CN102621332B - 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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- CN102621332B CN102621332B CN201210099532.6A CN201210099532A CN102621332B CN 102621332 B CN102621332 B CN 102621332B CN 201210099532 A CN201210099532 A CN 201210099532A CN 102621332 B CN102621332 B CN 102621332B
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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 of based on the coated Retinal-binding protein detection kit of latex particle.
[background technology]
RBP ELISA belongs to Lipocalin superfamily, relative molecular mass approximately 21 * 10
3, part is mainly alltrans retinol, i.e. vitamin A, Kd=20Nm, binding site 1.RBP is mainly responsible for the vitamin A in combination, transporting blood plasma.Ectogenic vitamin A enters after blood circulation system, is first combined with RBP, and retinol-RBP complex further forms ternary complex with transthyretin (transthyretin, TTR) and transported.
Liver is that vitamin A stores and the synthetic main place with secreting of RBP.The secretion of RBP is strictly subject to the impact of body retinol content.About the research of RBP mutant and knock out mice is found, the disappearance of RBP in blood, except causing visual function disorder, does not have a significant effect to other aspect functions of body, therefore, RBP may be just when Dietary Vitamin A lacks the metabolic stability for body retinol a kind of guarantee is provided.
Relation with disease: RBP a kind ofly can freely pass through glomerulus, the very high small molecular weight protein of heavy absorptivity, and in healthy human urine, content is very low.Just there is tubular injury in Diabetic Nephropathy patients, urine RBP is a more sensitive index of diagnosis early diabetic nephropathy in early days; In chronic renal failure (CRF) patient blood, RBP gathers in a large number.
RBP is synthesized by liver cell, and when hepatocellular injury, RBP is synthetic suppressed, therefore detect blood RBP level, can reflect sensitively the change of liver function.Put the method for exempting from and detect discovery, the serum RBP level of cirrhosis and acute hepatitis, chronic hepatitis is all starkly lower than Normal 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, its secretion is also strictly subject to the impact of body vitamin A content, a large amount of oral retinols can cause blood and liver RBP to decline.The shortage of vitamin A can change in blood RBP content and suppress hepatic secretion RBP, and when body malnutrition or stress reaction, RBP can decline rapidly.Because the half life period of RBP is relatively short, change higher early than prealbumin (PA), transferrins (TRF) etc. and sensitivity, therefore can be used as the index diagnostic method of judgement nutritional status stress reaction
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 (particle-enhanced turbidimetric immunoassay, PETIA).PETIA method is divided into two kinds substantially.A kind of is 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 is all the surface-crosslinked monoclonal antibody at polymer latex microballoon, after the crosslinked microballoon that has antibody is combined with antigen, can flock together rapidly at short notice, changed astigmatic performance or the light transmission of reactant liquor.And reactant liquor astigmatism performance or the change of light transmission (being absorbance) and the concentration of tested antigen have stronger correlativity, can reflect within the specific limits the concentration of tested antigen.PETIA detection method is in homogeneous reaction system, to carry out the mensuration of antigen, antibody response and result.After antigen, antibody response, directly the absorbance of assaying reaction liquid, has save ELISA method and has repeatedly hatched and washed the loaded down with trivial details operation stepss such as plate, and a few minutes just can obtain result, time saving and energy saving.In addition, the interference of the extraneous factors such as many manual operation factors and 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 more truly the content of measured matter.Although the sensitivity of immunoturbidimetry is more weaker than ELISA method, the lower limit of foot many marker proteins in human normal plasma being detected, can meet clinical detection requirement completely.
Retinal-binding protein detection method, by the qualitative detection of initial immunoelectrophoresis, to radio-immunity, fluorescence immunoassay and immunoenzymatic quantitative detection, and detects to the robotization of immunoturbidimetry, develops very rapid.At present, immunoturbidimetry is the most general general method both at home and abroad.But immunoturbidimetry principle in conjunction with forming turbidity, detects the concentration of RBP in serum based on antibody antigen immune response.Do not adopt detection signal method and technology, its lowest detectable limit can only reach 10mg/l.Exist simultaneously uncork stability bad, be subject to the problems such as haemolysis interference.Clinically can only be for the auxiliary diagnosis of renal function.
[summary of the invention]
The present invention is in order to overcome the deficiencies in the 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 a kind ofly based on the coated Retinal-binding protein detection kit of latex particle, has comprised reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 is 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 is coated with polystyrene latex particle sensitization particle; Phosphate buffer 35-60mmol/l, pH value is 7.2-7.6;
Reference calibrations product, 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%.
In described reagent R2, the diameter of polystyrene latex particle is 45-92nm.
In described reagent R1, polymkeric substance is ethylene glycol 6000-8000; Or be Macrogol 6000-8000, glucosan, polyoxyethylene polymer.
The coated polystyrene latex particle of described goat-anti human retinol-binding protein polyclonal antibody adopts physisorphtion, concrete steps are: goat-anti human retinol-binding protein polyclonal antibody and polystyrene latex are mixed, both are mixed to rear 37 ℃ of absorption 8 hours, the antibody not connecting is removed in dialysis afterwards, add confining liquid, seal 2 hours, the centrifugal supernatant that goes, uses latex diluted to 1.2-2.5%.
Described goat-anti human retinol-binding protein polyclonal antibody compares 1/20-5/10 with polystyrene latex mass particle.
Described confining liquid comprises skimmed milk power and glycocoll.
Described latex dilution comprises, 35-60mmol/l, and pH value is 7.2-7.6 phosphate buffer; 0.01%-0.1% skimmed milk power, 0.9% NaN3.
Co-occurrence technology of the present invention is compared, adopt the coated RBP ELISA polyclonal antibody of polystyrene latex, with the RBP ELISA generation association reaction in sample to be measured (serum or blood plasma), form immune complex, with light intensity in transmission, detect this variation, with the calibration curve of RBP ELISA standard items, draw the concentration of target detection thing RBP ELISA in sample.The present invention adopts Nano microsphere signal amplification technique, makes the inspection of reagent lowest detectable limit reach 1mg/l, and anti-haemoglobin disturbs and reached 500mg/dl, and uncork stability has reached 1 month.Clinically, not only can be for the auxiliary diagnosis of renal function disease, the while also can be for underfed auxiliary diagnosis.There is very high clinical value.
[accompanying drawing explanation]
Fig. 1 is the typical curve of the RBP ELISA normative reference of 5 kinds of different contents in reagent of the present invention.
Fig. 2 is the typical curve of the RBP ELISA normative reference of 5 kinds of different contents in common immunoturbidimetry reagent.
Fig. 3 is that reagent of the present invention contrasts common immunoturbidimetry reagent correlativity curve map.
[embodiment]
Below in conjunction with accompanying drawing, the invention will be further described.
Embodiment 1:
Reagent R1:
Above-mentioned various compositions are at room temperature added successively, or 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 is coated with polystyrene latex particle sensitization particle; Phosphate buffer 35-60mmol/l, pH value is 7.2-7.6; Its preparation method is as follows:
Take the polystyrene latex particles that diameter is 45nm, through over-richness, be 45mmol/l, pH value is after 7.4 Tris-Hcl buffer solution for cleaning, mix with the mass ratio of goat-anti human retinol-binding protein polyclonal antibody with 5: 10, both are mixed to rear 37 ℃ of absorption 8 hours, and the antibody not connecting is removed in dialysis afterwards.
Add confining liquid, seal 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, by 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 product calibration object:
By treated cow's serum, add BAS 0.1%, NaN30.9% obtains calibration object dilution.With restructuring RBP ELISA, be dissolved in the solution of the similar human serum matrix of preparation, prepare the standard items (0,5mg/l, 15mg/l, 30mg/l, 60mg/l, 120mg/l) of variable concentrations
The Retinal-binding protein detection kit that the present embodiment is described, is applicable to various types of full automatic biochemical apparatus, and Hitachi's 7170 full automatic biochemical apparatus of take are example, and it operates as table 1.Analytical approach: Two point end assay, i.e. reagent R1, the consumption of R2 is respectively 120ul and 120ul, sample size 3ul.120ul reagent R1 adds 3ul sample after 37C5min, to add 120ulR2, starts read point, after reaction 5min, reads another point; 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 represents the normative reference product of a content, and wherein X-axis represents RBP ELISA content (mg/L); Y-axis represents absorbance, and design parameter is referring to table 2 and table 3.
Table 2:
Table 3:
Experiment one: the correlation test that detects reagent;
Use this law invention reagent to contrast common immunoturbidimetry reagent, adopt automatic 7170 automatic clinical chemistry analyzers to measure by each autoregressive parameter 50 parts of human serums (comprising normal and monstrosity) simultaneously, measured value is carried out to correlation analysis.According to above-mentioned " RBP ELISA assay method " in parameter measure, measurement result is shown in Fig. 3, X, Y-axis is measured value (the content mg/L of RBP ELISA).
Result by Fig. 3 finds out, the relevant of two kinds of reagent is R2=0.9819, and regression equation is y=1.0758x.It is good that result shows that this reagent and import reagent are measured patients serum's correlativity, has good specificity and accuracy.
In addition, above 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, is also applicable to other full-automatic or semi-automatic biochemical analyzers.
Experiment two: lowest detectable limit test;
Experiment purpose is to detect the minimum check-up inducing degree of reagent when test clinical sample.
Adopt reagent, contrast agents, reference calibrations product, blank solution (being generally normal saline solution and Purified Water), normal human serum sample in 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, then 50% be diluted to 5 points, each test sample is 5 times together with zero point, and calculating mean value, tries to achieve SD numerical value.
Result is resolved: according to detecting data, calculates SD numerical value and CV numerical value, calculates respectively 1SD, and 2SD, from minimum, the numerical value of its mean value-2SD is exactly the minimum check-up inducing degree of reagent more than zero point mean value+2SD.
The demonstration of table 4 result, when reagent of the present invention is measured dilution 1/16,1/8,1/4,1/2 serum, the numerical value of measuring mean value-2SD is all greater than mean value+2SD at zero point, shows that reagent lowest detectable limit of the present invention at least can reach 0.8mg/l.
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 with zero point mean value+2SD big or small, wherein the numerical value of 1/16,1/8,1/4 dilution mean value-2SD is less than mean value+2SD at zero point, the numerical value of 1/2 dilution mean value-2SD is greater than mean value+2SD at zero point, shows that common immunoturbidimetry reagent lowest detection is limited to 12mg/l left and right.
Table 5:
Experiment three: sensitivity experiment;
This experiment purpose is to detect the absorbance changing value of reagent when test physiological saline and certain density management serum.
Adopt the reagent in embodiment 1, contrast agents, reference calibrations product, blank solution, 0.9% normal saline solution, the absorbance changing value during 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.
As shown in Table 6, when reagent of the present invention is measured physiological saline, absorbance is changed to-2.8 (1/10000A), and when theoretical concentration is 0.35mg/l serum sample, absorbance is changed to 323.6 (1/10000A).
Table 6:
And common immunoturbidimetry reagent, referring to table 7, while measuring physiological saline, absorbance is changed to-21.75 (10000A), when theoretical concentration is 0.35mg/l serum sample, absorbance is changed to 31.5 (1/10000A), shows that reagent sensitivity of the present invention will be significantly higher than common immunoturbidimetry reagent.
Table 7:
Experiment four: detect the test of reagent antijamming capability;
The present invention has carried out anti-interference test to detecting reagent and contrast agents simultaneously, and result is as shown in table 8 below, add after chaff interference, 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. based on the coated Retinal-binding protein detection kit of latex particle, it is characterized in that, comprise reagent R1, reagent R2 and calibration object, wherein:
Reagent R1 is 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 is coated with polystyrene latex particle sensitization particle; Phosphate buffer 35-60mmol/l, pH value is 7.2-7.6; Wherein, the coated polystyrene latex particle of described goat-anti human retinol-binding protein polyclonal antibody adopts physisorphtion, concrete steps are: goat-anti human retinol-binding protein polyclonal antibody and polystyrene latex are mixed, both are mixed to rear 37 ℃ of absorption 8 hours, the antibody not connecting is removed in dialysis afterwards, adds confining liquid, seals 2 hours, the centrifugal supernatant that goes, uses latex diluted to 1.2-2.5%;
Calibration object, comprises cow's serum matrix; According to the volumetric usage number percent of cow's serum matrix, also comprise antiseptic 0.2-2.2% and stabilizing agent 1-10%;
And described kit is the kit of measuring the RBP ELISA in serum or blood plasma.
2. according to claim 1 based on the coated Retinal-binding protein detection kit of latex particle, it is characterized in that: in described reagent R2, the diameter of polystyrene latex particle is 45-92nm.
3. according to claim 1 based on the coated Retinal-binding protein detection kit of latex particle, it is characterized in that: in described reagent R1, polymkeric substance is Macrogol 6000-8000, glucosan, or polyoxyethylene polymer.
4. according to claim 1 based on the coated Retinal-binding protein detection kit of latex particle, it is characterized in that: the composition of described latex dilution is: pH value is 7.4 phosphate buffer 45mmol/l, disodium ethylene diamine tetraacetate 10.2mmol/l, 0.05% skimmed milk power, 0.9%NaN
3.
5. according to claim 4 based on the coated Retinal-binding protein detection kit of latex particle, it is characterized in that: described goat-anti human retinol-binding protein polyclonal antibody compares 1/20-5/10 with polystyrene latex mass particle.
6. according to claim 4 based on the coated Retinal-binding protein detection kit of latex particle, it is characterized in that: described confining liquid comprises skimmed milk power and glycocoll.
7. according to claim 1ly based on the coated Retinal-binding protein detection kit of latex particle, it is characterized in that: described latex dilution comprises, 35-60mmol/l, pH value is 7.2-7.6 phosphate buffer; 0.01%-0.1% skimmed milk power, 0.9% NaN
3.
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| CN102841210A (en) * | 2012-08-30 | 2012-12-26 | 谢兵 | Retinol detection kit and preparation method thereof |
| CN103134934B (en) * | 2013-02-27 | 2014-11-12 | 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 |
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| 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 |
| 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 |
| CN106950363A (en) * | 2017-03-31 | 2017-07-14 | 四川迈克生物科技股份有限公司 | Suppress the latex enhancing immune of rheumatoid factor interference than turbid reagent |
| 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 |
| CN108627652B (en) * | 2018-05-31 | 2019-05-03 | 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 |
| CN112730848B (en) * | 2020-12-29 | 2022-03-22 | 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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