Retinol binding protein detection kit as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological detection, and particularly relates to a retinol binding protein detection kit as well as a preparation method and application thereof. The kit provided by the invention comprises a reagent R1, a reagent R2 and a standard substance, the composition and the concentration of each component are strictly limited, a calibration curve is drawn by using the standard substance with known concentration and the concentration, a sample with unknown concentration can be measured to obtain a signal value, and the corresponding concentration of the measured sample is obtained according to the corresponding signal value on the standard curve. The retinol binding protein detection kit provided by the invention is low in cost, simple in production process, convenient to operate, high in specificity, high in sensitivity and good in stability, can realize simultaneous detection of blood and urine, and can simultaneously detect the RBP content in the blood and the urine. The result of the embodiment shows that the linear range can reach 0.5-200mg / L in a blood mode, and the linear range can reach 0.3-20mg / L in a urine mode.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and specifically relates to a retinol binding protein detection kit and a preparation method and application thereof. Background Art

[0002] Retinol binding protein (RBP) is mainly synthesized by liver cells and widely distributed in human serum, cerebrospinal fluid, urine and other body fluids. RBP is a small molecule protein with a short half-life. It is a specific transporter of vitamin A in the blood and can sensitively respond to the body's condition. When kidney disease occurs, due to decreased glomerular filtration rate and tubular reabsorption disorders, RBP in serum and urine increases significantly. In the early stage, it is more sensitive than creatinine and urea nitrogen, and is not affected by dietary interference.

[0003] RBP is a sensitive and important parameter for evaluating renal function damage, and has important clinical value for the diagnosis of early renal function damage such as diabetic nephropathy and hypertensive nephropathy.

[0004] At present, the market usually adopts latex enhanced immunoturbidimetry to determine RBP, mainly including latex enhanced immunoturbidimetry with double particle size mixture, latex enhanced immunoturbidimetry with double monoclonal antibodies, and latex immunoturbidimetry with one monoclonal antibody and one polyclonal antibody. Although the sensitivity is high and the linearity can basically reach 200mg / L, it is not possible to detect blood and urine at the same time, and the production cost is high and the process is complicated, which is not conducive to mass production and even more not conducive to market promotion. Summary of the invention

[0005] The purpose of the present invention is to provide a retinol binding protein detection kit and its preparation method and application, so as to realize the simultaneous detection of blood and urine and quantitatively detect the concentration of retinol binding protein in a sample with strong specificity, high sensitivity and good stability.

[0006] The present invention provides a retinol binding protein detection kit, which comprises an R1 reagent, an R2 reagent and a standard; The R1 reagent includes 0.01-0.1 mol / L of a first buffer, 8-30 g / L of a first electrolyte, 0-20 g / L of a sensitizer, 0.5-5 g / L of a first surfactant, and 0.5-2 g / L of a first preservative. The R2 reagent includes 0.1-2 g / L anti-human retinol binding protein antibody coated latex particles, 0.01-0.1 mol / L second buffer, 8-30 g / L second electrolyte, 0.5-30 g / L first stabilizer, 0.5-2 g / L second preservative, 0-20 g / L second surfactant and 0-5 g / L blocking agent; The standard comprises 0.01-0.1 mol / L third buffer, 8-30 g / L third electrolyte, 0-40 g / L protective agent, 0-40 g / L second stabilizer, 0.5-5 g / L third preservative, 0-140 mg / L RBP antigen and 0-4 g / L third surfactant; The first stabilizer is trehalose; the second stabilizer is sucrose and trehalose; The mass ratio of trehalose to sucrose in the second stabilizer is 1:1.

[0007] Preferably, the first buffer, the second buffer and the third buffer respectively include one or more of PB buffer, Tris buffer, MES buffer, glycine buffer and HEPES buffer; The first electrolyte, the second electrolyte and the third electrolyte respectively include one or more of sodium chloride, calcium chloride, potassium chloride and magnesium chloride; The sensitizer includes one or more of PEG6000, PEG8000, PEG20000 and PEG35000; The first surfactant, the second surfactant and the third surfactant respectively include one or more of Tween-20, Tween-80, Triton-100, B66 and glycerol; The first preservative, the second preservative and the third preservative respectively include one or more of Proclin-300, sodium azide and thimerosal; The blocking agent includes one or more of BSA, casein, ethanolamine and CE510; The protective agent includes one or more of BSA, casein and gelatin; The RBP antigen is a recombinant RBP antigen or a natural RBP antigen.

[0008] Preferably, the first buffer is Tris buffer; the second buffer is glycine buffer; and the third buffer is Tris buffer.

[0009] Preferably, the sensitizer is PEG6000; The first surfactant is Tween-20; the second surfactant is glycerol; the third surfactant is glycerol and Triton-100; The first electrolyte, the second electrolyte and the third electrolyte are respectively sodium chloride; The first preservative, the second preservative and the third preservative are respectively sodium azide; The blocking agent is CE510; The protective agent is BSA.

[0010] Preferably, the anti-human retinol binding protein antibody comprises one or more of rabbit anti-human retinol binding protein antibody, mouse anti-human retinol binding protein antibody and goat anti-human retinol binding protein antibody.

[0011] Preferably, in the anti-human retinol binding protein antibody-coated latex particles, the mass ratio of latex microspheres to anti-human retinol binding protein antibodies is 60-100:1-3.

[0012] Preferably, the pH value of the R1 reagent is 6-8; the pH value of the R2 reagent is 6-8.

[0013] The present invention also provides a method for preparing the retinol binding protein detection kit described in the above technical solution, including the preparation of R1 reagent and the preparation of R2 reagent; The preparation method of the R1 reagent comprises the following steps: The first buffer solution and water are mixed, and the pH is adjusted to 6-8. Then, the first electrolyte, the sensitizer, the first surfactant and the first preservative are weighed, and the mixture is dissolved in water respectively, and stirred evenly to obtain the R1 reagent; The preparation method of the R2 reagent comprises the following steps: The second buffer solution is mixed with water, the pH is adjusted to 6-8, and then mixed with latex microspheres to obtain a first mixed solution; The first mixed solution and the newly prepared EDC solution are mixed to activate the latex for 10 to 20 minutes, and during the stirring process, anti-human retinol binding protein antibody is added and coupled for 1 to 2 hours to obtain anti-human retinol binding protein antibody-coated latex particles; The anti-human retinol binding protein antibody-coated latex particles and the blocking agent are mixed, blocked at 20-30° C. for 0.5-1 h, and then a second electrolyte, a first stabilizer, and a second surfactant are added. After stirring for 20 min, a second preservative is added, and the pH is adjusted to 6-8 to obtain the R2 reagent.

[0014] The present invention also provides the use of the retinol binding protein detection kit described in the above technical solution in the detection of retinol binding protein; The direct purpose of the retinol binding protein detection is non-diagnostic and non-therapeutic.

[0015] The present invention also provides a method for detecting retinol binding protein for non-diagnostic and non-therapeutic purposes, and the retinol binding protein detection kit based on the above technical solution comprises the following steps: (1) Mix the calibrator and R1 reagent, incubate at 37°C for 5 min, add R2 reagent and mix well; 45 s after adding R2 reagent, detect the absorbance A1 at a wavelength of 600 nm; 255 s after adding R2 reagent, detect the absorbance A2 at a wavelength of 600 nm. Draw a calibration curve with the absorbance change value of the calibrator △A=A2-A1 as the ordinate and its corresponding concentration C calibration as the abscissa; (2) Mix the sample to be tested and reagent R1, incubate at 37°C for 5 minutes, add reagent R2 and mix well; 45 seconds after adding reagent R2, detect the absorbance A3 at a wavelength of 600 nm; 255 seconds after adding reagent R2, detect the absorbance A4 at a wavelength of 600 nm; calculate the concentration of retinol binding protein in the sample to be tested using the absorbance change value ΔA=A4-A3 according to the standard curve drawn in step (1).

[0016] Beneficial effects: The kit provided by the present invention includes R1 reagent, R2 reagent and standard substance, and the composition and concentration of each component are strictly limited. A calibration curve is drawn using a standard substance of known concentration and concentration, and a sample of unknown concentration can be measured to obtain a signal value, and the corresponding concentration of the sample to be tested is obtained by the corresponding signal value on the standard curve. The retinol binding protein detection kit provided by the present invention has low cost, simple production process, can realize simultaneous detection of blood and urine, is easy to operate, has strong specificity, high sensitivity, good stability, and can simultaneously detect the RBP content in blood and urine. The results of the embodiment show that in the blood mode, the linear range can reach 0.5~200mg / L, and in the urine mode, the linear range can reach 0.3~20mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0018] Figure 1 is the calibration curve of the retinol binding protein detection kit in the blood mode; Figure 2 is the calibration curve of the retinol binding protein detection kit in urine mode; Figure 3 The linearity test result of the retinol binding protein detection kit of Example 1 in the blood mode; Figure 4 The linearity test result of the retinol binding protein detection kit of comparative example 1 in the blood mode; Figure 5 This is the linear detection result of the retinol binding protein detection kit of comparative example 2 in the blood mode. DETAILED DESCRIPTION

[0019] The present invention provides a retinol binding protein detection kit, which comprises an R1 reagent, an R2 reagent and a standard; The R1 reagent includes 0.01-0.1 mol / L of a first buffer, 8-30 g / L of a first electrolyte, 0-20 g / L of a sensitizer, 0.5-5 g / L of a first surfactant, and 0.5-2 g / L of a first preservative. The R2 reagent includes 0.1-2 g / L anti-human retinol binding protein antibody coated latex particles, 0.01-0.1 mol / L second buffer, 8-30 g / L second electrolyte, 0.5-30 g / L first stabilizer, 0.5-2 g / L second preservative, 0-20 g / L second surfactant and 0-5 g / L blocking agent; The standard comprises 0.01-0.1 mol / L third buffer, 8-30 g / L third electrolyte, 0-40 g / L protective agent, 0-40 g / L second stabilizer, 0.5-5 g / L third preservative, 0-140 mg / L RBP antigen and 0-4 g / L third surfactant; The first stabilizer is trehalose; the second stabilizer is sucrose and trehalose; The mass ratio of trehalose to sucrose in the second stabilizer is 1:1.

[0020] In the present invention, as an embodiment, the R1 reagent, the R2 reagent and the standard in the retinol binding protein detection kit are packaged separately.

[0021] In the present invention, the R1 reagent includes 0.01-0.1 mol / L of the first buffer. As an embodiment, the R1 reagent includes 0.02-0.08 mol / L of the first buffer; as another embodiment, the R1 reagent includes 0.05 mol / L of the first buffer. As an embodiment, the first buffer includes one or more of PB buffer, Tris buffer, MES buffer, glycine buffer and HEPES buffer; as another embodiment, the first buffer is Tris buffer.

[0022] In the present invention, the R1 reagent includes 8-30 g / L of the first electrolyte. As an embodiment, the R1 reagent includes 9-25 g / L of the first electrolyte; as another embodiment, the R1 reagent includes 10-20 g / L of the first electrolyte; as another embodiment, the R1 reagent includes 15 g / L of the first electrolyte. As an embodiment, the first electrolyte includes one or more of sodium chloride, calcium chloride, potassium chloride and magnesium chloride; as another embodiment, the first electrolyte is sodium chloride.

[0023] In the present invention, the R1 reagent includes 0-20 g / L of a sensitizer. As an embodiment, the R1 reagent includes 0.5-15 g / L of a sensitizer; as another embodiment, the R1 reagent includes 1.0-10 g / L of a sensitizer; as another embodiment, the R1 reagent includes 5-8 g / L of a sensitizer. As an embodiment, the sensitizer includes one or more of PEG6000, PEG8000, PEG20000, and PEG35000; as another embodiment, the sensitizer is PEG6000.

[0024] In the present invention, the R1 reagent includes 0.5-5 g / L of the first surfactant. As an embodiment, the R1 reagent includes 1-4 g / L of the first surfactant; as another embodiment, the R1 reagent includes 2 g / L of the first surfactant. As an embodiment, the first surfactant includes one or more of Tween-20, Tween-80, Triton-100, B66 and glycerol; as another embodiment, the first surfactant is Tween-20.

[0025] In the present invention, the R1 reagent includes 0.5-2 g / L of the first preservative. As an embodiment, the R1 reagent includes 0.8-1.5 g / L of the first preservative; as another embodiment, the R1 reagent includes 1.0 g / L of the first preservative. As an embodiment, the first preservative includes one or more of Proclin-300, sodium azide and thimerosal; as another embodiment, the first preservative is sodium azide.

[0026] In the present invention, as one embodiment, the pH value of the R1 reagent is 6-8; as another embodiment, the pH value of the R1 reagent is 7.

[0027] In the present invention, the R2 reagent includes 0.1~2g / L anti-human retinol binding protein antibody coated latex particles. As an embodiment, the R2 reagent includes 0.9g / L anti-human retinol binding protein antibody coated latex particles. As an embodiment, the anti-human retinol binding protein antibody includes one or more of rabbit anti-human retinol binding protein antibody, mouse anti-human retinol binding protein antibody and sheep anti-human retinol binding protein antibody; as another embodiment, the anti-human retinol binding protein antibody is rabbit anti-human retinol binding protein antibody. As an embodiment, in the anti-human retinol binding protein antibody coated latex particles, the mass ratio of latex microspheres to anti-human retinol binding protein antibodies is 60~100:1~3; as another embodiment, in the anti-human retinol binding protein antibody coated latex particles, the mass ratio of latex microspheres to anti-human retinol binding protein antibodies is 60:3. As an embodiment, the rabbit anti-human retinol binding protein antibody was purchased from Shanghai BoLeShi Biotechnology Co., Ltd. with the catalog number BLS-KY-00020Y.

[0028] In the present invention, the R2 reagent includes 0.01-0.1 mol / L of the second buffer. As an embodiment, the R2 reagent includes 0.02-0.08 mol / L of the second buffer; as another embodiment, the R2 reagent includes 0.05 mol / L of the second buffer. As an embodiment, the second buffer includes one or more of PB buffer, Tris buffer, MES buffer, glycine buffer and HEPES buffer; as another embodiment, the second buffer is glycine buffer.

[0029] In the present invention, the R2 reagent includes 8-30 g / L of the second electrolyte. As an embodiment, the R2 reagent includes 9-25 g / L of the second electrolyte; as another embodiment, the R2 reagent includes 10-20 g / L of the second electrolyte; as another embodiment, the R2 reagent includes 15 g / L of the electrolyte. As an embodiment, the second electrolyte includes one or more of sodium chloride, calcium chloride, potassium chloride and magnesium chloride; as another embodiment, the second electrolyte is sodium chloride.

[0030] In the present invention, the R2 reagent includes 0.5-30 g / L of the first stabilizer. As one embodiment, the R2 reagent includes 1.0-25 g / L of the first stabilizer; as another embodiment, the R2 reagent includes 5-20 g / L of the first stabilizer; as another embodiment, the R2 reagent includes 10-15 g / L of the stabilizer. The first stabilizer of the present invention is trehalose.

[0031] In the present invention, the R2 reagent includes 0.5-2 g / L of a second preservative. As an embodiment, the R2 reagent includes 0.8-1.5 g / L of a second preservative; as another embodiment, the R2 reagent includes 1.0 g / L of a second preservative. As an embodiment, the second preservative includes one or more of Proclin-300, sodium azide, and thimerosal; as another embodiment, the second preservative is sodium azide.

[0032] In the present invention, the R2 reagent includes 0-4 g / L of a second surfactant. As an embodiment, the R2 reagent includes 0.5-2 g / L of a second surfactant; as another embodiment, the R2 reagent includes 1.0 g / L of a second surfactant. As an embodiment, the second surfactant includes one or more of Tween-20, Tween-80, Triton-100, B66 and glycerol; as another embodiment, the second surfactant is glycerol.

[0033] In the present invention, the R2 reagent includes 0-5 g / L of a blocking agent. As an embodiment, the R2 reagent includes 1.0-4.0 g / L of a blocking agent; as another embodiment, the R2 reagent includes 2.5 g / L of a blocking agent. As an embodiment, the blocking agent in the R2 reagent includes one or more of BSA, casein, ethanolamine, and CE510; as another embodiment, the blocking agent in the R2 reagent is CE510.

[0034] In the present invention, as one embodiment, the pH value of the R2 reagent is 6-8; as another embodiment, the pH value of the R2 reagent is 7-7.5.

[0035] In the present invention, the standard comprises 0.01-0.1 mol / L of the third buffer. As an embodiment, the standard comprises 0.02-0.08 mol / L of the third buffer; as another embodiment, the standard comprises 0.05 mol / L of the third buffer. As an embodiment, the third buffer comprises one or more of PB buffer, Tris buffer, MES buffer, glycine buffer and HEPES buffer; as another embodiment, the third buffer is Tris buffer.

[0036] In the present invention, the standard includes 8-30 g / L of the third electrolyte. As an embodiment, the standard includes 9-25 g / L of the third electrolyte; as another embodiment, the standard includes 10-20 g / L of the third electrolyte; as another embodiment, the standard includes 15 g / L of the electrolyte. As an embodiment, the third electrolyte includes one or more of sodium chloride, calcium chloride, potassium chloride and magnesium chloride; as another embodiment, the third electrolyte is sodium chloride.

[0037] In the present invention, the standard includes 0-40 g / L of protective agent. As an embodiment, the standard includes 5-30 g / L of protective agent; as another embodiment, the standard includes 10-20 g / L of protective agent; as another embodiment, the standard includes 15 g / L of protective agent. As an embodiment, the protective agent includes one or more of BSA, casein and gelatin; as another embodiment, the protective agent is BSA.

[0038] In the present invention, the standard includes 0-40 g / L of the second stabilizer. As one embodiment, the standard includes 1.0-30 g / L of the second stabilizer; as another embodiment, the standard includes 5-25 g / L of the second stabilizer; as another embodiment, the standard includes 10-20 g / L of the stabilizer. The second stabilizer of the present invention is trehalose and sucrose, and the mass ratio of trehalose to sucrose in the second stabilizer is 1:1.

[0039] In the present invention, the standard includes 0.5-5 g / L of the third preservative. As an embodiment, the standard includes 0.8-1.5 g / L of the third preservative; as another embodiment, the standard includes 1.0 g / L of the third preservative. As an embodiment, the third preservative includes one or more of Proclin-300, sodium azide and thimerosal; as another embodiment, the third preservative is sodium azide.

[0040] In the present invention, the standard includes 0-4 g / L of the third surfactant. As an embodiment, the standard includes 0.5-2 g / L of the third surfactant; as another embodiment, the standard includes 1.0 g / L of the third surfactant. As an embodiment, the third surfactant includes one or more of Tween-20, Tween-80, Triton-100, B66 and glycerol; as another embodiment, the third surfactant is Triton-100.

[0041] In the present invention, the standard includes 0~140mg / L RBP antigen. As an embodiment, the standard includes blood standard and / or urine standard; as another embodiment, the standard is a blood standard and a urine standard. As an embodiment, the blood standard includes 0~140mg / L RBP antigen; as another embodiment, the blood standard includes 17.5mg / L RBP antigen, 35mg / L RBP antigen, 70mg / L RBP antigen and 140mg / L RBP antigen. As an embodiment, the urine standard includes 0~20mg / L RBP antigen; as another embodiment, the urine standard includes 1mg / L RBP antigen, 5mg / L RBP antigen, 10mg / L RBP antigen and 20mg / L RBP antigen. As an embodiment, the RBP antigen is a recombinant RBP antigen or a natural RBP antigen; as another embodiment, the RBP antigen is a recombinant RBP antigen. As an embodiment, the recombinant RBP antigen is purchased from Guilin Yingmei Biotechnology Co., Ltd., with the product number KAY0024.CR.

[0042] The present invention also provides a method for preparing the retinol binding protein detection kit described in the above technical solution, including the preparation of R1 reagent and the preparation of R2 reagent.

[0043] As an embodiment, the preparation method of the R1 reagent of the present invention comprises the following steps: mixing a first buffer solution and water, adjusting the pH to 6-8, then weighing a first electrolyte, a sensitizer, a first surfactant and a first preservative, dissolving them in water respectively, and stirring evenly to obtain the R1 reagent. The present invention limits the order of adding raw materials in the preparation process of the R1 reagent to reduce errors.

[0044] As an embodiment, the preparation method of the R2 reagent described in the present invention includes the following steps: mixing a second buffer solution and water, adjusting the pH to 6-8 and mixing with latex microspheres to obtain a first mixed solution; mixing the first mixed solution and the freshly prepared EDC solution, activating the latex for 10-20 minutes, adding anti-human retinol binding protein antibodies during stirring, coupling for 1-2 hours, and obtaining anti-human retinol binding protein antibody-coated latex particles; mixing the anti-human retinol binding protein antibody-coated latex particles and a sealing agent, sealing at 20-30° C. for 0.5-1 hour, adding a second electrolyte, a first stabilizer, and a second surfactant, stirring for 20 minutes, adding a second preservative, adjusting the pH to 6-8, and obtaining the R2 reagent.

[0045] In the present invention, as an embodiment, the time for activating the latex is 15 minutes. As an embodiment, the time for coupling is 1.5 hours. As an embodiment, the temperature for sealing is 25°C; the time for sealing is 0.8 hours. As an embodiment, the sealing uses a shaking table; the rotation speed of the shaking table is 100-300 r / min; as another embodiment, the rotation speed of the shaking table is 200 r / min.

[0046] The present invention uses anti-human retinol binding protein antibody coated latex particles as a component of the R2 reagent, and the anti-human retinol binding protein antibody is marked on the surface of the latex microspheres. In the R1 reagent system, the anti-human retinol binding protein antibody coated latex particles of the R2 reagent react with the antigen to be tested to form an antigen-antibody-microsphere complex, and the complex causes turbidity in the reagent, and the change in turbidity is reflected by the change in the instrument signal value, thereby detecting RBP. The retinol binding protein detection kit provided by the present invention is easy to operate, low in cost, and can be mass-produced; it can realize the simultaneous detection of blood and urine. The results of the embodiment show that in the blood mode, the linear range can reach 0.5~200mg / L, and in the urine mode, the linear range can reach 0.3~20mg / L.

[0047] In view of the advantages of the anti-human retinol binding protein antibody provided by the present invention, the application of the retinol binding protein detection kit in retinol binding protein detection also falls within the protection scope of the present invention. The direct purpose of the retinol binding protein detection of the present invention is non-diagnosis and non-treatment.

[0048] The present invention also provides a method for detecting retinol binding protein for non-diagnostic and non-therapeutic purposes, and the retinol binding protein detection kit based on the above technical solution comprises the following steps: (1) Mix the calibrator and R1 reagent, incubate at 37°C for 5 min, add R2 reagent and mix well; 45 s after adding R2 reagent, detect the absorbance A1 at a wavelength of 600 nm; 255 s after adding R2 reagent, detect the absorbance A2 at a wavelength of 600 nm. Draw a calibration curve with the absorbance change value of the calibrator △A=A2-A1 as the ordinate and its corresponding concentration C calibration as the abscissa; (2) Mix the sample to be tested and reagent R1, incubate at 37°C for 5 minutes, add reagent R2 and mix well; 45 seconds after adding reagent R2, detect the absorbance A3 at a wavelength of 600 nm; 255 seconds after adding reagent R2, detect the absorbance A4 at a wavelength of 600 nm; calculate the concentration of retinol binding protein in the sample to be tested using the absorbance change value ΔA=A4-A3 according to the standard curve drawn in step (1).

[0049] The present invention uses a known concentration of a standard substance to draw a calibration curve with the concentration, measures a sample of unknown concentration to obtain a signal value, and obtains the corresponding concentration of the sample to be tested by the corresponding signal value on the standard curve. The retinol binding protein detection kit provided by the present invention has low cost, simple production process, can realize simultaneous detection of blood and urine, is easy to operate, has high sensitivity, and can simultaneously detect the RBP content in blood and urine.

[0050] To further illustrate the present invention, a retinol binding protein detection kit provided by the present invention and its preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1 A retinol binding protein detection kit, comprising R1 reagent, R2 reagent, blood standard product, blood quality control product, urine standard product and urine quality control product, specifically: (1) R1 reagent: 0.1 mol / L phosphate buffer, 9 g / L NaCl, 0.5 g / L Tween-20, 0.5 g / L polyethylene glycol-6000, 1 g / L sodium azide, the solvent is purified water; the pH value of R1 reagent is 7; (2) R2 reagent: 0.1 mol / L glycine buffer, 9 g / L NaCl, 0.5 g / L glycerol, 0.5 g / L trehalose, 2.5 g / L CE510, 1 g / L sodium azide, 0.06% latex microspheres, 300 mg / L RBP antibody, the solvent is purified water; the pH value of R2 reagent is 8.

[0052] (3) Blood standards: 0.00, 17.50, 35.00, 70.00, and 140.00 mg / L antigen solutions, the solvent being the antigen diluent.

[0053] (4) Urine standard: 0.00, 1.00, 5.00, 10.00, 20.00 mg / L antigen solution, the solvent is antigen diluent.

[0054] (5) Blood quality control: 40.00 and 80.00 mg / L antigen solution, the solvent is antigen diluent; (6) Urine quality control: 0.5 and 2.6 mg / L antigen solution, the solvent is antigen diluent; The compositions of the antigen diluents described in (3) to (6) are: 0.05 mol / L Tris, 9 g / L NaCl, 20 g / L sucrose, 20 g / L trehalose, 5 g / L BSA, 5 g / L glycerol, 2 g / L Triton-100 and 1 g / L sodium azide. The solvent is purified water with a pH value of 8.0. The antigens were purchased from Guilin Inmet Biotechnology Co., Ltd., catalog number KAY0024.CR).

[0055] The preparation method of R1 reagent is as follows: dissolve phosphate in water, adjust the pH to 7 to obtain phosphate buffer, then weigh NaCl, Tween-20, PEG6000, and sodium azide in turn and dissolve them in the phosphate buffer, stir evenly, and obtain R1 reagent.

[0056] The preparation method of R2 reagent is: (1) Dissolve glycine in pure water to prepare 0.1 mol / L glycine buffer, adjust the pH to 8, dissolve the latex microspheres in the glycine buffer, and stir evenly; (2) Add the freshly prepared EDC solution, place at 25°C, shake at 200 r / min, and activate the latex for 10-20 min; (3) During the stirring process, add RBP antibody and place in a shaker at 25°C, 200 r / min, and couple for 2 h; (4) Add CE510, place in a shaker at 25°C, 200 r / min, and seal for 1 h; (5) After sealing, take out and place on a stirrer for stirring. Add NaCl, trehalose, and glycerol respectively and stir for 20 minutes. Add a preservative and adjust the pH to 8.0. Continue stirring for 10 minutes to obtain the R2 reagent.

[0057] Comparative Example 1 A retinol binding protein detection kit similar to Example 1, the only difference being that the pH value of the R2 reagent is 7.5.

[0058] Comparative Example 2 A retinol binding protein detection kit similar to Example 1, with the following differences: (1) The concentration of phosphate buffer in reagent R1 is 0.05 mol / L; (2) The concentration of glycine buffer in R2 reagent is 0.05 mol / L, and the pH value of R2 reagent is 7.

[0059] Comparative Example 3 A retinol binding protein detection kit similar to Example 1, the only difference being that glycerol is not added to the R2 reagent.

[0060] Comparative Example 4 A retinol binding protein detection kit similar to Example 1, the only difference being that the content of glycerol in the R2 reagent is 20 g / L.

[0061] Comparative Example 5 A retinol binding protein detection kit similar to Example 1, the only difference being that trehalose is not added to the R2 reagent.

[0062] Comparative Example 6 A retinol binding protein detection kit similar to Example 1, the only difference being that the content of trehalose in the R2 reagent is 20 g / L.

[0063] Comparative Example 7 A retinol binding protein detection kit similar to Example 1, the only difference being that the content of NaCl in the R1 reagent is 7 g / L.

[0064] Test Example 1 (1) Establishment of standard curve in blood mode According to the parameters in Table 1, the blood standard products in the retinol binding protein detection kits of Example 1 and Comparative Examples 1-2 were tested, and the results are shown in Tables 2 and Figure 1 shown.

[0065] Table 1 Calibration operating parameters in blood mode

[0066] Table 2 Calibration results in blood mode

[0067] According to Table 2 and Figure 1 It can be seen that the retinol binding protein detection kits of Comparative Examples 1 and 2 have higher sensitivity, but the rising trend of the curve is not as good as that of Example 1.

[0068] (2) Establishment of standard curve in urine mode According to the parameters in Table 3, the urine standard in the retinol binding protein detection kit of Example 1 and Comparative Examples 1-2 was tested, and the results are shown in Tables 4 and Figure 2 shown.

[0069] Table 3 Calibration operating parameters in urine mode

[0070] Table 4 Calibration results in urine mode

[0071] According to Table 4, and Figure 2 It can be seen that the retinol binding protein detection kits of Comparative Examples 1 and 2 have higher sensitivity, but the rising trend of the curve is not as good as that of Example 1.

[0072] Test Example 2 RBP linear range detection in blood mode (1) Draw a calibration curve based on the concentration and absorbance of the standard sample in the blood mode of Test Example 1.

[0073] (2) According to the parameters in Table 1 of Test Example 1, 200 mg / L of RBP antigen was diluted to 1 / 32, and the retinol binding protein detection kit of Example 1 and Comparative Examples 1-2 was used for detection. The detected absorbance was substituted into the calibration curve of step (1) to determine the concentration of retinol binding protein. The results are shown in Tables 5 and Figures 3-5 shown.

[0074] Table 5 Detection results of standard curve in blood mode

[0075] According to Table 5 and Figures 3-5 It can be seen that within the linear concentration range of 0.5-200 mg / L, the retinol binding protein detection kit of Example 1 has a wide linear range and has sufficient advantages.

[0076] Test Example 3 Opening stability in blood mode (1) According to the parameters in Table 1 of Test Example 1, the blood standards in the retinol binding protein detection kits of Comparative Examples 3 to 6 were tested. The results are shown in Table 6.

[0077] Table 6 Calibration results of comparative examples 3 to 6

[0078] (2) According to the parameters in Table 1 of Test Example 1, the blood quality control product 1 (retinol binding protein concentration is 40 mg / L) was tested using the retinol binding protein detection kits of Example 1 and Comparative Examples 3 to 6 with different bottle opening times. The results are shown in Table 7.

[0079] Table 7 Retinol binding protein test results at different bottle opening times (mg / L)

[0080] According to Table 7, it can be seen that the test results of the quality control of the kit in Example 1 are not obvious as time goes by, while the test results of the quality control values ​​of the kits in Comparative Examples 3 and 5 decrease significantly as time goes by; the test kits in Example 1 and Comparative Examples 4 and 6 have a quality control value deviation of less than 5% after opening the bottle for 14 days, and the deviation of Example 1 is smaller. The addition of glycerol and trehalose to the R2 reagent in the retinol binding protein detection kit provided in Example 1 of the present invention can prolong the opening stability of RBP.

[0081] Test Example 4 (1) According to the parameters in Table 1 and Table 3 in Test Example 1, the blood standard and urine standard in the retinol binding protein detection kit of Comparative Example 7 were tested respectively. The results are shown in Table 8.

[0082] Table 8 Calibration results of comparative example 7

[0083] (2) According to the parameters in Table 1 and Table 3 of Test Example 1, the retinol binding protein detection kits of Example 1 and Comparative Example 7 were used to detect blood quality control 1 (retinol binding protein concentration of 40 mg / L), blood quality control 2 (retinol binding protein concentration of 80 mg / L), urine quality control 1 (retinol binding protein concentration of 0.5 mg / L) and urine quality control 2 (retinol binding protein concentration of 2.6 mg / L), respectively, and the precision CV was calculated. The results are shown in Tables 9 and 10.

[0084] Table 9 Precision test results of Example 1

[0085] Table 10 Precision test results of Comparative Example 7

[0086] It can be seen from Tables 9 and 10 that the retinol binding protein detection kit of Example 1 was used to detect high and low quality controls 10 times each, and the precision CV was <6.0%; while the retinol binding protein detection kit of Comparative Example 7 was used to detect high and low quality controls 10 times each, and the precision CV was >6.0%. The precision of the retinol binding protein detection kit of Example 1 is better than that of Comparative Example 7.

[0087] It can be seen from the above content that the kit provided by the present invention can realize the simultaneous detection of blood and urine, and quantitatively detect the concentration of retinol binding protein in the sample, with strong specificity, high sensitivity and good stability.

[0088] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A retinol binding protein detection kit, characterized in that: The retinol binding protein detection kit comprises R1 reagent, R2 reagent and standard substance; The R1 reagent includes 0.01-0.1 mol / L of a first buffer, 8-30 g / L of a first electrolyte, 0-20 g / L of a sensitizer, 0.5-5 g / L of a first surfactant, and 0.5-2 g / L of a first preservative. The R2 reagent includes 0.1-2 g / L anti-human retinol binding protein antibody coated latex particles, 0.01-0.1 mol / L second buffer, 8-30 g / L second electrolyte, 0.5-30 g / L first stabilizer, 0.5-2 g / L second preservative, 0-20 g / L second surfactant and 0-5 g / L blocking agent; The standard comprises 0.01-0.1 mol / L third buffer, 8-30 g / L third electrolyte, 0-40 g / L protective agent, 0-40 g / L second stabilizer, 0.5-5 g / L third preservative, 0-140 mg / L RBP antigen and 0-4 g / L third surfactant; The first stabilizer is trehalose; the second stabilizer is sucrose and trehalose; The mass ratio of trehalose to sucrose in the second stabilizer is 1:

1.

2. The retinol binding protein detection kit according to claim 1, characterized in that: The first buffer, the second buffer and the third buffer respectively include one or more of PB buffer, Tris buffer, MES buffer, glycine buffer and HEPES buffer; The first electrolyte, the second electrolyte and the third electrolyte respectively include one or more of sodium chloride, calcium chloride, potassium chloride and magnesium chloride; The sensitizer includes one or more of PEG6000, PEG8000, PEG20000 and PEG35000; The first surfactant, the second surfactant and the third surfactant respectively include one or more of Tween-20, Tween-80, Triton-100, B66 and glycerol; The first preservative, the second preservative and the third preservative respectively include one or more of Proclin-300, sodium azide and thimerosal; The blocking agent includes one or more of BSA, casein, ethanolamine and CE510; The protective agent includes one or more of BSA, casein and gelatin; The RBP antigen is a recombinant RBP antigen or a natural RBP antigen.

3. The retinol binding protein detection kit according to claim 1, characterized in that: The first buffer is a Tris buffer; the second buffer is a glycine buffer; and the third buffer is a Tris buffer.

4. The retinol binding protein detection kit according to claim 2, characterized in that: The sensitizer is PEG6000; The first surfactant is Tween-20; the second surfactant is glycerol; the third surfactant is glycerol and Triton-100; The first electrolyte, the second electrolyte and the third electrolyte are respectively sodium chloride; The first preservative, the second preservative and the third preservative are respectively sodium azide; The blocking agent is CE510; The protective agent is BSA.

5. The retinol binding protein detection kit according to claim 1, characterized in that: The anti-human retinol binding protein antibody includes one or more of rabbit anti-human retinol binding protein antibody, mouse anti-human retinol binding protein antibody and goat anti-human retinol binding protein antibody.

6. The retinol binding protein detection kit according to claim 1 or 5, characterized in that: In the anti-human retinol binding protein antibody-coated latex particles, the mass ratio of latex microspheres to the anti-human retinol binding protein antibody is 60-100:1-3.

7. The retinol binding protein detection kit according to claim 1, characterized in that: The pH value of the R1 reagent is 6-8; the pH value of the R2 reagent is 6-8.

8. The method for preparing the retinol binding protein detection kit according to any one of claims 1 to 7, characterized in that: Including the preparation of R1 reagent and the preparation of R2 reagent; The preparation method of the R1 reagent comprises the following steps: The first buffer solution and water are mixed, and the pH is adjusted to 6-8. Then, the first electrolyte, the sensitizer, the first surfactant and the first preservative are weighed, and the mixture is dissolved in water respectively, and stirred evenly to obtain the R1 reagent; The preparation method of the R2 reagent comprises the following steps: The second buffer solution is mixed with water, the pH is adjusted to 6-8, and then mixed with latex microspheres to obtain a first mixed solution; The first mixed solution and the newly prepared EDC solution are mixed to activate the latex for 10 to 20 minutes, and during the stirring process, anti-human retinol binding protein antibody is added and coupled for 1 to 2 hours to obtain anti-human retinol binding protein antibody-coated latex particles; The anti-human retinol binding protein antibody-coated latex particles and the blocking agent are mixed, blocked at 20-30° C. for 0.5-1 h, and then a second electrolyte, a first stabilizer, and a second surfactant are added. After stirring for 20 min, a second preservative is added, and the pH is adjusted to 6-8 to obtain the R2 reagent.

9. Use of the retinol binding protein detection kit according to any one of claims 1 to 7 or the retinol binding protein detection kit prepared by the preparation method according to claim 8 in the detection of retinol binding protein; The direct purpose of the retinol binding protein detection is non-diagnostic and non-therapeutic.

10. A method for detecting retinol binding protein for non-diagnostic and non-therapeutic purposes, characterized in that: The retinol binding protein detection kit according to any one of claims 1 to 7 comprises the following steps: (1) Mix the calibrator and R1 reagent, incubate at 37°C for 5 min, add R2 reagent and mix well; 45 s after adding R2 reagent, detect the absorbance A1 at a wavelength of 600 nm; 255 s after adding R2 reagent, detect the absorbance A2 at a wavelength of 600 nm. Draw a calibration curve with the absorbance change value of the calibrator △A=A2-A1 as the ordinate and its corresponding concentration C calibration as the abscissa; (2) Mix the sample to be tested and reagent R1, incubate at 37°C for 5 minutes, add reagent R2 and mix well; 45 seconds after adding reagent R2, detect the absorbance A4 at a wavelength of 600 nm; 255 seconds after adding reagent R2, detect the absorbance A3 at a wavelength of 600 nm; calculate the concentration of retinol binding protein in the sample to be tested using the absorbance change value ΔA=A4-A3 according to the standard curve drawn in step (1).

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