A reagent for detecting trace protein in urine and its application

Abstract

The present application relates to the field of medical detection technology, in particular to a reagent for detecting trace protein in urine and its application. A reagent for detecting trace protein in urine, comprising reagent R1 and reagent R2; the reagent R1 comprises: 0.05-0.15 mol / L buffer; 20-30 ml / L polyethylene glycol; 2-4 g / L ammonium sulfate; 1-5 g / L polyelectrolyte; 0.5-1.5 g / L preservative; 1-3 g / L surfactant; the reagent R2 comprises: 1-3 g / L latex microspheres bound to sheep anti-human urine microalbumin antibody; 0.05-0.15 mol / L buffer; 0.5-1.5 g / L preservative; 1-3 g / L surfactant; and 0.5-1.5 g / L stabilizer. 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 0.01-1 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 prevalent protein in human plasma, maintaining the body's nutrition and osmotic pressure. Under normal circumstances, only a small amount of albumin can be filtered through the glomerular basement membrane, while 95% is reabsorbed in the proximal convoluted tubules. The albumin present in urine is called microalbuminuria. If the barrier function of the glomerular basement membrane is damaged, the amount of albumin filtered through the basement membrane increases significantly, and the amount of microalbuminuria will also rise significantly. Microalbuminuria is the earliest biochemical marker of diabetic nephropathy and hypertensive nephropathy. Its measurement is of revolutionary significance for the early diagnosis and improved prognosis of diabetic nephropathy. It also has important diagnostic value for hypertensive nephropathy, eclampsia, and renal damage caused by various toxic substances.

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

[0004] The most commonly used method for urine microalbumin detection 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 method for measuring antigen-antibody binding. Its basic principle is that when antigen and antibody react in a specific dilution system at the appropriate ratio, the resulting soluble immune complex, under the action of a coagulation promoter in the dilution system, precipitates from the liquid phase, forming microparticles and causing turbidity in the reaction solution. When the antibody concentration is fixed, the amount of immune complex formed increases with 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 antigen content in the test sample can be calculated.

[0006] Regarding the existing immunoturbidimetric method, the applicant found that the linear range of detection is small and it is necessary to conduct research to expand the linear range of 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 reagent R1 and reagent R2;

[0010] The reagent R1 includes:

[0011] Buffer 0.05-0.15 mol / L;

[0012] Polyethylene glycol 20-30 ml / L;

[0013] Ammonium sulfate 2-4g / L;

[0014] Polyelectrolytes 1-5 g / L;

[0015] Preservative 0.5-1.5g / L;

[0016] Surfactant 1-3g / L;

[0017] The reagent R2 includes:

[0018] Latex microspheres conjugated with goat anti-human urine microalbumin antibody 1-3 g / l;

[0019] Buffer 0.05-0.15 mol / L;

[0020] Preservative 0.5-1.5g / L;

[0021] Surfactant 1-3g / L;

[0022] Stabilizer 0.5-1.5g / L.

[0023] By adopting the above technical solution, goat anti-human urinary microalbumin antibodies and urinary microalbumin react in a specifically diluted system to form a soluble immune complex. Under the combined action of polyethylene glycol, ammonium sulfate, and polyelectrolytes in the dilution system, particles are directly formed from the liquid, causing turbidity in the reaction solution, from which the urinary microalbumin content in the sample can be calculated. Polyethylene glycol can promote the aggregation of urinary microalbumin and goat anti-human urinary microalbumin antibodies, increasing the sensitivity of the test; ammonium sulfate promotes the precipitation of the urinary microalbumin and goat anti-human urinary microalbumin antibody complex by changing the ionic strength of the solution, thereby increasing turbidity; and polyelectrolytes promote the formation of the urinary microalbumin and goat anti-human urinary microalbumin antibody complex through charge neutralization. While ensuring the sensitivity of the test, the linear range of the test is maximized, reaching a linear range of 0.01-1g / L.

[0024] Furthermore, the latex microspheres bound to goat anti-human urinary microalbumin antibodies include first microspheres with a particle size of 100-150 nm and second microspheres with a particle size of 300-400 nm.

[0025] Furthermore, the weight ratio of the first latex microspheres to the second latex microspheres is (2-3):1.

[0026] By adopting this technical solution, the large specific surface area of ​​the 100-150 nm first microspheres facilitates the adsorption and coupling of biorecognition molecules, increasing the binding efficiency between the microspheres and urinary microalbumin, thereby improving the sensitivity of the method. Microspheres of 100-150 nm bind more effectively to urine with low urinary microalbumin concentrations, while microspheres of 300-400 nm bind more effectively to urine with high urinary microalbumin concentrations. Combining these two microspheres expands the linear range of the method.

[0027] Furthermore, the preparation method of the latex microspheres bound to the goat anti-human urine microalbumin antibody is as follows:

[0028] 1) Styrene, divinylmethylbenzene, water, tert-butyl peroxide, and vinyl bisstearamide were mixed, heated to 130-150°C, and then nitrogen was continuously introduced and reacted for 2-3 hours to obtain a styrene-divinylmethylbenzene suspension;

[0029] 2) adding 3-aminopropyltriethoxysilane to the obtained suspension and heating it to 180-200° C. to obtain amino-modified polystyrene microspheres;

[0030] 3) activating the goat anti-human urine microalbumin antibody with an activating agent to obtain an activated goat anti-human urine microalbumin antibody;

[0031] 4) Mixing the activated goat anti-human urine microalbumin antibody with amino-modified polystyrene microspheres in a buffer solution 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;

[0032] 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;

[0033] 6) Centrifugation, filtration, and washing.

[0034] Furthermore, the activator is N-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide in a weight ratio of 1:1.

[0035] Furthermore, the weight ratio of the activated goat anti-human urinary microalbumin antibody to the amino-modified polystyrene microspheres is (15-20):1.

[0036] Furthermore, the polyelectrolyte is polyacrylic acid and chitosan in a weight ratio of 1:(4-7).

[0037] Furthermore, the buffer solution is PBS buffer solution.

[0038] In a second aspect, the present application provides an application of a reagent for detecting trace protein in urine, adopting the following technical solution.

[0039] An application of a reagent for detecting trace protein in urine comprises the following steps:

[0040] S1. Detection was performed on an automated biochemical analyzer at 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, followed by a first photometric measurement. Then, 70 μL of reagent R2 was added and incubated for 300 seconds, followed by a second photometric measurement.

[0041] S2. Create a calibration curve using six concentration series of calibrators.

[0042] S3. The urine microalbumin concentration in the sample is read from the calibration curve.

[0043] In summary, this application has the following beneficial effects:

[0044] This application uses polyethylene glycol to promote the aggregation of urinary microalbumin and goat anti-human urinary microalbumin antibodies, thereby increasing the sensitivity of the test. Ammonium sulfate promotes the precipitation of the complex of urinary microalbumin and goat anti-human urinary microalbumin antibodies by changing the ionic strength of the solution, thereby increasing turbidity. Polyelectrolytes promote the formation of the complex of urinary microalbumin and goat anti-human urinary microalbumin antibodies through charge neutralization. While ensuring the sensitivity of the test, the linear range of the test is widened to the greatest extent, and the linear range of the test can reach 0.01-1g / L. DETAILED DESCRIPTION

[0045] The present application is further described in detail below with reference to the embodiments.

[0046] Preparation examples of raw materials and intermediates

[0047] raw material

[0048] The raw materials in the examples of this application can be obtained commercially:

[0049] Buffer, PBS buffer 0.01 mol / L;

[0050] Polyethylene glycol, PEG-2000, analytical grade;

[0051] Ammonium sulfate, analytical grade;

[0052] preservative, sodium azide;

[0053] surfactant, ethylphenyl polyethylene glycol;

[0054] Stabilizer, disodium EDTA;

[0055] Styrene, analytical grade;

[0056] Divinylmethylbenzene, analytical grade;

[0057] Tert-butyl peroxide, analytical grade;

[0058] Vinyl bisstearamide, analytical grade;

[0059] 3-Aminopropyltriethoxysilane, analytical grade;

[0060] N-Hydroxysuccinimide, analytical grade;

[0061] Ethyl-3-(3-dimethylaminopropyl)carbodiimide, analytical grade;

[0062] Polyacrylic acid, average molecular weight 2000;

[0063] Chitosan, analytical grade.

[0064] Preparation Example

[0065] Preparation Example 1

[0066] A latex microsphere combined with sheep anti-human urine microalbumin antibody, the preparation method of which is as follows:

[0067] 1) Mix 15 kg of styrene, 15 kg of divinylmethylbenzene, 40 kg of water, 15 kg of tert-butyl peroxide, and 15 kg of vinyl bisstearamide, heat to 140°C, and continuously introduce nitrogen at a flow rate of 10 ml / min. React for 2 hours to obtain a styrene-divinylmethylbenzene suspension.

[0068] 2) Add 10 kg of 3-aminopropyltriethoxysilane to the obtained suspension, raise the temperature to 200°C, and react for 10 minutes to obtain amino-modified polystyrene microspheres;

[0069] 3) Activate the goat anti-human urine microalbumin antibody with an activator to obtain activated goat anti-human urine microalbumin antibody:

[0070] 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;

[0071] 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 activators, and the mixture was mixed and reacted at 25°C for 30 minutes to convert the lysine amino groups of the antibody into active ester groups;

[0072] After the reaction is completed, the unreacted activator and by-products are removed by dialysis;

[0073] 4) Mix 15 kg of activated goat anti-human urine microalbumin antibody with 1 kg of amino-modified polystyrene microspheres in PBS buffer (pH 7.2) and incubate at 25°C for 3 hours to allow the active groups of the antibody to react with the amino groups on the microsphere surface to form covalent bonds, resulting in goat anti-human urine microalbumin antibody-bound latex microspheres.

[0074] 5) Incubate the latex microspheres bound to goat anti-human urine microalbumin antibody in PBS buffer (pH 7.2) containing 3 mg / ml bovine serum albumin to block unreacted amino groups.

[0075] 6) Centrifugation, filtration, and washing;

[0076] The particle size of the obtained latex microspheres bound to the goat anti-human urinary microalbumin antibody was 120 nm.

[0077] Preparation Example 2

[0078] A latex microsphere combined with sheep anti-human urine microalbumin antibody, the preparation method of which is as follows:

[0079] 1) Mix 18 kg of styrene, 14 kg of divinylmethylbenzene, 46 kg of water, 10 kg of tert-butyl peroxide, and 12 kg of vinyl bisstearamide, heat to 140°C, and continuously introduce nitrogen at a flow rate of 10 ml / min. React for 2 hours to obtain a styrene-divinylmethylbenzene suspension.

[0080] The rest is the same as Preparation Example 1;

[0081] The particle size of the obtained latex microspheres bound to the goat anti-human urinary microalbumin antibody was 350 nm.

[0082] Preparation Example 3

[0083] A latex microsphere combined with sheep anti-human urine microalbumin antibody, the preparation method of which is as follows:

[0084] 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 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 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

[0085] Examples 1-3

[0086] 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.

[0087] Table 1 Ingredients of Examples 1-3

[0088]

[0089] The latex microspheres bound with goat anti-human urinary microalbumin antibody are from Preparation Example 1, and the polyelectrolyte is chitosan.

[0090] Example 4

[0091] The difference from Example 2 is that the latex microspheres bound to the goat anti-human urine microalbumin antibody in Example 4 come from Preparation Example 2.

[0092] Example 5

[0093] Different from Example 2, the latex microspheres bound to goat anti-human urine microalbumin antibody in Example 5 are latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 2:1.

[0094] Example 6

[0095] Different from Example 2, the latex microspheres bound to goat anti-human urine microalbumin antibody in Example 6 are latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 3:1.

[0096] Example 7

[0097] Different from Example 2, the latex microspheres bound to goat anti-human urine microalbumin antibody in Example 7 are latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 4:1.

[0098] Example 8

[0099] Different from Example 2, the latex microspheres bound to goat anti-human urine microalbumin antibody in Example 8 are latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 1 and latex microspheres bound to goat anti-human urine microalbumin antibody from Preparation Example 2 in a weight ratio of 1:3.

[0100] Example 9

[0101] Different from Example 6, the polyelectrolyte in Example 9 is polyacrylic acid.

[0102] Example 10

[0103] Different from Example 6, the polyelectrolyte in Example 10 is polyacrylic acid and chitosan in a weight ratio of 1:4.

[0104] Example 11

[0105] Different from Example 6, the polyelectrolyte in Example 11 is polyacrylic acid and chitosan in a weight ratio of 1:7.

[0106] Example 12

[0107] Different from Example 6, the polyelectrolyte in Example 12 is polyacrylic acid and chitosan in a weight ratio of 5:1.

[0108] Comparative Example

[0109] Comparative Example 1

[0110] The difference from Example 1 is that in Comparative Example 1, an equal amount of polyethylene glycol was used to replace ammonium sulfate.

[0111] Comparative Example 2

[0112] The difference from Example 1 is that in Comparative Example 2, chitosan was replaced by an equal amount of polyethylene glycol.

[0113] Application Examples

[0114] Application Example 1

[0115] A method for using a reagent for detecting trace protein in urine comprises the following steps:

[0116] S1. Detection was performed on a Beckman AU5800 fully automated 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 from Example 1 for 300 seconds, followed by a first photometric measurement. Then, 70 μL of reagent R2 from Example 1 was added and incubated for 300 seconds, followed by a second photometric measurement.

[0117] S2. Carefully reconstitute the lyophilized calibrator with 1 mL of purified water. Dilute the reconstituted calibrator into six series of calibrators as shown in Table 2 and plot a calibration curve.

[0118] Table 2 Concentration of calibrator series

[0119] ;

[0120] S3. The urine microalbumin concentration in the sample is read from the calibration curve.

[0121] Application Example 2-12

[0122] Different from Application Example 1, the urine trace protein detection reagents in Application Examples 2-12 are from Examples 2-12, respectively.

[0123] Comparative Application Examples

[0124] Comparative Application Examples 1-2

[0125] Different from Application Example 1, the urine trace protein detection reagents in Comparative Application Examples 1-2 are from Comparative Examples 1-2, respectively.

[0126] Performance testing

[0127] Linear Relationship Analysis: Following the methods described in the Application Examples and Comparative Examples, a 1 g / L high-albuminuria urine sample was used as the test sample. The urine sample was diluted to seven different concentrations: 10 mg / L, 100 mg / L, 200 mg / L, 400 mg / L, 600 mg / L, 800 mg / L, and 1000 mg / L. Each concentration of the sample was tested using the detection methods described in the Application Examples and Comparative Examples. Each test value was the average of the five concentrations obtained by repeating the test five times. The relative deviation of each test value from the theoretical value was 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.

[0128] Table 3 Test results of application example 1

[0129]

[0130] Table 4 Test results of application example 2

[0131]

[0132] Table 5 Test results of application example 3

[0133]

[0134] Table 6 Test results of application example 4

[0135]

[0136] Table 7 Test results of application example 5

[0137]

[0138] Table 8 Test results of application example 6

[0139]

[0140] Table 9 Test results of application example 7

[0141]

[0142] Table 10 Test results of application example 8

[0143]

[0144] Table 11 Test results of application example 9

[0145]

[0146] Table 12 Test results of application example 10

[0147]

[0148] Table 13 Test results of application example 11

[0149]

[0150] Table 14 Test results of application example 12

[0151]

[0152] Table 15 Comparative test results of application example 1

[0153]

[0154] Table 16 Comparative test results of application example 2

[0155]

[0156] 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.

[0157] Combining Application Example 1 with Comparative Example 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 Example 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.

[0158] 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 beneficial to broadening the linear range of detection and ensuring the accuracy of detection. This may be because the first microspheres of 100-150nm have a large specific surface area, which is beneficial 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 on urine with low-concentration urinary microalbumin, and the microspheres of 300-400nm have a better binding effect on urine with high-concentration urinary microalbumin. The combination of the two expands the linear range of the method.

[0159] 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 non-creative modifications to the present embodiment as needed, but as long as they are within the scope of the claims of the present application, they are 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 includes: Buffer 0.05-0.15 mol / L; Polyethylene glycol 20-30 ml / L; Ammonium sulfate 2-4g / L; Polyelectrolytes 1-5 g / L; Preservative 0.5-1.5g / L; Surfactant 1-3g / L; The reagent R2 includes: Latex microspheres conjugated with goat anti-human urine microalbumin antibody 1-3 g / l; Buffer 0.05-0.15 mol / L; Preservative 0.5-1.5g / L; Surfactant 1-3g / L; Stabilizer 0.5-1.5g / L; The latex microspheres bound to goat anti-human urinary microalbumin antibodies include first microspheres with a particle size of 100-150 nm and second microspheres with a particle size of 300-400 nm; The weight ratio of the first microspheres to the second microspheres is (2-3):1; The preparation method of the latex microspheres combined with goat anti-human urine microalbumin antibody is as follows: 1) Styrene, divinylmethylbenzene, water, tert-butyl peroxide, and vinyl bisstearamide were mixed, heated to 130-150°C, and then nitrogen was continuously introduced and reacted for 2-3 hours to obtain a styrene-divinylmethylbenzene suspension; 2) adding 3-aminopropyltriethoxysilane to the obtained suspension and heating it to 180-200° C. to obtain amino-modified polystyrene microspheres; 3) activating the goat anti-human urine microalbumin antibody with an activating agent to obtain an activated goat anti-human urine microalbumin antibody; 4) Mixing the activated goat anti-human urine microalbumin antibody with amino-modified polystyrene microspheres in a buffer solution 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; 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; The polyelectrolyte is polyacrylic acid and chitosan in a weight ratio of 1:(4-7); The buffer solution is PBS buffer; The surfactant is ethylphenyl polyethylene glycol; The stabilizer is disodium edetate.

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

1.

3. A urine trace protein detection reagent according to claim 1, characterized in that: The weight ratio of the activated goat anti-human urinary microalbumin antibody to the amino-modified polystyrene microspheres is (15-20):

1.

4. A use of a urine trace protein detection reagent according to any one of claims 1 to 3, characterized in that: The following steps are involved: S1. Detection was performed on an automated biochemical analyzer at 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, followed by a first photometric measurement. Then, 70 μL of reagent R2 was added and incubated for 300 seconds, followed by a second photometric measurement. S2. Create a calibration curve using six concentration series of calibrators. S3. The urine microalbumin concentration in the sample is read from the calibration curve; The application is for non-diagnostic purposes.

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