CN102621332A - Retinol binding protein assay kit based on latex particle coating - Google Patents
Retinol binding protein assay kit based on latex particle coating Download PDFInfo
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Abstract
The invention relates to the field of medical immonological in vitro diagnosis and in particular relates to a retinol binding protein assay kit based on latex particle coating. The assay kit comprises a reagent R1, a reagent R2 and a calibrator, wherein the reagent R1 is a Tris-HCl buffer system, and comprises a Tris-HCl buffer solution, a polymer and ethylene diamine tetraacetic acid; the reagent R2 comprises goat anti human retinol binding protein polyclonal antibody coated polystyrene latex particle sensitized granules and a phosphate buffer solution; and the reference calibrator comprises bovine serum matrix, as well as 0.2-2.2% of antiseptic and 1-10% of stabilizer according to the volume percentage of the bovine serum matrix. Compared with the prior art, the assay kit can be used for assistant diagnosis of kidney diseases and assistant diagnosis of mal-nutrition, and has high clinical application value.
Description
[technical field]
The present invention relates to medical immunology in-vitro diagnosis field, particularly a kind ofly encapsulate the Retinal-binding protein detection kit based on latex particle.
[background technology]
RBP ELISA belongs to the Lipocalin superfamily, relative molecular mass about 21 * 10
3, part mainly is an alltrans retinol, i.e. vitamin A, Kd=20Nm, binding site 1.RBP mainly is responsible for combining, transporting the vitamin A in the blood plasma.Ectogenic vitamin A at first combines with RBP after getting into blood circulation system, and further (transthyretin TTR) forms ternary complex to retinol-RBP complex and quilt is transported with transthyretin.
Liver is that vitamin A stores and RBP is synthetic and the main place of secretion.The secretion strictness of RBP receives the influence of body retinol content.About discovering of RBP mutant and knock out mice; The disappearance of RBP in the blood is except causing visual function disorder, to the not obviously influence of other aspect functions of body; Therefore, RBP possibly just be that the metabolic stability of body retinol provides a kind of guarantee when the meals VAD.
Relation with disease: RBP a kind ofly can freely pass through glomerulus, the very high small molecular weight protein of heavy absorptivity, and content is very low in the healthy human urine.Tubular injury just appears in Diabetic Nephropathy patients in early days, and urine RBP is a sensitive indicators of diagnosis early diabetic nephropathy; RBP gathers in a large number in chronic renal failure (CRF) the patient blood.
RBP is synthetic by liver cell, and when hepatocellular injury, RBP is synthetic to be suppressed, so detect blood RBP level, can reflect the change of liver function sensitively.Put the method for exempting from and detect discovery; The serum RBP level of cirrhosis and acute and chronic hepatitis all is starkly lower than the normal control group; The nutritional status of acute viral hepatitis early stage blood serum RBP content suppression ratio more obvious RBP in late period and body: RBP not only participates in the running of retinol; The also strict influence that receives the body vitamin A content of its secretion, a large amount of oral retinols can cause blood and liver RBP to descend.The shortage of vitamin A can change in the blood RBP content and suppress hepatic secretion RBP, and RBP can descend rapidly when body malnutrition or stress reaction.Because half life period of RBP is shorter relatively, changes higher early than prealbumin (PA), transferrins (TRF) etc. and sensitivity, so can be used as the index diagnostic method of judgement nutritional status stress reaction
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.
The Retinal-binding protein detection method, 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.At present, immunoturbidimetry is the most general method in common both at home and abroad.But the immunoturbidimetry principle combines to form turbidity based on the antibody antigen immune response, detects the concentration of RBP in the serum.Do not adopt the detection signal method and technology, its LDL can only reach 10mg/l.Exist simultaneously uncork stability bad, be subject to problems such as haemolysis interference.The auxiliary diagnosis that can only be used for renal function clinically.
[summary of the invention]
The present invention is in order to overcome the deficiency of prior art, and a kind of high sensitivity, good stability, accuracy is high, antijamming capability is strong Retinal-binding protein detection kit are provided.
To achieve these goals, the present invention has designed and has a kind ofly encapsulated the Retinal-binding protein detection kit based on latex particle, comprises reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 is the Tris-HCl buffer system, comprises Tris-HCl damping fluid 30-60mmol/l, and pH value is 7.2-7.6; Polymkeric substance 60-95mmol/l; Disodium ethylene diamine tetraacetate 6-13mmol/l;
Reagent R2 comprises that goat-anti human retinol-binding protein polyclonal antibody encapsulates polystyrene latex particle sensitization particle; Phosphate buffer 35-60mmol/l, pH value are 7.2-7.6;
The reference calibrations article comprise cow's serum matrix; Volumetric usage number percent according to cow's serum matrix also comprises antiseptic 0.2-2.2%; Stabilizing agent 1-10%.
Among the said reagent R2, the diameter of polystyrene latex particle is 45-92nm.
Among the said reagent R1, polymkeric substance is monoethylene glycol 6000-8000; Or be Macrogol 6000-8000, glucosan, polyoxyethylene polymer.
Said goat-anti human retinol-binding protein polyclonal antibody encapsulates the polystyrene latex particle and adopts physisorphtion; Concrete steps are: goat-anti human retinol-binding protein polyclonal antibody and polystyrene latex are mixed, and with 37 ℃ of absorption behind both mixings 8 hours, the antibody that does not connect was removed in dialysis afterwards; Add confining liquid; Sealed 2 hours, the centrifugal supernatant that goes, with the latex diluted to 1.2-2.5%.
Said goat-anti human retinol-binding protein polyclonal antibody compares 1/20-5/10 with the polystyrene latex mass particle.
Described confining liquid comprises skimmed milk power and glycocoll.
Said latex dilution comprises, 35-60mmol/l, pH value are the 7.2-7.6 phosphate buffer; The 0.01%-0.1% skimmed milk power, 0.9% NaN3.
The present invention is with showing compared with techniques; Adopt polystyrene latex to encapsulate the RBP ELISA polyclonal antibody; With the RBP ELISA generation association reaction in the sample to be measured (serum or blood plasma); Form immune complex, detect this variation, draw the concentration of target detection thing RBP ELISA in the sample with the calibration curve of RBP ELISA standard items with the light intensity in transmission.The present invention adopts the Nano microsphere signal amplification technique, makes the inspection of reagent LDL reach 1mg/l, and anti-haemoglobin disturbs and reached 500mg/dl, and uncork stability has reached 1 month.Clinically, not only can be used for the auxiliary diagnosis of renal function disease, also can be used for underfed auxiliary diagnosis simultaneously.Has very high clinical value.
[description of drawings]
Fig. 1 is the typical curve of the RBP ELISA normative reference of 5 kinds of different contents in the reagent of the present invention.
Fig. 2 is the typical curve of the RBP ELISA normative reference of 5 kinds of different contents in the common immunoturbidimetry reagent.
Fig. 3 is common immunoturbidimetry reagent correlativity curve map for reagent of the present invention contrasts.
[embodiment]
Below in conjunction with accompanying drawing the present invention is further described.
Embodiment 1:
Reagent R1:
Above-mentioned various compositions are at room temperature added successively, perhaps add simultaneously, or respectively separately packing and with detect before in instant preparation.
Reagent R2 comprises that goat-anti human retinol-binding protein polyclonal antibody encapsulates polystyrene latex particle sensitization particle; Phosphate buffer 35-60mmol/l, pH value are 7.2-7.6; Its preparation method is following:
Take by weighing the polystyrene latex particles that diameter is 45nm; Be 45mmol/l through over-richness; PH value is after 7.4 the Tris-Hcl buffer solution for cleaning; Mix with the mass ratio of goat-anti human retinol-binding protein polyclonal antibody with 5: 10, with 37 ℃ of absorption behind both mixings 8 hours, the antibody on connecting was removed not in dialysis afterwards.
Add confining liquid, sealed 2 hours, the principal ingredient of confining liquid is the glycocoll of 0.1% skimmed milk power and 0.2mm.
The centrifugal supernatant that goes, with latex diluted to 1.5%, the principal ingredient pH value of latex dilution is 7.4 phosphate buffer 45mmol/l, disodium ethylene diamine tetraacetate 10.2mmol/l, 0.05% skimmed milk power, 0.9%NaN3.
The preparation of reference calibrations article calibration object:
With treated cow's serum, add BAS 0.1%, NaN30.9% obtains the calibration object dilution.Be dissolved in the solution of the similar human serum matrix of preparation with the reorganization RBP ELISA, the standard items of preparation variable concentrations (0,5mg/l, 15mg/l, 30mg/l, 60mg/l, 120mg/l)
The Retinal-binding protein 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 120ul and 120ul, sample size 3ul.120ul reagent R1 adds the 3ul sample and behind 37C5min, adds 120ulR2, promptly begins to read a little, reads another point behind the reaction 5min; Detect wavelength predominant wavelength 570nm commplementary wave length 800nm respectively.
Table 1:
Adopt this reagent and said determination method; Contrast common immunoturbidimetry reagent; The curve of the RBP ELISA standard items (self-control) of 5 kinds of different contents that employing Hitachi 7170 Biochemical Analyzers record; As illustrated in fig. 1 and 2, each point is represented the normative reference article of a content, and wherein the X axle is represented RBP ELISA content (mg/L); The Y axle is represented absorbance, and concrete parameter is referring to table 2 and table 3.
Table 2:
Table 3:
Experiment one: the correlation test of detectable;
Use this law invention reagent to contrast common immunoturbidimetry reagent, adopt automatic 7170 automatic clinical chemistry analyzers that 50 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 " RBP ELISA assay method " in parameter measure, measure the result and see Fig. 3, X, Y axle are measured value (the content mg/L of RBP ELISA).
Result by Fig. 3 finds out that the facies relationship of two kinds of reagent is R2=0.9819, and regression equation is y=1.0758x.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 to adopt 7170 full automatic biochemical apparatus of Hitachi, Ltd to 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.
Experiment two: LDL test;
Experiment purpose is that the minimum of detectable when the test clinical sample detects sensitivity.
Adopt reagent, contrast agents, reference calibrations article, blank solution (generally being normal saline solution and Purified Water), normal human serum sample among the embodiment 1, 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 each test sample 5 times together then, 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 4 result shows 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 0.8mg/l at least.
Table 4:
And contrast common immunoturbidimetry reagent; As shown in table 5; Measure dilution 1/16,1/8,1/4,1/2 serum, and relatively serum mean value-2SD and mean value+2SD size at zero point, wherein 1/16,1/8,1/4 dilute mean value-2SD numerical value less than mean value+2SD at zero point; The numerical value of 1/2 dilution mean value-2SD shows that greater than mean value+2SD at zero point common immunoturbidimetry reagent lowest detection is limited to about 12mg/l.
Table 5:
Experiment 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 among the embodiment 1, the absorbance changing value when contrast agents, reference calibrations article, blank solution, 0.9% normal saline solution, 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.
Show that like table 6 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.
Table 6:
And common immunoturbidimetry reagent; Referring to table 7; Absorbance is changed to-21.75 (10000A) when measuring physiological saline, and 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 common immunoturbidimetry reagent.
Table 7:
Experiment four: detectable antijamming capability test;
The present invention has carried out the anti-interference test simultaneously to detectable and contrast agents, and the result is as shown in table 8 below, add chaff interference after, to the relative error of various chaff interferences all in 10%.Contrast agents all surpasses 10% to the relative error of various chaff interferences.
Table 8:
Claims (7)
1. one kind encapsulates the Retinal-binding protein detection kit based on latex particle, comprises reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 is the Tris-HCl buffer system, comprises Tris-HCl damping fluid 30-60mmol/l, and pH value is 7.2-7.6; Polymkeric substance 60-95mmol/l; Disodium ethylene diamine tetraacetate 6-13mmol/l;
Reagent R2 comprises that goat-anti human retinol-binding protein polyclonal antibody encapsulates polystyrene latex particle sensitization particle; Phosphate buffer 35-60mmol/l, pH value are 7.2-7.6;
The reference calibrations article comprise cow's serum matrix; Volumetric usage number percent according to cow's serum matrix also comprises antiseptic 0.2-2.2%; Stabilizing agent 1-10%.
2. according to claim 1ly encapsulate the Retinal-binding protein detection kit based on latex particle, it is characterized in that: among the said reagent R2, the diameter of polystyrene latex particle is 45-92nm.
3. according to claim 1ly encapsulate the Retinal-binding protein detection kit based on latex particle, it is characterized in that: among the said reagent R1, polymkeric substance is monoethylene glycol 6000-8000; Or be Macrogol 6000-8000, glucosan, polyoxyethylene polymer.
4. according to claim 1ly encapsulate the Retinal-binding protein detection kit based on latex particle; It is characterized in that: said goat-anti human retinol-binding protein polyclonal antibody encapsulates the polystyrene latex particle and adopts physisorphtion; Concrete steps are: goat-anti human retinol-binding protein polyclonal antibody and polystyrene latex are mixed, and with 37 ℃ of absorption behind both mixings 8 hours, the antibody that does not connect was removed in dialysis afterwards; Add confining liquid; Sealed 2 hours, the centrifugal supernatant that goes, with the latex diluted to 1.2-2.5%.
5. according to claim 4ly encapsulate the Retinal-binding protein detection kit based on latex particle, it is characterized in that: said goat-anti human retinol-binding protein polyclonal antibody compares 1/20-5/10 with the polystyrene latex mass particle.
6. according to claim 4ly encapsulate the Retinal-binding protein detection kit based on latex particle, it is characterized in that: described confining liquid comprises skimmed milk power and glycocoll.
7. according to claim 4ly encapsulate the Retinal-binding protein detection kit based on latex particle, it is characterized in that: said latex dilution comprises, 35-60mmol/l, pH value are the 7.2-7.6 phosphate buffer; The 0.01%-0.1% skimmed milk power, 0.9% NaN3.
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Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102841210A (en) * | 2012-08-30 | 2012-12-26 | 谢兵 | Retinol detection kit and preparation method thereof |
| 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 |
| CN103344763A (en) * | 2013-06-18 | 2013-10-09 | 华东师范大学 | Latex-reinforced AFP detection kit and its preparation method and use |
| CN105486875A (en) * | 2016-01-26 | 2016-04-13 | 宁波天康生物科技有限公司 | Retinol conjugated protein detection kit |
| CN105891501A (en) * | 2015-03-31 | 2016-08-24 | 北京科美生物技术有限公司 | Colloidal gold immunocolorimetry kit for detecting retinol conjugated protein (RBP) and preparation method of kit |
| CN106093423A (en) * | 2016-05-31 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring retinol binding protein and preparation method thereof |
| CN106383234A (en) * | 2016-08-31 | 2017-02-08 | 上海科华生物工程股份有限公司 | Coating method for retinol-binding protein detection reagent |
| CN106814191A (en) * | 2015-11-30 | 2017-06-09 | 山东博科生物产业有限公司 | A kind of RBP ELISA immunoturbidimetry detection kit |
| CN106932589A (en) * | 2015-12-30 | 2017-07-07 | 上海复星长征医学科学有限公司 | Determine kit of human serum RBP ELISA content and preparation method thereof |
| CN107490696A (en) * | 2017-08-10 | 2017-12-19 | 迈克生物股份有限公司 | A kind of Retinal-binding protein detection kit and detection method |
| CN107656065A (en) * | 2017-03-31 | 2018-02-02 | 迈克生物股份有限公司 | Suppress the RBP ELISA latex enhancing immune of rheumatoid factor interference than turbid reagent |
| CN108627652A (en) * | 2018-05-31 | 2018-10-09 | 宁波海壹生物科技有限公司 | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen |
| CN112730848A (en) * | 2020-12-29 | 2021-04-30 | 中生北控生物科技股份有限公司 | Kit and method for detecting retinol binding protein |
| CN116359489A (en) * | 2022-11-30 | 2023-06-30 | 广州杰博生物科技有限公司 | Immunodetection kit and preparation method and application thereof |
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Cited By (16)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102841210A (en) * | 2012-08-30 | 2012-12-26 | 谢兵 | Retinol detection kit and preparation method thereof |
| 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 |
| CN103344763A (en) * | 2013-06-18 | 2013-10-09 | 华东师范大学 | Latex-reinforced AFP detection kit and its preparation method and use |
| CN105891501A (en) * | 2015-03-31 | 2016-08-24 | 北京科美生物技术有限公司 | Colloidal gold immunocolorimetry kit for detecting retinol conjugated protein (RBP) and preparation method of kit |
| CN106814191A (en) * | 2015-11-30 | 2017-06-09 | 山东博科生物产业有限公司 | A kind of RBP ELISA immunoturbidimetry detection kit |
| CN106932589A (en) * | 2015-12-30 | 2017-07-07 | 上海复星长征医学科学有限公司 | Determine kit of human serum RBP ELISA content and preparation method thereof |
| CN105486875A (en) * | 2016-01-26 | 2016-04-13 | 宁波天康生物科技有限公司 | Retinol conjugated protein detection kit |
| CN106093423A (en) * | 2016-05-31 | 2016-11-09 | 安徽伊普诺康生物技术股份有限公司 | A kind of test kit measuring retinol binding protein and preparation method thereof |
| CN106383234A (en) * | 2016-08-31 | 2017-02-08 | 上海科华生物工程股份有限公司 | Coating method for retinol-binding protein detection reagent |
| CN106383234B (en) * | 2016-08-31 | 2017-11-24 | 上海科华生物工程股份有限公司 | The method for coating of Retinal-binding protein detection reagent |
| CN107656065A (en) * | 2017-03-31 | 2018-02-02 | 迈克生物股份有限公司 | Suppress the RBP ELISA latex enhancing immune of rheumatoid factor interference than turbid reagent |
| CN107656065B (en) * | 2017-03-31 | 2020-06-12 | 迈克生物股份有限公司 | Retinol binding protein latex enhanced immunoturbidimetry reagent for inhibiting rheumatoid factor interference |
| CN107490696A (en) * | 2017-08-10 | 2017-12-19 | 迈克生物股份有限公司 | A kind of Retinal-binding protein detection kit and detection method |
| CN108627652A (en) * | 2018-05-31 | 2018-10-09 | 宁波海壹生物科技有限公司 | It is a kind of to detect based on simple grain diameter and simultaneously the kit of RBP ELISA in serum and urine specimen |
| CN112730848A (en) * | 2020-12-29 | 2021-04-30 | 中生北控生物科技股份有限公司 | Kit and method for detecting retinol binding protein |
| CN116359489A (en) * | 2022-11-30 | 2023-06-30 | 广州杰博生物科技有限公司 | Immunodetection kit and preparation method and application thereof |
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