Urine transferrin detection kit and preparation method

Abstract

The present invention provides a urine transferrin detection kit and a preparation method thereof, and relates to the technical field of biological detection. The urine transferrin detection kit comprises a reagent R 1 and reagent R 2 ; The reagent R 1 The reagent R comprises: citric acid buffer, sodium azide, polyethylene glycol and surfactant A; 2 The invention comprises: a citric acid buffer, latex particles coated with rabbit anti-human transferrin antibody, sodium azide, a surfactant B and a stabilizer; the surfactant A comprises sorbitan fatty acid ester, the surfactant B comprises Tween-20 and lauryl alcohol polyoxyethylene ether, and the stabilizer comprises disodium ethylenediaminetetraacetate, mannitol and Frank's gum. The kit of the invention has excellent precision and stability in detecting urine transferrin.

Description

Technical Field

[0001] The invention relates to the technical field of biological detection, and in particular to a urine transferrin detection kit and a preparation method thereof. Background Art

[0002] Transferrin is a protein formed in the liver with a relatively large molecular weight. It is generally not filtered through the glomerular basement membrane. If the kidney is damaged early and the glomerulus is damaged, the permeability of the filtration membrane will increase, and the transferrin in the blood can be excreted through the damaged basement membrane and can be detected in the urine. Increased urinary transferrin confirms that the kidney is damaged and the damage is relatively serious. Quantitative detection of urinary transferrin can more sensitively reflect the damage to the glomerular charge barrier.

[0003] At present, the methods for detecting urine transferrin include enzyme-linked immunosorbent assay, immunoturbidimetry, colloidal gold method and radioimmunoassay, among which, the enzyme-linked immunosorbent assay has low sensitivity, the colloidal gold method has low sensitivity and is prone to false positives, and the radioimmunoassay is prone to radioactive pollution and damage to the human body. Therefore, in comparison, the immunoturbidimetry is widely used due to its high stability and repeatability. The prior art, such as patent CN116359513A, discloses a kit for detecting urine transferrin, including: reagent 1 and reagent 2; wherein reagent 1 includes tris(hydroxymethyl)aminomethane, sodium chloride, Triton X-100, bovine serum albumin, and liquid biological preservative; reagent 2 includes tris(hydroxymethyl)aminomethane, sodium chloride, anti-human transferrin antibody latex particles, and bovine serum albumin. In addition, reagent 2 also includes trehalose, dihydroquercetin and hydroxyproline. The repeatability and uniformity of the detection of the kit are excellent, and the linear range is relatively wide. However, this invention focused on the repeatability, batch difference and linear range of the kit, but did not focus on the precision and stability of the kit.

[0004] For another example, patent CN105044352A discloses a urine transferrin immunoturbidimetric reagent and a preparation method thereof, wherein the kit includes freeze-dried R1 reagent and R2 reagent fixed in the same container, wherein the freeze-dried R1 reagent and R2 reagent are freeze-dried by adding excipients and protective agents to liquid R1 reagent and R2 reagent; wherein the liquid R1 reagent is a buffer solution containing PEG, and the liquid R2 reagent is a latex coupled with anti-human transferrin polyclonal or paired monoclonal antibodies, or colloidal gold coupled with anti-human transferrin polyclonal antibodies or paired monoclonal antibodies. The invention is easy to operate, saves time, and is convenient for transportation and storage, thereby solving the transportation problem in cold areas and increasing the stability of the reagent. The above invention focuses on the fact that most of the current immunoturbidimetric detection kits are liquid double reagents, but freezing and thawing will affect the performance of the reagents, thereby causing problems in their transportation and storage. In addition to adding some protective agents, the important coping method focuses on freeze-drying the kit to achieve this, which has high requirements for instruments and equipment and high costs.

[0005] In view of the problems existing in the prior art, it is necessary to find a urine transferrin detection kit and preparation method with excellent overall performance such as precision and stability. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides a urine transferrin detection kit (latex enhanced immunoturbidimetry) and a preparation method thereof. The kit has excellent precision and stability in detecting urine transferrin.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a urine transferrin detection kit, comprising a reagent R1 and a reagent R2;

[0009] The reagent R1 comprises: citric acid buffer, sodium azide, polyethylene glycol and surfactant A;

[0010] The reagent R2 comprises: a citric acid buffer, latex particles coated with rabbit anti-human transferrin antibodies, sodium azide, a surfactant B and a stabilizer;

[0011] The surfactant A comprises sorbitan fatty acid ester, the surfactant B comprises Tween-20 and lauryl alcohol polyoxyethylene ether, and the stabilizer comprises disodium ethylenediaminetetraacetate, mannitol and Frank's gum.

[0012] Furthermore, the polyethylene glycol is polyethylene glycol 6000.

[0013] Furthermore, the reagent R1 comprises: 40-60 mmol / L of citric acid buffer, 1-3 g / L of sodium azide, 5-50 g / L of polyethylene glycol and 5-20 g / L of surfactant A.

[0014] Preferably, the reagent R1 comprises: 50 mmol / L citric acid buffer, 1 g / L sodium azide, 20 g / L polyethylene glycol and 10 g / L surfactant A.

[0015] Furthermore, the reagent R2 includes: 40-60 mmol / L of citric acid buffer, 1-4 g / L of latex particles coated with rabbit anti-human transferrin antibodies, 1-3 g / L of sodium azide, 10-30 g / L of surfactant B and 10-50 g / L of stabilizer.

[0016] Preferably, the reagent R2 comprises: 50 mmol / L of citric acid buffer, 2 g / L of latex particles coated with rabbit anti-human transferrin antibody, 1 g / L of sodium azide, 20 g / L of surfactant B and 20 g / L of stabilizer.

[0017] Furthermore, the urine transferrin detection kit also includes a calibrator and a quality control product, and the calibrator and the quality control product both include phosphate buffer, sodium azide and TRF antigen (urine transferrin).

[0018] Furthermore, the calibrator includes 40 mmol / L phosphate buffer, 2 g / L sodium azide and 0-200 mmol / L TRF antigen (urinary transferrin), and the quality control includes 40 mmol / L phosphate buffer, 2 g / L sodium azide and 5-100 mmol / L TRF antigen (urinary transferrin).

[0019] Furthermore, the surfactant B comprises Tween-20 and lauryl alcohol polyoxyethylene ether in a weight ratio of (3-6):(1-2).

[0020] Preferably, the surfactant B comprises Tween-20 and lauryl alcohol polyoxyethylene ether in a weight ratio of 4:1.

[0021] Furthermore, the stabilizer comprises disodium ethylenediaminetetraacetic acid, mannitol and Frank's gum in a weight ratio of (1-3):(2-4):1.

[0022] Preferably, the stabilizer comprises disodium ethylenediaminetetraacetic acid, mannitol and Frank's gum in a weight ratio of 2:2:1.

[0023] Furthermore, the present invention also provides a method for preparing the above-mentioned urine transferrin detection kit (latex enhanced immunoturbidimetry), comprising the following steps:

[0024] Prepare reagent R1: mix citric acid buffer, sodium azide, polyethylene glycol and surfactant A;

[0025] Preparation of reagent R2: Mix citric acid buffer, latex particles coated with rabbit anti-human transferrin antibody, sodium azide, surfactant B and stabilizer.

[0026] In some specific embodiments, the preparation method of the urine transferrin detection kit (latex enhanced immunoturbidimetry) comprises the following steps:

[0027] Prepare reagent R1: mix citric acid buffer, sodium azide, polyethylene glycol and sorbitan fatty acid ester;

[0028] Preparation of reagent R2: Mix citric acid buffer, latex particles coated with rabbit anti-human transferrin antibody, sodium azide, Tween-20, polyoxyethylene lauryl alcohol ether, disodium ethylenediaminetetraacetic acid, mannitol and Frank's gel.

[0029] Furthermore, the preparation method also includes the configuration of standard products and quality control products, and the specific method is to mix the raw materials in the standard products or quality control products.

[0030] The technical effects achieved by the present invention are:

[0031] The kit of the present invention adopts latex enhanced immunoturbidimetry and is used for quantitatively determining the content of transferrin in human urine in vitro to assist clinical diagnosis. Specifically, in the reaction system, the transferrin reagent is a polystyrene latex particle suspension of uniform size, and the particle surface is coated with IgG specific for human transferrin. When a sample containing transferrin is mixed with the suspension, an obvious coagulation reaction occurs, and the content of transferrin in the sample can be calculated by measuring the change in absorbance at 570nm. In addition, the present invention further optimizes the double reagents R1 and R2 in the kit, so that the kit finally obtained has excellent precision and stability in detecting urine transferrin. DETAILED DESCRIPTION

[0032] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0033] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing specific embodiments rather than for limiting the scope of protection of the present invention.

[0034] When the embodiment gives a numerical range, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those of ordinary skill in the art to which the present invention belongs.

[0035] It is worth noting that the latex particles coated with rabbit anti-human transferrin antibodies used in the present invention were purchased from Shaoxing Chuangye Biotechnology Co., Ltd., and the remaining raw materials are common commercial products, so their sources are not specifically limited.

[0036] Example 1

[0037] A urine transferrin detection kit (latex enhanced immunoturbidimetric method) comprises reagent R1, reagent R2, quality control products and calibration products.

[0038] Specifically, reagent R1 contains: 40 mmol / L of citric acid buffer, 1 g / L of sodium azide, 5 g / L of polyethylene glycol 6000, and 5 g / L of sorbitan fatty acid ester.

[0039] Reagent R2 contains: 40 mmol / L citric acid buffer, 1 g / L latex particles coated with rabbit anti-human transferrin antibody, 1 g / L sodium azide, 7.55 g / L Tween-20, 2.5 g / L lauryl alcohol polyoxyethylene ether surfactant, 12.5 g / L disodium ethylenediaminetetraacetic acid, 25 g / L mannitol and 12.5 g / L Frank's gel.

[0040] The calibrator contains: phosphate buffer 40mmol / L, sodium azide 2g / L and TRF antigen (urinary transferrin) 5mmol / L.

[0041] The quality control product contains: phosphate buffer 40mmol / L, sodium azide 2g / L and TRF antigen (urinary transferrin) 5mmol / L.

[0042] The preparation method of the above-mentioned kit is specifically as follows:

[0043] Prepare reagent R1: mix citric acid buffer, sodium azide, polyethylene glycol and sorbitan fatty acid ester;

[0044] Preparation of reagent R2: Mix citric acid buffer, latex particles coated with rabbit anti-human transferrin antibody, sodium azide, Tween-20, polyoxyethylene lauryl alcohol ether, disodium ethylenediaminetetraacetic acid, mannitol and Frank's gel.

[0045] Example 2

[0046] A urine transferrin detection kit (latex enhanced immunoturbidimetric method) comprises reagent R1, reagent R2, quality control products and calibration products.

[0047] Specifically, reagent R1 contains: 60 mmol / L of citric acid buffer, 3 g / L of sodium azide, 50 g / L of polyethylene glycol 6000, and 20 g / L of sorbitan fatty acid ester.

[0048] Reagent R2 contains: 60 mmol / L of citric acid buffer, 4 g / L of latex particles coated with rabbit anti-human transferrin antibody, 3 g / L of sodium azide, 22.5 g / L of Tween-20, 7.5 g / L of polyoxyethylene lauryl alcohol ether, 18.75 g / L of disodium ethylenediaminetetraacetic acid, 25 g / L of mannitol and 6.25 g / L of Frank's gel.

[0049] The calibrator contains: phosphate buffer 40mmol / L, sodium azide 2g / L and TRF antigen (urinary transferrin) 200mmol / L.

[0050] The quality control product contains: phosphate buffer 40mmol / L, sodium azide 2g / L and TRF antigen (urinary transferrin) 100mmol / L.

[0051] The preparation method of the above-mentioned kit is specifically as follows:

[0052] Prepare reagent R1: mix citric acid buffer, sodium azide, polyethylene glycol and sorbitan fatty acid ester;

[0053] Preparation of reagent R2: Mix citric acid buffer, latex particles coated with rabbit anti-human transferrin antibody, sodium azide, Tween-20, polyoxyethylene lauryl alcohol ether, disodium ethylenediaminetetraacetic acid, mannitol and Frank's gel.

[0054] Preparation of calibrant: Mix phosphate buffer, sodium azide and TRF antigen (urinary transferrin).

[0055] Preparation of quality control product: Mix phosphate buffer, sodium azide and TRF antigen (urinary transferrin).

[0056] Example 3

[0057] A urine transferrin detection kit (latex enhanced immunoturbidimetric method) comprises reagent R1, reagent R2, quality control products and calibration products.

[0058] Specifically, reagent R1 contains: 50 mmol / L of citric acid buffer, 1 g / L of sodium azide, 20 g / L of polyethylene glycol, and 10 g / L of sorbitan fatty acid ester.

[0059] Reagent R2 contains: 50 mmol / L citric acid buffer, 2 g / L latex particles coated with rabbit anti-human transferrin antibody, 1 g / L sodium azide, 16 g / L Tween-20, 4 g / L lauryl alcohol polyoxyethylene ether, 8 g / L disodium ethylenediaminetetraacetic acid, 8 g / L mannitol and 2 g / L Frank's gel.

[0060] The calibrator contains: phosphate buffer 40mmol / L, sodium azide 2g / L and TRF antigen (urinary transferrin) 100mmol / L.

[0061] The quality control product contains: phosphate buffer 40mmol / L, sodium azide 2g / L and TRF antigen (urinary transferrin) 50mmol / L.

[0062] The preparation method of the above-mentioned kit is specifically as follows:

[0063] Prepare reagent R1: mix citric acid buffer, sodium azide, polyethylene glycol and sorbitan fatty acid ester;

[0064] Preparation of reagent R2: Mix citric acid buffer, latex particles coated with rabbit anti-human transferrin antibody, sodium azide, Tween-20, polyoxyethylene lauryl alcohol ether, disodium ethylenediaminetetraacetic acid, mannitol and Frank's gel.

[0065] Comparative Example 1

[0066] The only difference from Example 3 is that the stabilizer contains only disodium edetate (the content is consistent with the total weight of disodium edetate, mannitol and Frank's gum in Example 3).

[0067] That is, reagent R2 contains: 50 mmol / L of citric acid buffer, 2 g / L of latex particles coated with rabbit anti-human transferrin antibody, 1 g / L of sodium azide, 16 g / L of Tween-20, 4 g / L of polyoxyethylene lauryl alcohol ether and 18 g / L of disodium ethylenediaminetetraacetate.

[0068] The preparation method was adaptively adjusted, and the rest was consistent with Example 3.

[0069] Comparative Example 2

[0070] The only difference from Example 3 is that the stabilizer contains only mannitol (the content is consistent with the total weight of disodium edetate, mannitol and Frank's gum in Example 3).

[0071] That is, reagent R2 contains: 50 mmol / L of citric acid buffer, 2 g / L of latex particles coated with rabbit anti-human transferrin antibody, 1 g / L of sodium azide, 16 g / L of Tween-20, 4 g / L of polyoxyethylene lauryl alcohol ether, and 18 g / L of mannitol.

[0072] The preparation method was adaptively adjusted, and the rest was consistent with Example 3.

[0073] Comparative Example 3

[0074] The only difference from Example 3 is that the stabilizer contains only Frank's gum (the content is consistent with the total weight of disodium ethylenediaminetetraacetic acid, mannitol and Frank's gum in Example 3).

[0075] That is, reagent R2 contains: 50 mmol / L of citric acid buffer, 2 g / L of latex particles coated with rabbit anti-human transferrin antibody, 1 g / L of sodium azide, 16 g / L of Tween-20, 4 g / L of polyoxyethylene lauryl alcohol ether and 18 g / L of Frank's gel.

[0076] The preparation method was adaptively adjusted, and the rest was consistent with Example 3.

[0077] Comparative Example 4

[0078] The only difference from Example 3 is that the surfactant B in reagent R2 is replaced by an equal amount of surfactant A.

[0079] That is, reagent R2 contains: 50 mmol / L citric acid buffer, 2 g / L latex particles coated with rabbit anti-human transferrin antibody, 1 g / L sodium azide, 16 g / L Tween-20, 4 g / L lauryl alcohol polyoxyethylene ether and 18 g / L sorbitan fatty acid ester.

[0080] The rest are consistent with Example 3.

[0081] Test example:

[0082] 1. Test principle: Latex particles coated with transferrin antibodies can produce agglutination reaction with transferrin in urine to form antigen-antibody complexes, the turbidity of which is proportional to UTRF in urine when a certain amount of antibody is present. The concentration of UTRF in urine can be calculated by measuring the absorbance value at a specific wavelength and referring to the calibration curve.

[0083] 2. Sample requirements: Random midstream urine. Samples stored at 2-8°C should be measured within 24 hours. If the sample is stored for more than 24 hours, it should be frozen below -20°C and centrifuged after thawing to avoid repeated freezing and thawing.

[0084] 3. Inspection method:

[0085] 3.1 Reagent preparation:

[0086] This reagent is liquid and can be used directly.

[0087] 3.2 Determination of the conditions and reagents:

[0088] Table 1 Determination of conditions and operating steps

[0089]

[0090] 3.3 Calibration Procedure

[0091] (1) Multi-point calibration is generally used during use;

[0092] (2) The calibrator should be operated according to its instructions;

[0093] (3) The biochemical analyzer can automatically draw a calibration curve based on the calibration results;

[0094] (4) Recalibration is recommended every 2 weeks or when the following situations occur: the reagent batch number is changed; the quality control value shifts significantly; the biochemical analyzer undergoes major maintenance.

[0095] 3.4 Quality Control Procedures

[0096] The quality control products should be operated according to their instructions. It is recommended to conduct a quality control test once a day.

[0097] 4. Calculation of results

[0098] This product adopts a 6-point nonlinear calibration method to measure the absorbance of calibrants at 6 concentration levels in turn, draw a standard curve with ΔA as the ordinate and concentration as the abscissa, and correct the standard curve. The result is automatically calculated by the fully automatic biochemical analyzer.

[0099] 5. Kit performance:

[0100] 5.1 Precision

[0101] Batch difference: Use 3 parallel batches of the kits of each embodiment and comparative example to repeatedly test urine samples (sample 1: transferrin concentration in urine = 0.2 mg / L, sample 2: transferrin concentration in urine = 24 mg / L) 10 times, calculate the mean and standard deviation of the test results (retain 2 decimal places), and calculate the coefficient of variation CV value, wherein the CV value should be ≤15%.

[0102] Table 2 Precision results

[0103]

[0104] According to the above results, the kit of the present invention can detect transferrin at a concentration of 0.2-24 mg / L, and the detection precision is high, and the CV values ​​are all ≤15%. In comparison, the detection CV values ​​of the kits of Comparative Examples 1-4 are all worse than those of Example 3, and in the detection of low-concentration samples, the CV values ​​of the kits of Comparative Examples 1, 2 and 4 do not meet the CV standard. In the detection of high-concentration samples, the CV value of the kit of Comparative Example 4 does not meet the CV standard, indicating that the components of reagent R1 and reagent R2 in the kit of the present invention have a greater impact on the precision of the kit.

[0105] 5.2 Stability

[0106] Test method: Reagent R1 and reagent R2 of each embodiment and comparative example were respectively placed at 37°C, relative humidity of 75% and stored at 4°C (refrigerated condition), and tested on the 0th day and the 8th day, respectively, to detect the content of transferrin in urine containing a specific concentration of transferrin (transferrin concentration in urine = 20 mg / L), and each group was tested 3 times in parallel to calculate the average value. At the same time, the accelerated test results at 37°C and relative humidity of 75% were compared with the experimental results at 4°C, and the relative deviation was calculated (retaining 1 decimal place), and the results were statistically reported in the following table.

[0107] Table 3 Stability results

[0108]

[0109] According to the above results, the kit of the present invention has good stability. At the end of the accelerated stability experiment, the relative deviation is smaller than that of the refrigerated kit, and the relative deviation difference between the 8th day and the 0th day is not obvious. In comparison, the relative deviation of the reagents in the kit of the comparative example after the accelerated stability experiment is significantly higher than that of the embodiment groups. It can be seen that the key role of the specific reagents in the kit of the present invention.

[0110] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A urine transferrin detection kit, characterized in that: comprising reagent R1 and reagent R2; The reagent R1 comprises: citric acid buffer, sodium azide, polyethylene glycol and surfactant A; The reagent R2 comprises: a citric acid buffer, latex particles coated with rabbit anti-human transferrin antibodies, sodium azide, a surfactant B and a stabilizer; The surfactant A comprises sorbitan fatty acid ester, the surfactant B comprises Tween-20 and lauryl alcohol polyoxyethylene ether, and the stabilizer comprises disodium ethylenediaminetetraacetic acid, mannitol and Frank gum; The reagent R1 comprises: 40-60 mmol / L of citric acid buffer, 1-3 g / L of sodium azide, 5-50 g / L of polyethylene glycol and 5-20 g / L of surfactant A; The reagent R2 comprises: 40-60 mmol / L of citric acid buffer, 1-4 g / L of latex particles coated with rabbit anti-human transferrin antibody, 1-3 g / L of sodium azide, 10-30 g / L of surfactant B and 10-50 g / L of stabilizer; The surfactant B comprises Tween-20 and lauryl alcohol polyoxyethylene ether in a weight ratio of 3-6:1-2; The stabilizer comprises disodium ethylenediaminetetraacetic acid, mannitol and Frank's gum in a weight ratio of 1-3: 2-4:

1.

2. The urine transferrin detection kit according to claim 1, characterized in that: The reagent R1 includes: 50 mmol / L of citric acid buffer, 1 g / L of sodium azide, 20 g / L of polyethylene glycol and 10 g / L of surfactant A.

3. The urine transferrin detection kit according to claim 1, characterized in that: The reagent R2 comprises: 50 mmol / L of citric acid buffer, 2 g / L of latex particles coated with rabbit anti-human transferrin antibody, 1 g / L of sodium azide, 20 g / L of surfactant B and 20 g / L of stabilizer.

4. The urine transferrin detection kit according to claim 1, characterized in that: Also included are calibrators and quality control products, both of which include phosphate buffer, sodium azide and TRF antigen.

5. The urine transferrin detection kit according to claim 4, characterized in that: The calibration product includes 40mmol / L phosphate buffer, 2g / L sodium azide and 0-200mmol / L TRF antigen, and the quality control product includes 40mmol / L phosphate buffer, 2g / L sodium azide and 5-100mmol / L TRF antigen.

6. The method for preparing a urine transferrin detection kit according to any one of claims 1 to 5, characterized in that: The following steps are involved: Prepare reagent R1: mix citric acid buffer, sodium azide, polyethylene glycol and surfactant A; Preparation of reagent R2: Mix citric acid buffer, latex particles coated with rabbit anti-human transferrin antibody, sodium azide, surfactant B and stabilizer.

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