Urine microalbumin immunoturbidimetry kit and detection method

Abstract

The invention discloses a urine microalbumin immunoturbidimetry kit and a detection method, and belongs to the technical field of biology. The invention relates to a urine microalbumin immunoturbidimetry kit, which comprises a reagent R1, a reagent R2, a reagent R3 and a reagent R4, wherein the reagent R1 comprises trihydroxymethyl aminomethane, a coagulant, a blocking agent, sodium chloride, bovine serum albumin, trehalose, a preservative and ultrapure water; the reagent R2 is prepared from hydroxyethyl piperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, glycerol, trehalose, a preservative, a mALB monoclonal antibody and ultrapure water; the mALB monoclonal antibody is prepared by mixing a model number of UDA 9107-1, a model number of UDA 9107-2, a model number of UDA 9107-3 and a model number of UDA 9107-4 according to a mass ratio of (0.8 to 1.2): (0.8 to 1.2): (0.8 to 1.2): (0.8 to 1.2). A mixed antibody of four mALB monoclonal antibodies is specifically combined with a urine microalbumin antigen in a sample to form an antigen-antibody compound, and the antigen-antibody compound has high specificity and high sensitivity; the method has a relatively high detection range, can effectively avoid a hook effect of an ultrahigh-value sample, and can realize rapid and convenient automatic detection.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a urine microalbumin immunoturbidimetric kit and a detection method. Background Art

[0002] Urine microalbumin (mALB) refers to the presence of trace amounts of albumin in the urine. Albumin is a normal protein in the blood, but under physiological conditions, only very small amounts of albumin appear in the urine. Urine microalbumin reflects abnormal protein leakage from the kidneys. Urine microalbumin measurements reflect early kidney disease and kidney damage. Pathological increases are seen in diabetic nephropathy, hypertension, and preeclampsia during pregnancy. In the early stages of urine microalbumin is an early signal and precursor to kidney disease. At this time, kidney damage is still reversible. If treated promptly, the progression of kidney disease can be stopped or reversed. Urine microalbumin testing can be used as an indicator of renal function in response to systemic or local inflammatory reactions, such as early kidney lesions caused by urinary tract infections and other reasons; it is a predictive indicator of complications of acute pancreatitis; and people taking medications that affect renal function can also have their urine microalbumin tested to facilitate early observation of renal function and early action.

[0003] Patent application publication number CN 107741493 A discloses a kit for measuring urinary microalbumin using an immunocompetitive turbidimetric method. The kit includes anti-human albumin antibodies, human albumin-labeled latex microspheres, a buffer, and a device for separating the anti-human albumin antibodies and the human albumin-labeled latex microspheres. This kit reduces false negatives caused by high-concentration samples through competitive binding between antibodies and antigens; however, the antibodies and latex microspheres must be stored separately, adding additional steps to the procedure.

[0004] There are many methods for measuring urine mALB. Most of the early methods have been eliminated due to their complex operation, low sensitivity and poor specificity. With the development of experimental medicine, many new methods have emerged. There are four commonly used detection methods in clinical practice, as follows: (1) Radioimmunoassay (RIA) using labeled antigen (Ag + ) competes with unlabeled antigen (Ag) to bind to limited antibody (Ab) 125I is a commonly used labeling radionuclide. Its sensitivity reaches the picogram level and is the gold standard for microalbuminuria detection, but it has radioactive hazards, cross-reactions and sample processing defects, and relies on special instruments. (2) Enzyme-linked immunosorbent assay (ELISA) uses a solid phase carrier to fix antigen / antibody and uses enzyme-labeled substances to catalyze color development for quantitative detection. Methods include double antibody sandwich method, indirect method, etc. It has high sensitivity (picogram level) and is non-radioactive, but there is a hook effect and the detection time is long. (3) Immunoturbidimetry is based on the quantitative analysis of turbidity changes of antigen-antibody complexes. It is divided into three categories: transmission turbidimetry, scattering turbidimetry (rate / endpoint method) and latex turbidimetry. It is simple to operate, fast and can be automated. The transmission method requires a spectrophotometer, and the scattering method is more sensitive but requires special equipment. (4) Chemiluminescence immunoassay (ECLI) combines chemiluminescence and immune reaction. It is highly sensitive, safe, fast and can be automated, but it is expensive and has a limited linear range. Summary of the Invention

[0005] The present invention aims to provide a urine microalbumin immunoturbidimetric kit and a detection method to improve the sensitivity and detection range of the reagent detection.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] A urine microalbumin immunoturbidimetric kit comprising:

[0008] R1 reagent: tris(hydroxymethyl)aminomethane, coagulant, blocking agent, sodium chloride, bovine serum albumin, trehalose, preservative and ultrapure water;

[0009] R2 reagents: hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, glycerol, trehalose, preservatives, mALB monoclonal antibody, and ultrapure water;

[0010] Calibrator and quality control diluents: hydroxyethylpiperazine ethanesulfonic acid, trehalose, dimethyl sulfoxide, bovine serum albumin, sodium chloride, glycerol, gentamicin sulfate, and ultrapure water;

[0011] The mALB monoclonal antibodies are models UDA9107-1, UDA9107-2, UDA9107-3 and UDA9107-4, mixed in a mass ratio of (0.8-1.2): (0.8-1.2): (0.8-1.2).

[0012] Furthermore, the pH of the R1 reagent, R2 reagent, and the calibration and quality control dilution solutions is 7.0-7.4.

[0013] Furthermore, the mass ratio of tris(hydroxymethyl)aminomethane, coagulant, blocker, sodium chloride, bovine serum albumin, trehalose, preservative and ultrapure water in the R1 reagent is (0.5-0.8):(6-9):(0.01-0.03):(0.5-2):(0.05-0.2):(0.3-0.8):(0.05-0.2):(90-120).

[0014] Furthermore, the mass ratio of hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, glycerol, trehalose, preservative, mALB monoclonal antibody and ultrapure water in the R2 reagent is (1-1.5): (0.5-1.5): (0.05-0.2): (4-6): (1-3): (0.05-0.2): (0.4-0.8): (90-120).

[0015] Furthermore, the mass ratio of hydroxyethylpiperazine ethanesulfonic acid, trehalose, dimethyl sulfoxide, bovine serum albumin, sodium chloride, glycerol, gentamicin sulfate and ultrapure water in the calibration and quality control diluents is (0.2-0.5): (1-3): (8-12): (1-3): (0.5-1.4): (2-8): (0.01-0.03): (90-120).

[0016] Furthermore, the coagulant is one or a combination of PEG8000 and PEG20000.

[0017] Furthermore, the blocker is one or a combination of GU Blocker-MM1, GUBlocker-MM2, GU Blocker-MM3, GU Blocker-MM4, GU Blocker-MM5, GU Blocker-MM6, GUBlocker-MM7 and Beijing Jingda HBR-5 produced by Anhui Global Gene Technology Co., Ltd.

[0018] Furthermore, the preservative is Proclin 300.

[0019] A method for preparing a urine microalbumin immunoturbidimetric kit comprises the following steps:

[0020] Respectively mix the components of R1 reagent, R2 reagent and calibrator and quality control diluent, stir evenly at 800-1200 rpm at room temperature, and adjust the pH to obtain R1 reagent, R2 reagent and calibrator and quality control diluent.

[0021] A method for detecting urine microalbumin using an immunoturbidimetric kit comprises the following steps:

[0022] S1. Centrifuge the sample to be tested at 2-6°C at 10,000-12,000 rpm for 5-10 min, and collect the supernatant to obtain the sample;

[0023] S2. Establish a standard curve using calibrators and quality control products. Mix the sample with R1 reagent and incubate for 5-10 minutes. Then add R2 reagent. The mALB mixed monoclonal antibody will bind and aggregate with the mALB in the sample, and the OD value A1 will be detected. After 5-10 minutes of reaction, the OD value A2 will be detected again. Calculate the reaction degree ΔA = A2 - A1. Substitute the reaction degree ΔA into the calibration curve established with the calibrators and quality control products to estimate the concentration.

[0024] Furthermore, the calibrator and control diluents diluted the mALB antigen to 0 mg / L, 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, 1000 mg / L, 30 mg / L, and 200 mg / L.

[0025] Beneficial effects of the present invention:

[0026] (1) The present invention significantly improves reagent performance through the synergistic effect of multiple antibodies. A mixed antibody of four mALB monoclonal antibodies from Anhui Global Gene Technology Co., Ltd. specifically binds to the urine microalbumin antigen in the sample to form an antigen-antibody complex with high specificity. The complex can accurately detect urine microalbumin (mALB) in low, medium, and high concentration ranges with a lower detection limit and high sensitivity. The complex can be detected in a biochemical analyzer by nephelometry. A concentration-OD value calibration curve is established by calibrating the quality control product. The OD value of the complex detected by nephelometry is substituted into the calibration curve to calculate the urine microalbumin content in the sample.

[0027] (2) The four mALB monoclonal antibody mixtures used in the present invention have high sensitivity, specificity, and detection range, and can specifically detect urine microalbumin antigens at different levels, including high, medium, and low, effectively avoiding the hook effect of ultra-high-value samples. In addition, the immunoturbidimetric method can be applied to biochemical analyzers, enabling rapid and convenient automated detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] The present invention will be further described below with reference to the accompanying drawings.

[0029] Figure 1 It is a schematic diagram of the correlation curve between the detection value of the reagent of the present invention on the urine sample and the detection result of the control reagent. DETAILED DESCRIPTION

[0030] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments.

[0031] Example 1

[0032] This embodiment provides a urine microalbumin immunoturbidimetric kit, which is prepared by the following steps:

[0033] S1. Weigh the following components: R1 reagent: 0.6 parts of tris (hydroxymethyl)aminomethane, 8 parts of PEG 8000, 10.02 parts of GU Blocker-MM, 1 part of sodium chloride, 0.1 parts of bovine serum albumin, 0.5 parts of trehalose, 0.1 parts of Proclin 3000, and 100 parts of ultrapure water;

[0034] R2 reagent: 1.2 parts of hydroxyethylpiperazine ethanesulfonic acid, 1 part of sodium chloride, 0.1 part of bovine serum albumin, 5 parts of glycerol, 2 parts of trehalose, 0.1 part of Proclin 3000, 0.5 part of mALB monoclonal antibody and 100 parts of ultrapure water;

[0035] Calibrator and quality control diluent: 1.2 parts of hydroxyethylpiperazine ethanesulfonic acid, 2 parts of trehalose, 10 parts of dimethyl sulfoxide, 2 parts of bovine serum albumin, 0.9 parts of sodium chloride, 6 parts of glycerol, 0.02 parts of gentamicin sulfate and 100 parts of ultrapure water;

[0036] The mALB monoclonal antibodies are models UDA9107-1, UDA9107-2, UDA9107-3 and UDA9107-4 mixed in a mass ratio of 1:1:1:1;

[0037] S2. Uniformly mix the components of R1 reagent, R2 reagent, and calibrator and control diluents, stir at 1000 rpm at room temperature, and adjust the pH of each mixture to 7.4 to obtain R1 reagent, R2 reagent, and calibrator and control diluents. Use the calibrator and control diluents to dilute mALB antigen to 0 mg / L, 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, 1000 mg / L, 30 mg / L, and 200 mg / L.

[0038] Example 2

[0039] Compared with Example 1, the dosage of components R1 and R2 in this example is adjusted. The specific implementation steps are as follows:

[0040] S1. Weigh the following components: R1 reagent: 0.8 parts of tris (hydroxymethyl)aminomethane, 7 parts of PEG 8000, 10.01 parts of GU Blocker-MM, 0.8 parts of sodium chloride, 0.1 parts of bovine serum albumin, 0.5 parts of trehalose, 0.1 parts of Proclin 3000, and 100 parts of ultrapure water;

[0041] R2 reagent: 1 part hydroxyethylpiperazine ethanesulfonic acid, 1 part sodium chloride, 0.1 part bovine serum albumin, 5 parts glycerol, 2 parts trehalose, 0.1 part Proclin 3000, 0.4 part mALB monoclonal antibody and 100 parts ultrapure water;

[0042] Calibrator and quality control diluent: 1.2 parts of hydroxyethylpiperazine ethanesulfonic acid, 2 parts of trehalose, 10 parts of dimethyl sulfoxide, 2 parts of bovine serum albumin, 0.9 parts of sodium chloride, 6 parts of glycerol, 0.02 parts of gentamicin sulfate and 100 parts of ultrapure water;

[0043] The mALB monoclonal antibodies are models UDA9107-1, UDA9107-2, UDA9107-3 and UDA9107-4 mixed in a mass ratio of 1:1:1:1;

[0044] S2. Uniformly mix the components of R1 reagent, R2 reagent, and calibrator and control diluents, stir at 1000 rpm at room temperature, and adjust the pH of each mixture to 7.4 to obtain R1 reagent, R2 reagent, and calibrator and control diluents. Use the calibrator and control diluents to dilute mALB antigen to 0 mg / L, 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, 1000 mg / L, 30 mg / L, and 200 mg / L.

[0045] The remaining raw materials and preparation process remain the same as in Example 1.

[0046] Example 3

[0047] Compared with Example 1, the dosage of components R1 and R2 in this example is adjusted. The specific implementation steps are as follows:

[0048] S1. Weigh the following components: R1 reagent: 0.5 parts of tris (hydroxymethyl)aminomethane, 8 parts of PEG 8000, 10.03 parts of GU Blocker-MM, 1 part of sodium chloride, 0.1 parts of bovine serum albumin, 0.5 parts of trehalose, 0.1 parts of Proclin 3000, and 100 parts of ultrapure water;

[0049] R2 reagent: 1.5 parts of hydroxyethylpiperazine ethanesulfonic acid, 1.5 parts of sodium chloride, 0.1 parts of bovine serum albumin, 5 parts of glycerol, 2 parts of trehalose, 0.1 parts of Proclin 3000, 0.8 parts of mALB monoclonal antibody and 100 parts of ultrapure water;

[0050] Calibrator and quality control diluent: 1.2 parts of hydroxyethylpiperazine ethanesulfonic acid, 2 parts of trehalose, 10 parts of dimethyl sulfoxide, 2 parts of bovine serum albumin, 0.9 parts of sodium chloride, 6 parts of glycerol, 0.02 parts of gentamicin sulfate and 100 parts of ultrapure water;

[0051] The mALB monoclonal antibodies are models UDA9107-1, UDA9107-2, UDA9107-3 and UDA9107-4 mixed in a mass ratio of 1:1:1:1;

[0052] S2. Uniformly mix the components of R1 reagent, R2 reagent, and calibrator and control diluents, stir at 1000 rpm at room temperature, and adjust the pH of each mixture to 7.4 to obtain R1 reagent, R2 reagent, and calibrator and control diluents. Use the calibrator and control diluents to dilute mALB antigen to 0 mg / L, 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, 1000 mg / L, 30 mg / L, and 200 mg / L.

[0053] The remaining raw materials and preparation process remain the same as in Example 1.

[0054] Example 4

[0055] This embodiment provides a method for detecting urine microalbumin using an immunoturbidimetric kit, comprising the following steps:

[0056] S1. Centrifuge the sample at 12000 rpm at 4°C for 5 min, collect the supernatant, and obtain the sample.

[0057] S2. Establish a standard curve using calibrators and quality control products. Mix the sample with R1 reagent and incubate for 5 minutes. Then add R2 reagent. The mALB mixed monoclonal antibody will bind and aggregate with the mALB in the sample, and the OD value A1 will be measured. After 5 minutes of reaction, the OD value A2 will be measured again. The reaction degree ΔA = A2 - A1 is calculated. The reaction degree ΔA is substituted into the calibration curve established with the calibrators and quality control products to estimate the concentration.

[0058] The calibrator and quality control diluents were used to dilute mALB antigen to 0 mg / L, 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, 1000 mg / L, 30 ± 6 mg / L, and 200 ± 40 mg / L.

[0059] Performance Testing

[0060] 1. The urine microalbumin immunoturbidimetric kit prepared by the present invention was used to compare the urine sample test values ​​according to the above detection method. The urine sample test values ​​and the control reagent test results are shown in Table 1, and the correlation curve diagram is shown in Figure 1 As shown:

[0061] Table 1

[0062]

[0063]

[0064] Combined with Table 1 and Figure 1 It can be seen that the test values ​​of the reagents in this method have good clinical correlation, R 2 =0.9946, which is highly consistent with the detection results of the control reagent and is suitable for quantitative screening and dynamic monitoring of urine microalbumin.

[0065] 2. Reagent Accuracy Test: The urine microalbumin immunoturbidimetric kit prepared by the present invention was tested for repeatability of quality control products according to the above-mentioned detection method. The results are shown in Table 2:

[0066] Table 2

[0067]

[0068]

[0069] From the data in Table 2, it can be concluded that the low-value quality control: CV = 6%, which meets clinical requirements; the high-value quality control: CV = 5%, has excellent stability and is suitable for high-concentration sample detection; this indicates that the reagent of the present invention has good precision.

[0070] The above disclosures are only a few specific embodiments of the present invention. However, the embodiments of the present invention are not limited thereto. Any changes that can be conceived by those skilled in the art should fall within the scope of protection of the present invention.

Claims

1. A urine microalbumin immunoturbidimetric kit, characterized in that: include: R1 reagent: tris(hydroxymethyl)aminomethane, coagulant, blocking agent, sodium chloride, bovine serum albumin, trehalose, preservative and ultrapure water; R2 reagents: hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, glycerol, trehalose, preservatives, mALB monoclonal antibody, and ultrapure water; Calibrator and quality control diluents: hydroxyethylpiperazine ethanesulfonic acid, trehalose, dimethyl sulfoxide, bovine serum albumin, sodium chloride, glycerol, gentamicin sulfate, and ultrapure water; The mALB monoclonal antibodies are models UDA9107-1, UDA9107-2, UDA9107-3 and UDA9107-4, mixed in a mass ratio of (0.8-1.2): (0.8-1.2): (0.8-1.2).

2. A urine microalbumin immunoturbidimetric kit according to claim 1, characterized in that: The pH of the R1 reagent, R2 diluent, and calibrator and quality control diluents is 7.0-7.

4.

3. A urine microalbumin immunoturbidimetric kit according to claim 1, characterized in that: The mass ratio of tris(hydroxymethyl)aminomethane, coagulant, blocking agent, sodium chloride, bovine serum albumin, trehalose, preservative and ultrapure water in the R1 reagent is (0.5-0.8):(6-9):(0.01-0.03):(0.5-2):(0.05-0.2):(0.3-0.8):(0.05-0.2):(90-120).

4. A urine microalbumin immunoturbidimetric kit according to claim 1, characterized in that: The mass ratio of hydroxyethylpiperazine ethanesulfonic acid, sodium chloride, bovine serum albumin, glycerol, trehalose, preservative, mALB monoclonal antibody and ultrapure water in the R2 diluent is (1-1.5): (0.5-1.5): (0.05-0.2): (4-6): (1-3): (0.05-0.2): (0.4-0.8): (90-120).

5. A urine microalbumin immunoturbidimetric kit according to claim 1, characterized in that: The mass ratio of hydroxyethylpiperazine ethanesulfonic acid, trehalose, dimethyl sulfoxide, bovine serum albumin, sodium chloride, glycerol, gentamicin sulfate and ultrapure water in the calibration and quality control diluents is (0.2-0.5): (1-3): (8-12): (1-3): (0.5-1.4): (2-8): (0.01-0.03): (90-120).

6. A urine microalbumin immunoturbidimetric kit according to claim 1, characterized in that: The coagulant is one or a combination of PEG8000 and PEG20000.

7. A urine microalbumin immunoturbidimetric kit according to claim 1, characterized in that: The blocker is one or a combination of GU Blocker-MM1, GU Blocker-MM2, GU Blocker-MM3, GUBlocker-MM4, GU Blocker-MM5, GU Blocker-MM6, GU Blocker-MM7 and Beijing Jingda HBR-5 produced by Anhui Global Gene Technology Co., Ltd.

8. A urine microalbumin immunoturbidimetric kit according to claim 1, characterized in that: The preservative is Proclin 300.

9. A method for preparing the urine microalbumin immunoturbidimetric kit according to any one of claims 1 to 8, characterized in that: The following steps are involved: Respectively mix the components of R1 reagent, R2 reagent and calibrator and quality control diluent, stir evenly at 800-1200 rpm at room temperature, and adjust the pH to obtain R1 reagent, R2 reagent and calibrator and quality control diluent.

10. A method for detecting urine microalbumin using an immunoturbidimetric kit, characterized in that: The following steps are involved: S1. Centrifuge the sample to be tested at 2-6°C at 10,000-12,000 rpm for 5-10 min, and collect the supernatant to obtain the sample; S2. Establish a standard curve using calibrators and quality control products. Mix the sample with R1 reagent and incubate for 5-10 minutes. Then add R2 reagent. The mALB mixed monoclonal antibody will bind and aggregate with the mALB in the sample, and the OD value A1 will be measured. After 5-10 minutes of reaction, the OD value A2 will be measured again. The reaction degree ΔA is calculated as A2-A1. Substitute the reaction degree ΔA into the calibration curve established with the calibrators and quality control products to estimate the concentration. The calibrator and control diluents were used to dilute mALB antigen to 0 mg / L, 30 mg / L, 60 mg / L, 120 mg / L, 240 mg / L, 1000 mg / L, 30 mg / L, and 200 mg / L.

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