method thereof |
| CN109358009A (en) * | 2018-10-26 | 2019-02-19 | 武汉百德瑞康生物技术有限公司 | Bladder chalone C determining reagent kit and preparation method thereof and detection method |
| CN109406771A (en) * | 2018-10-26 | 2019-03-01 | 安徽大千生物工程有限公司 | Cystatin C latex-enhanced turbidimetry detection kit and its preparation application method |
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102809654A (en) * | 2012-08-23 | 2012-12-05 | 上海睿康生物科技有限公司 | Double-particle compounded C-reactive protein detection kit |
| CN102818903A (en) * | 2012-08-23 | 2012-12-12 | 上海睿康生物科技有限公司 | Double-particle compounded Lp-a detection kit |
| CN103018465A (en) * | 2012-12-16 | 2013-04-03 | 北京利德曼生化股份有限公司 | Human cystatin C chemiluminescence quantitative detection method |
| CN103134934A (en) * | 2013-02-27 | 2013-06-05 | 宁波美康生物科技股份有限公司 | Kit for simultaneously detecting retinol-binding protein (RBP) in urine sample and serum sample |
| CN104049084A (en) * | 2013-03-11 | 2014-09-17 | 南京澳林生物科技有限公司 | Cystine protease inhibitor C detection kit |
| CN103645323A (en) * | 2013-08-02 | 2014-03-19 | 浙江夸克生物科技有限公司 | Cystatin C detection kit and preparation method therefor |
| CN103389383A (en) * | 2013-08-07 | 2013-11-13 | 上海睿康生物科技有限公司 | Detection kit for measuring content of myohemoglobin in serum |
| CN103389385A (en) * | 2013-08-07 | 2013-11-13 | 上海睿康生物科技有限公司 | Latex-coated troponin detection kit |
| CN105061599A (en) * | 2015-08-05 | 2015-11-18 | 海奥斯生物科技镇江有限公司 | Method for producing goat-anti-human cystatin C protein antiserum |
| CN105738617A (en) * | 2016-04-01 | 2016-07-06 | 武汉生之源生物科技股份有限公司 | Cystatin C latex-particle-enhanced turbidimetry detection reagent kit and application thereof |
| CN106932594A (en) * | 2017-03-14 | 2017-07-07 | 吉林省富生医疗器械有限公司 | A kind of preferred cystatin C detection kit and preparation method thereof |
| CN109358009A (en) * | 2018-10-26 | 2019-02-19 | 武汉百德瑞康生物技术有限公司 | Bladder chalone C determining reagent kit and preparation method thereof and detection method |
| CN109406771A (en) * | 2018-10-26 | 2019-03-01 | 安徽大千生物工程有限公司 | Cystatin C latex-enhanced turbidimetry detection kit and its preparation application method |
| CN109358009B (en) * | 2018-10-26 | 2021-08-10 | 武汉百德瑞康生物技术有限公司 | Cystatin C determination kit, preparation method and detection method thereof |
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Application publication date: 20120815 |