CN111239404B - Detection kit capable of simultaneously detecting retinol binding protein in urine sample and serum sample - Google Patents
Detection kit capable of simultaneously detecting retinol binding protein in urine sample and serum sample Download PDFInfo
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- CN111239404B CN111239404B CN201910254700.6A CN201910254700A CN111239404B CN 111239404 B CN111239404 B CN 111239404B CN 201910254700 A CN201910254700 A CN 201910254700A CN 111239404 B CN111239404 B CN 111239404B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
Abstract
A detection kit capable of simultaneously detecting retinol binding protein in a urine sample and a serum sample, which comprises the following components: the kit comprises a reagent R1, a reagent R2 and a retinol binding protein standard, wherein the reagent R1 is a buffer solution containing a sample dilution function, the reagent R2 is a buffer solution containing a latex antibody conjugate, the latex antibody conjugate comprises a large-particle-size latex marked with anti-retinol binding protein antibody white transparent latex and a small-particle-size latex containing black and white latexes, the large-particle-size latex adopts anti-retinol binding protein polyclonal antibody, and the small-particle-size latex adopts a mixture of anti-retinol binding protein monoclonal antibodies for coupling.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to a detection kit for simultaneously detecting retinol binding protein in a urine sample and a serum sample.
Background
Retinol Binding Protein (RBP) is a small lipocalin (molecular weight 21000 daltons) synthesized in the liver. Retinol binding protein, when synthesized in the liver, transports all-trans retinol (vitamin a) to epithelial tissues, providing vitamin a to the retina. Research shows that RBP exists not only in retina, but also in human blood, urine and other body fluids, and the content of RBP is closely related to the health condition of the kidney and the viscera. Currently, serum RBP detection is an early indicator for the assessment of liver function impairment and glomerular filtration dysfunction, while urine RBP detection is an early marker for the assessment of proximal tubular dysfunction. At present, enzyme-linked immunosorbent assay (ELISA), immunoturbidimetric assay, radioimmunoassay (IRMA), immunoelectrophoresis and the like are used as detection methods of retinol binding protein. The immunoturbidimetry utilizes a full-automatic biochemical analyzer for detection, has no radioactive source, is relatively environment-friendly, has good stability, precision and accuracy, and is widely applied to clinical routine detection.
According to the results of the investigation of the use of the RBP kit, it can be found that a considerable part of laboratories are (or will be) detecting RBPs, and that sRBP is the main factor. Then, while the pass rate of sRBP is high in the survey, it does not mean that the quality of the index test has satisfied the clinical requirement, because the evaluation criterion of + -30% of the target value is relatively loose. In addition, there are cases where the difference in results between different laboratories is more than 3-fold for the same reagent method. The quality control product for investigation is derived from human serum, contains various proteins such as transthyretin and the like besides sRBP, and the difference of detection results among laboratories is probably related to the performance of a reagent method, such as specificity, anti-interference capability and the like. The results of the examination of uRBP are not satisfactory, and the overall passing rate is not 25% even when evaluated as. + -. 30% of the target value. Like sRBP, the results of the same reagent in different laboratories are greatly different and more prominent, and the CV reaches 123.8%. The uRBP quality control product is derived from urine, albumin with different concentrations is added besides the uRBP4, and reagent manufacturers and laboratories consider whether the problems of reagent method specificity, linearity and albumin interference exist. Reflecting some problems of RBP4 detection, the commercially available sRBP4 and uRBP4 detection kits need to be subjected to methodology research, and the quality of detection needs to be improved.
Disclosure of Invention
The present invention is provided to solve the above problems.
The invention is realized by the following technical scheme: a detection kit capable of simultaneously detecting retinol binding protein in a urine sample and a serum sample, which comprises the following components: the kit comprises a reagent R1, a reagent R2 and a retinol binding protein standard, wherein the reagent R1 is a buffer solution containing sample dilution, and the reagent R2 is a buffer solution containing a latex antibody conjugate. The latex antibody conjugate comprises large-particle-size latex marked with anti-human retinol conjugated protein antibody white transparent latex and mixed small-particle-size latex with black and white colors, wherein the large-particle-size latex adopts anti-human retinol conjugated protein polyclonal antibody, and the small-particle-size latex adopts a mixture of two matched anti-human retinol conjugated protein monoclonal antibodies for marking.
The small-particle-size latex contains white latex and black latex with the number ratio of particles (20-5): 1; the small-particle-size latex is polystyrene latex with the particle size of 50-280nm, and the mass fraction of the small-particle-size latex in the reagent R2 is 0.05-0.15%; the large-particle-size latex is white transparent polystyrene microspheres with the particle size of 500-900nm, and the mass fraction of the large-particle-size latex in the reagent R2 is 0.15-0.2%; the small-particle-size latex contains white latex and black latex with the number ratio of particles of 10: 1; the small-particle-size latex is preferably polystyrene latex with the particle size of 100-180 nm; the large-particle-size latex is polystyrene latex with the particle size of 550-750nm being preferred.
The components of the kit are preferably: the reagent R1 comprises the following components: 1% of NaCl, 7% of PEG-2000, 0.05% of egg white protein, 2% of propylene glycol alginate and 0.5M Tris-HCl buffer solution with the pH value of 6.5; the reagent R2 component was 3% (w/v) of antibody coated latex particles suspended in reagent R1, where the latex particles were as previously shown. Black latex particles were purchased from shanghai jukebox co. Other latexes are available from fasttime (shanghai) commercial limited.
Drawings
FIG. 1 comparison of import kit and kit of the invention
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and embodiments. The following examples are only exemplary and are intended to illustrate the technical solutions of the present invention in further detail, and it should be understood by those skilled in the art that modifications or substitutions to the technical solutions without departing from the spirit and scope of the technical solutions of the present invention should be covered by the claims of the present invention.
Example 1
The preparation method of the RBP kit comprises the following steps:
1. small particle size latex conjugated antibody:
(1) activation of the latex: weighing 0.05g of black latex with the particle size of 150nm, weighing 0.5g of white latex with the same particle size, mixing two polystyrene latex microspheres, adding the mixture into 0.1M Tris-HCl buffer solution with the pH value of 7.4 and an activating agent (EDC/NHS) into the buffer solution, uniformly mixing at room temperature, carrying out oscillation reaction for a period of time, centrifuging, washing and coating a precipitate with PBS (phosphate buffer solution), carrying out ultrasonic treatment to obtain activated polystyrene microspheres, collecting supernatant, and measuring the concentration of unreacted antibody protein in the supernatant by using an ultraviolet absorption method.
(2) Coupling of the antibody: and (3) taking the activated microsphere solution and RBP antibody with a certain concentration to perform oscillation reaction in a buffer solution for several hours, centrifuging, collecting supernatant, and washing the precipitate with PBS for multiple times of collateral washing, oscillation and ultrasonic treatment to obtain the coupled immune microsphere.
(3) And (3) sealing:
a) weighing 0.1g of glycine, dissolving in 8mL of PBS, adding immune microspheres, mixing, fixing the volume to 18mL, ultrasonically mixing uniformly, preserving the glycine content in the solution to be 3.3% (W/V), and stirring at a low speed of 300rpm at room temperature for 1 hour.
b) Weighing 2mL PBS buffer mask, weighing 0.4g BSA, mixing well, adding into the solution, stirring at low speed of 300rpm for 1 h. After the separation, Tween-PBS buffer solution is added into the sediment for ultrasonic cleaning twice, and the supernatant is removed.
C) Adding BSA-Tween-PBS buffer solution into the centrifuged precipitate, and performing ultrasonic dispersion uniformly to obtain the immune latex.
2. Antibody coupling of large particle size latex:
the procedure is coupled with small particle size latex microspheres, except that step 1 is activated with 1.5g by weight of 550nm large particle size latex.
Selecting an activating agent of EDC/NHS, an activating time of 30min, a small-particle-size latex mixture, an antibody protein ratio of 10:1 and a large-particle-size latex: the ratio of antibody protein is 5:1, the particle size of the polystyrene microsphere is 100nm, the reaction system is MES buffer solution with pH6.1, the reaction time is 2h, and the concentration of PEG6000 is 2%, the latex microsphere antibody protein coupling is most suitable.
The RBP detection kit can be obtained by configuring R1 and R2 reagents according to the following components of the kit:
the detection reagent comprises the following components:
the reagent R1 comprises the following components: 1% of NaCl, 7% of PEG-2000, 0.05% of egg white protein, 2% of propylene glycol alginate and 0.5M Tris-HCl buffer solution with the pH value of 6.5; reagent R2 component was antibody coated latex particle 3% (w/v) suspended by reagent R1, where the polyclonal antibody in the latex particle was coupled to 550nm large particle size latex: the ratio of the double monoclonal antibody mixed coupled 150nm small-particle size latex to the antibody coupled black latex is 5: 3: the white latex ratio was 1: 10.
Comparative example 1
The detection reagent comprises the following components:
the reagent R1 comprises the following components: 1% of NaCl, 7% of PEG-2000, 0.05% of egg white protein, 2% of propylene glycol alginate and 0.5M Tris-HCl buffer solution with the pH value of 6.5; reagent R2 component was antibody coated latex particle 3% (w/v) suspended by reagent R1, where the polyclonal antibody in the latex particle was coupled to 550nm large particle size latex: the ratio of the latex with the small particle size of 150nm in the mixed coupling of the double monoclonal antibodies is 5: 3.
The RBP kit is used by taking Hitachi 7180 biochemical analyzer as an example, measuring wavelength of 570nm, firstly adding R1 reagent 150 μ L, reacting at 37 ℃ for 30s, then adding standard substance 20 μ L, reacting for 1min, then adding R2 reagent 75 μ L, measuring absorbance value of reaction (A1, A2), and calculating delta A-A2-A1. And (3) repeatedly measuring each tube for 2 times, taking the absorbance value difference delta A measured by each tube as a vertical coordinate, taking the corresponding concentration as a horizontal coordinate, making a concentration-absorbance value standard curve, and measuring the absorbance value of the sample by the same method to obtain the concentration of the retinol binding protein in the sample.
Experimental example 2
Performance test of test sample with kit
1. Sensitivity of the probe
RBP standard was diluted to 6 low concentration samples of 0.2, 0.4, 0.6, 0.8, 1.0, 1.5mg/L accurately according to the judgment criteria of detection limit specified in JJCLA (Japan clinical laboratory Automation society) document, and the measurement was repeated 10 times under the same conditions with the same reagent. For counting the matrix liquid
Of values and results of measurements
Value of wherein
Greater than the matrix liquid
The lowest value of (d) is the detection limit. The detection limit of the detection reagent is 0.6 mg/L.
2. Linear range
According to the requirements of EP6, which is a standard document issued by CLSI, a serum sample of about 150mg/L is diluted with physiological saline by 0, 1/5, 2/5, 3/5, 4/5 and 5/5 (primary times), each dilution is used as a sample, and the measurement is carried out by the standard experimental operation steps, wherein the average value (yi) of the detection result of each dilution is respectively obtained by 3 times of each dilution. The diluted concentration (xi) was an independent variable, and the average value (yi) of the measurement results was a dependent variable, to obtain a linear regression equation. The correlation coefficient (r) of the linear regression is calculated, and the relative or absolute deviation. The results show that there is a good linear relationship between the two, and it is believed that the RBP reagent of the present invention is linear well in the range of 2-150 mg/L.
In the same manner, a 10mg/L urine specimen was diluted with physiological saline at 0, 1/5, 2/5, 3/5, 4/5 and 5/5 (original fold), and each dilution was used as a specimen, and the deviation was measured, and the following data were obtained:
it can be seen that the kit of the present invention is significantly superior to the kit prepared in comparative example 1 in terms of the deviation of linearity.
2. Correlation analysis
The RBP detection kit of the present invention and the RBP detection kit of Nippon Japan K.K. were used to carry out clinical specimen detection, and the results of the detection of both were analyzed by comparison, the result of the detection by the reagent of Nippon Japan K.K. was X-axis, and the result of the detection by the reagent of the present invention was Y-axis, and a scattergram was drawn, as shown in FIG. 1. The results show that the correlation between the two detection reagents is high, and after linear regression statistical analysis, the equation is obtained as Y-0.96943X +0.25843, r-0.987, wherein k-0.96943, b-0.25843, k is close to 1, b is close to 0, and r > 0.95, and the two detection reagents are considered to be highly correlated (p < 0.01).
Claims (9)
1. A detection kit capable of simultaneously detecting retinol binding protein in a urine sample and a serum sample, which comprises the following components: reagent R1, reagent R2 and retinol binding protein standard substance, reagent R1 is the buffer solution containing sample dilution, reagent R2 is the buffer solution containing latex antibody conjugate, its characteristic is: the latex antibody conjugate comprises white transparent large-particle-size latex marked with an anti-human retinol binding protein antibody and mixed small-particle-size latex with black and white colors, wherein the large-particle-size latex is marked by an anti-human retinol binding protein polyclonal antibody, and the small-particle-size latex is marked by a mixture of two anti-human retinol binding protein monoclonal antibodies.
2. The kit according to claim 1, wherein the kit is for simultaneously detecting retinol binding protein in a urine sample and a serum sample, and comprises: the polyclonal antibody against human retinol binding protein is an intact mammalian or avian antibody or an active antibody fragment or a recombinant antibody.
3. The kit according to claim 2, wherein the kit is for simultaneously detecting retinol binding protein in a urine sample and a serum sample, and comprises: the polyclonal anti-human retinol binding protein antibody is a humanized antibody.
4. The kit of claim 1, wherein: the small-particle-size latex contains a ratio of the number of white latex particles to the number of black latex particles of (20-5): 1.
5. The kit of claim 1, wherein: the small-particle-size latex is polystyrene latex with the particle size of 50-280nm, and the mass fraction of the small-particle-size latex in the reagent R2 is 0.05-0.15%.
6. The kit of claim 1, wherein: the large-particle-size latex is white transparent polystyrene microspheres with the particle size of 500-900nm, and the mass fraction of the large-particle-size latex in the reagent R2 is 0.15-0.2%.
7. The kit of claim 4, wherein: the small particle size latex contained a ratio of white latex to black latex particles of 10: 1.
8. The kit of claim 1, wherein: the small-particle-size latex is polystyrene latex with the particle size of 100-180 nm; the large-particle-size latex is polystyrene latex with the particle size of 550-750 nm.
9. A kit according to claim 1, wherein: the reagent R1 comprises the following components: 1% of NaCl, 7% of PEG-2000, 0.05% of egg white protein, 2% of propylene glycol alginate and 0.5M Tris-HCl buffer solution with the pH value of 6.5; the reagent R2 component was 3% (w/v) of antibody-coated latex particles suspended in reagent R1.
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| CN112763724A (en) * | 2020-12-26 | 2021-05-07 | 北京安图生物工程有限公司 | Reagent combination and kit for simultaneously determining content of retinol binding protein in serum and urine |
| 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 |
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| 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 |
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| WO2002079782A1 (en) * | 2001-03-30 | 2002-10-10 | Mitsubishi Kagaku Iatron, Inc. | Reagent and method for immunoanalysis of elastase 1 and method of detecting pancreatic disease |
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| JPH01214760A (en) * | 1988-02-23 | 1989-08-29 | Teikoku Hormone Mfg Co Ltd | Simple immunological measuring 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 |
| CN104215770A (en) * | 2014-08-14 | 2014-12-17 | 上海睿康生物科技有限公司 | Two-particle-based retinol binding protein 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 |
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