Reagent for urine trace protein detection and application thereof

Abstract

The invention relates to the technical field of medical detection, in particular to a urine trace protein detection reagent and application thereof. The invention discloses a reagent for detecting trace protein in urine. The reagent comprises a reagent R1 and a reagent R2, the reagent R1 is prepared from 0.05 mol / L to 0.15 mol / L of a buffer solution; 20 to 30 ml / L of polyethylene glycol; 2-4g / L of ammonium sulfate; 1-5 g / L of a polyelectrolyte; 0.5 to 1.5 g / L of preservative; 1-3 g / L of a surfactant; the reagent R2 comprises 1-3g / l of latex microspheres combined with a goat anti-human urine microalbumin antibody; the concentration of the buffer solution is 0.05 to 0.15 mol / L; 0.5 to 1.5 g / L of preservative; 1-3 g / L of a surfactant; and 0.5-1.5 g / L of a stabilizing agent. Under the condition that the detection sensitivity is guaranteed, the detection linear range is widened to the maximum extent, and the detection linear range can reach 0.01-1 g / L.

Description

Technical Field

[0001] The present application relates to the field of medical detection technology, and in particular to a reagent for detecting trace protein in urine and its application. Background Art

[0002] Albumin is the most important protein in human plasma, which maintains the body's nutrition and osmotic pressure. Under normal circumstances, only a small amount of albumin can be filtered through the glomerular basement membrane, and 95% of the albumin is reabsorbed in the proximal tubules. The albumin that appears in the urine is called urine microalbuminuria. If the barrier function of the glomerular basement membrane is damaged, the amount of albumin filtered through the basement membrane will increase significantly, and the urine microalbuminuria will also increase significantly. Urinary microalbuminuria is the earliest biochemical indicator of diabetic nephropathy and hypertensive nephropathy. The determination of urine microalbuminuria is of epoch-making significance for the early diagnosis and improvement of prognosis of diabetic nephropathy, and it has important diagnostic value for hypertensive nephropathy, eclampsia and renal damage caused by various toxic substances.

[0003] The main methods for detecting urine microalbumin are immunoturbidimetry, immunofluorescence, radioimmunoassay, enzyme-linked immunosorbent assay, etc.

[0004] The most commonly used method for detecting urine microalbumin in China is enzyme-linked immunosorbent assay (ELISA), but the ELISA has many operating steps and therefore has poor repeatability.

[0005] Immunoturbidimetry is a dynamic determination method for antigen-antibody binding. Its basic principle is: when the antigen and antibody react in a special dilution system and the ratio is appropriate, the soluble immune complex formed will precipitate from the liquid phase under the action of the polymerization promoter in the dilution system to form particles, causing turbidity in the reaction solution. When the antibody concentration is fixed, the amount of immune complex formed increases with the increase of the amount of antigen in the test sample, and the turbidity of the reaction solution also increases accordingly. By measuring the turbidity of the reaction solution and comparing it with a series of standards, the content of the antigen in the test sample can be calculated.

[0006] Regarding the existing immunoturbidimetric method, the applicant found that the linear range of the detection is small, and it is necessary to conduct research to expand the linear range of the detection. Summary of the invention

[0007] In order to broaden the linear range of urine microalbumin detection and improve the applicability of detection, the present application provides a reagent for urine microalbumin detection and its application.

[0008] In a first aspect, the present application provides a reagent for detecting trace protein in urine, which adopts the following technical solution.

[0009] A reagent for detecting trace protein in urine, comprising a reagent R1 and a reagent R2; The reagent R1 comprises: Buffer 0.05-0.15 mol / L; Polyethylene glycol 20-30ml / L; Ammonium sulfate 2-4g / L; Polyelectrolytes 1-5 g / L; Preservatives 0.5-1.5 g / L; Surfactant 1-3g / L; The reagent R2 comprises: Latex microspheres conjugated with goat anti-human urine microalbumin antibody 1-3 g / l; Buffer 0.05-0.15 mol / L; Preservatives 0.5-1.5 g / L; Surfactant 1-3g / L; Stabilizer 0.5-1.5g / L.

[0010] By adopting the above technical scheme, sheep anti-human urine microalbumin antibody and urine microalbumin are reacted in a specific dilution system to form a soluble immune complex, and under the compounding action of polyethylene glycol, ammonium sulfate and polyelectrolytes in the dilution system, particles are directly formed from the liquid state, so that the reaction solution appears turbid, so that the content of urine microalbumin in the sample can be calculated. Polyethylene glycol can promote the aggregation of urine microalbumin and sheep anti-human urine microalbumin antibody, increasing the sensitivity of detection; ammonium sulfate promotes the precipitation of urine microalbumin and sheep anti-human urine microalbumin antibody complex by changing the ionic strength of the solution, thereby increasing turbidity; polyelectrolytes promote the formation of urine microalbumin and sheep anti-human urine microalbumin antibody complex through charge neutralization. While ensuring the sensitivity of the detection, the linear range of the detection is widened to the greatest extent, and the linear range of the detection can reach 0.01-1g / L.

[0011] Furthermore, the latex microspheres combined with sheep anti-human urinary microalbumin antibody include first microspheres with a particle size of 100-150 nm and second microspheres with a particle size of 300-400 nm.

[0012] Furthermore, the weight ratio of the first latex microspheres to the second latex microspheres is (2-3):1.

[0013] By adopting the above technical solution, the specific surface area of ​​the first microsphere of 100-150nm is large, which is conducive to the adsorption and coupling of biorecognition molecules, and can improve the binding efficiency of the microspheres and urinary microalbumin, thereby improving the sensitivity of the method. The microspheres of 100-150nm have a better binding effect with urine with low concentration of urinary microalbumin, and the microspheres of 300-400nm have a better binding effect with urine with high concentration of urinary microalbumin. The combination of the two expands the linear range of the method.

[0014] Furthermore, the preparation method of the latex microspheres combined with sheep anti-human urine microalbumin antibody is: 1) Styrene, divinylmethylbenzene, water, tert-butyl peroxide and vinyl bisstearamide are mixed, the temperature is raised to 130-150°C, and then nitrogen is continuously introduced and reacted for 2h-3h to obtain a styrene-divinylmethylbenzene suspension; 2) adding 3-aminopropyltriethoxysilane to the obtained suspension, heating to 180-200° C., and obtaining amino-modified polystyrene microspheres; 3) activating the goat anti-human urine microalbumin antibody with an activator to obtain an activated goat anti-human urine microalbumin antibody; 4) Mixing the activated goat anti-human urine microalbumin antibody and the amino-modified polystyrene microspheres in a buffer solution, so that the active groups of the antibody react chemically with the amino groups on the surface of the microspheres to form covalent bonds, thereby obtaining latex microspheres bound with the goat anti-human urine microalbumin antibody; 5) Incubate the latex microspheres bound to goat anti-human urine microalbumin antibody in a buffer containing bovine serum albumin to block unreacted amino groups; 6) Centrifugation, filtration and washing.

[0015] Furthermore, the activator is N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a weight ratio of 1:1.

[0016] Furthermore, the weight ratio of the activated sheep anti-human urinary microalbumin antibody to the amino-modified polystyrene microspheres is (15-20):1.

[0017] Furthermore, the polyelectrolyte is polyacrylic acid and chitosan in a weight ratio of 1:(4-7).

[0018] Furthermore, the buffer is PBS buffer.

[0019] In a second aspect, the present application provides an application of a reagent for detecting trace protein in urine, using the following technical solution.

[0020] An application of a reagent for detecting trace protein in urine comprises the following steps: S1. The test was performed on an automatic biochemical analyzer at a reaction temperature of 37°C and a wavelength of 340 nm. First, 8 μL of urine sample was incubated with 280 μL of reagent R1 for 300 seconds and then the light was measured for the first time. Then, 70 μL of reagent R2 was added and incubated for 300 seconds and then the light was measured for the second time. S2. Use six series of concentrations of calibrators to draw a calibration curve; S3. The urine microalbumin concentration in the sample is read from the calibration curve.

[0021] In summary, this application has the following beneficial effects: The present application promotes the aggregation of urine microalbumin and goat anti-human urine microalbumin antibody by polyethylene glycol, thereby increasing the sensitivity of detection; ammonium sulfate promotes the precipitation of the complex of urine microalbumin and goat anti-human urine microalbumin antibody by changing the ionic strength of the solution, thereby increasing the turbidity; polyelectrolytes promote the formation of the complex of urine microalbumin and goat anti-human urine microalbumin antibody by charge neutralization. While ensuring the sensitivity of the detection, the linear range of the detection is widened to the greatest extent, and the linear range of the detection can reach 0.01-1g / L. DETAILED DESCRIPTION

[0022] The present application is further described in detail below with reference to the embodiments.

[0023] Raw materials and intermediate preparation examples raw material The raw materials in the examples of this application can be obtained from the market: Buffer, PBS buffer 0.01 mol / L; Polyethylene glycol, PEG-2000, analytical grade; Ammonium sulfate, analytical grade; preservative, sodium azide; Surfactant, ethylphenyl polyethylene glycol; Stabilizer, disodium EDTA; Styrene, analytical grade; Divinylmethylbenzene, analytical grade; Tert-butyl peroxide, analytical grade; Vinyl bisstearamide, analytical grade; 3-Aminopropyltriethoxysilane, analytical grade; N-Hydroxysuccinimide, analytical grade; Ethyl-3-(3-dimethylaminopropyl)carbodiimide, analytical grade; Polyacrylic acid, average molecular weight 2000; Chitosan, analytical grade.

[0024] Preparation Example Preparation Example 1 A latex microsphere combined with sheep anti-human urine microalbumin antibody, the preparation method of which is: 1) Mix 15kg of styrene, 15kg of divinylmethylbenzene, 40kg of water, 15kg of tert-butyl peroxide, and 15kg of vinyl bisstearamide, heat to 140°C, and then continuously introduce nitrogen at a flow rate of 10ml / min. React for 2h to obtain a styrene-divinylmethylbenzene suspension; 2) Add 10 kg of 3-aminopropyltriethoxysilane to the obtained suspension, heat to 200° C., and react for 10 min to obtain amino-modified polystyrene microspheres; 3) Activate the goat anti-human urine microalbumin antibody with an activator to obtain activated goat anti-human urine microalbumin antibody: 20 kg of goat anti-human urine microalbumin antibody was added to PBS buffer at pH 7.2 to maintain the stability and activity of the antibody; N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in a weight ratio of 1:1 were added to the above-mentioned buffer containing the antibody as an activator, and mixed and reacted at 25° C. for 30 minutes to convert the lysine amino group of the antibody into an active ester group; After the reaction is completed, the unreacted activator and by-products are removed by dialysis; 4) 15 kg of activated goat anti-human urine microalbumin antibody and 1 kg of amino-modified polystyrene microspheres were mixed in a PBS buffer solution at pH 7.2 and reacted at 25°C for 3 hours to allow the active groups of the antibody to react chemically with the amino groups on the surface of the microspheres to form covalent bonds, thereby obtaining latex microspheres bound to goat anti-human urine microalbumin antibody; 5) Incubate the latex microspheres bound to goat anti-human urine microalbumin antibody in PBS buffer containing 3 mg / ml bovine serum albumin, pH 7.2, to block unreacted amino groups; 6) Centrifugation, filtration and washing; The particle size of the obtained latex microspheres bound with goat anti-human urinary microalbumin antibody was 120 nm.

[0025] Preparation Example 2 A latex microsphere combined with sheep anti-human urine microalbumin antibody, the preparation method of which is: 1) Mix 18kg of styrene, 14kg of divinylmethylbenzene, 46kg of water, 10kg of tert-butyl peroxide, and 12kg of vinyl bisstearamide, heat to 140°C, and then continuously introduce nitrogen at a flow rate of 10ml / min. React for 2h to obtain a styrene-divinylmethylbenzene suspension; The rest is the same as Preparation Example 1; The particle size of the obtained latex microspheres bound with goat anti-human urinary microalbumin antibody was 350 nm.

[0026] Preparation Example 3 A latex microsphere combined with sheep anti-human urine microalbumin antibody, the preparation method of which is: Different from Preparation Example 1, in step 4) of Preparation Example 3, 20 kg of activated goat anti-human urine microalbumin antibody and 1 kg of amino-modified polystyrene microspheres are mixed in a PBS buffer solution at pH 7.2, and reacted at 25° C. for 3 hours to allow the active groups of the antibody to chemically react with the amino groups on the surface of the microspheres to form covalent bonds, thereby obtaining latex microspheres bound to the goat anti-human urine microalbumin antibody. Example

[0027] Examples 1-3 A reagent for detecting trace protein in urine includes reagent R1 and reagent R2. The components of reagent R1 and reagent R2 are shown in Table 1.

[0028] Table 1 Ingredients of Examples 1-3

[0029] The latex microspheres combined with goat anti-human urinary microalbumin antibody are from Preparation Example 1, and the polyelectrolyte is chitosan.

[0030] Example 4 Different from Example 2, the latex microspheres bound with goat anti-human urine microalbumin antibody in Example 4 come from Preparation Example 2.

[0031] Example 5 Different from Example 2, the latex microspheres bound to sheep anti-human urine microalbumin antibody in Example 5 are latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 2:1.

[0032] Example 6 Different from Example 2, the latex microspheres bound to sheep anti-human urine microalbumin antibody in Example 6 are latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 3:1.

[0033] Example 7 Different from Example 2, the latex microspheres bound to sheep anti-human urine microalbumin antibody in Example 7 are latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 4:1.

[0034] Example 8 Different from Example 2, the latex microspheres bound to sheep anti-human urine microalbumin antibody in Example 8 are latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to sheep anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 1:3.

[0035] Example 9 Different from Example 6, the polyelectrolyte in Example 9 is polyacrylic acid.

[0036] Example 10 Different from Example 6, the polyelectrolyte in Example 10 is polyacrylic acid and chitosan in a weight ratio of 1:4.

[0037] Embodiment 11 Different from Example 6, the polyelectrolyte in Example 11 is polyacrylic acid and chitosan in a weight ratio of 1:7.

[0038] Example 12 Different from Example 6, the polyelectrolyte in Example 12 is polyacrylic acid and chitosan in a weight ratio of 5:1.

[0039] Comparative Example Comparative Example 1 The difference from Example 1 is that in Comparative Example 1, an equal amount of polyethylene glycol is used to replace ammonium sulfate.

[0040] Comparative Example 2 Different from Example 1, in Comparative Example 2, chitosan was replaced by an equal amount of polyethylene glycol.

[0041] Application Examples Application Example 1 A method for using a reagent for detecting trace protein in urine comprises the following steps: S1. The test was performed on Beckman's AU5800 fully automatic biochemical analyzer, with a reaction temperature of 37° C. and a wavelength of 340 nm. First, 8 μL of urine sample was incubated with 280 μL of reagent R1 of Example 1 for 300 seconds and then the photometry was performed for the first time. Then, 70 μL of reagent R2 of Example 1 was added and incubated for 300 seconds and then the photometry was performed for the second time. S2. Carefully reconstitute the freeze-dried calibrator with 1 mL of purified water, dilute the reconstituted calibrator into 6 series of calibrators according to Table 2, and draw a calibration curve; Table 2 Concentration of calibrator series ; S3. The urine microalbumin concentration in the sample is read from the calibration curve.

[0042] Application Example 2-12 Different from Application Example 1, the urine trace protein detection reagents in Application Examples 2-12 are from Examples 2-12 respectively.

[0043] Comparative Application Examples Comparative Application Example 1-2 Different from Application Example 1, the urine trace protein detection reagents in Comparative Application Examples 1-2 come from Comparative Examples 1-2 respectively.

[0044] Performance Testing Linear relationship analysis: According to the method in the application example and comparative example, a 1g / L high albumin urine sample was used as the test sample, and the urine sample was diluted into 7 different concentrations, namely 10mg / L, 100 mg / L, 200 mg / L, 400mg / L, 600mg / L, 800mg / L, and 1000mg / L, and each concentration of the above samples was tested using the detection method in the application example and comparative application example, where each test value is the average of five concentrations obtained by repeating the test five times. The relative deviation of each test value from the theoretical value is calculated. The relative deviation is the ratio of the absolute value of the difference between the test value and the theoretical value to the theoretical value. The results are shown in Table 3-16.

[0045] Table 3 Test results of application example 1

[0046] Table 4 Test results of application example 2

[0047] Table 5 Test results of application example 3

[0048] Table 6 Test results of application example 4

[0049] Table 7 Test results of application example 5

[0050] Table 8 Test results of application example 6

[0051] Table 9 Test results of application example 7

[0052] Table 10 Test results of application example 8

[0053] Table 11 Test results of application example 9

[0054] Table 12 Test results of application example 10

[0055] Table 13 Test results of application example 11

[0056] Table 14 Test results of application example 12

[0057] Table 15 Comparative test results of application example 1

[0058] Table 16 Comparative Application Example 2 Test Results

[0059] In combination with Application Examples 1-12 and Tables 3-14, it can be seen that the accuracy of detecting low-concentration samples in Application Examples 1-3 is relatively high, the accuracy of detecting high-concentration samples in Application Example 4 is relatively high, and the accuracy of detecting samples in the concentration range of 10-1000 mg / L in Application Examples 5-12 is relatively high, indicating that the present application has excellent linearity in the range of 10-1000 mg / L.

[0060] Combining Application Example 1 with Comparative Application Examples 1-2, and combining Table 3 with Tables 15-16, it can be seen that the standard deviation of the detection in Application Example 1 is smaller than that in Comparative Application Examples 1-2, which indicates that the detection accuracy of the reagent used in Application Example 1 is higher. This may be because polyethylene glycol can promote the aggregation of urine microalbumin and goat anti-human urine microalbumin antibodies, thereby increasing the sensitivity of detection; ammonium sulfate promotes the precipitation of the complex of urine microalbumin and goat anti-human urine microalbumin antibodies by changing the ionic strength of the solution, thereby increasing the turbidity; polyelectrolytes promote the formation of the complex of urine microalbumin and goat anti-human urine microalbumin antibodies through charge neutralization, thereby improving the accuracy of detection and broadening the detection range.

[0061] Combining Application Example 2, Application Example 4 and Application Example 5, and combining Table 4, Table 6 and Table 7, it can be seen that the accuracy of detecting low-concentration samples in Application Example 2 is relatively high, the accuracy of detecting high-concentration samples in Application Example 4 is relatively high, and the accuracy of detecting samples in the concentration range of 10-1000 mg / L in Application Example 5 is relatively high, which shows that microspheres of different particle sizes are conducive to broadening the linear range of detection and ensuring the accuracy of detection. This may be because the first microsphere of 100-150nm has a large specific surface area, which is conducive to the adsorption and coupling of biorecognition molecules, and can improve the binding efficiency of microspheres with urinary microalbumin, thereby improving the sensitivity of the method. The microspheres of 100-150nm have a better binding effect with urine with low-concentration urinary microalbumin, and the microspheres of 300-400nm have a better binding effect with urine with high-concentration urinary microalbumin. The combination of the two expands the linear range of the method.

[0062] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed. However, as long as it is within the scope of the claims of the present application, it shall be protected by the patent law.

Claims

1. A reagent for detecting trace protein in urine, characterized in that: Comprising reagent R1 and reagent R2; The reagent R1 comprises: Buffer 0.05-0.15 mol / L; Polyethylene glycol 20-30ml / L; Ammonium sulfate 2-4g / L; Polyelectrolytes 1-5 g / L; Preservatives 0.5-1.5g / L; Surfactant 1-3g / L; The reagent R2 comprises: Latex microspheres conjugated with goat anti-human urine microalbumin antibody 1-3 g / l; Buffer 0.05-0.15 mol / L; Preservatives 0.5-1.5 g / L; Surfactant 1-3g / L; Stabilizer 0.5-1.5g / L.

2. A reagent for detecting trace protein in urine according to claim 1, characterized in that: The latex microspheres combined with goat anti-human urinary microalbumin antibody include first microspheres with a particle size of 100-150 nm and second microspheres with a particle size of 300-400 nm.

3. A reagent for detecting trace protein in urine according to claim 2, characterized in that: The weight ratio of the first latex microspheres to the second latex microspheres is (2-3):

1.

4. A reagent for detecting trace protein in urine according to claim 1, characterized in that: The preparation method of the latex microspheres combined with sheep anti-human urine microalbumin antibody is as follows: 1) Styrene, divinylmethylbenzene, water, tert-butyl peroxide and vinyl bisstearamide are mixed, the temperature is raised to 130-150°C, and then nitrogen is continuously introduced and reacted for 2h-3h to obtain a styrene-divinylmethylbenzene suspension; 2) adding 3-aminopropyltriethoxysilane to the obtained suspension, heating to 180-200° C., and obtaining amino-modified polystyrene microspheres; 3) activating the goat anti-human urine microalbumin antibody with an activator to obtain an activated goat anti-human urine microalbumin antibody; 4) Mixing the activated goat anti-human urine microalbumin antibody and the amino-modified polystyrene microspheres in a buffer solution, so that the active groups of the antibody react chemically with the amino groups on the surface of the microspheres to form covalent bonds, thereby obtaining latex microspheres bound with the goat anti-human urine microalbumin antibody; 5) Incubate the latex microspheres bound to goat anti-human urine microalbumin antibody in a buffer containing bovine serum albumin to block unreacted amino groups; 6) Centrifugation, filtration and washing.

5. A reagent for detecting trace protein in urine according to claim 4, characterized in that: The activator is N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a weight ratio of 1:

1.

6. A reagent for detecting trace protein in urine according to claim 4, characterized in that: The weight ratio of the activated sheep anti-human urinary microalbumin antibody to the amino-modified polystyrene microspheres is (15-20):

1.

7. A reagent for detecting trace protein in urine according to claim 1, characterized in that: The polyelectrolyte is polyacrylic acid and chitosan in a weight ratio of 1:(4-7).

8. A reagent for detecting trace protein in urine according to claim 1, characterized in that: The buffer is PBS buffer.

9. Use of a reagent for detecting trace protein in urine according to any one of claims 1 to 8, characterized in that: The following steps are involved: S1. The test was performed on an automatic biochemical analyzer at a reaction temperature of 37°C and a wavelength of 340 nm. First, 8 μL of urine sample was incubated with 280 μL of reagent R1 for 300 seconds and then the light was measured for the first time. Then, 70 μL of reagent R2 was added and incubated for 300 seconds and then the light was measured for the second time. S2. Use six series of concentrations of calibrators to draw a calibration curve; S3. The urine microalbumin concentration in the sample is read from the calibration curve.

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