A cerebrospinal fluid or urine total protein detection kit

Abstract

This invention discloses a cerebrospinal fluid or urine total protein detection kit, belonging to the technical field of detection kits. The kit includes reagent R1 and reagent R2, wherein reagent R1 includes a buffer stabilizer Tris to maintain the stability of reagent R1 during the opening process. This invention solves the problems of easy pH drop and poor stability of existing turbidimetric total protein assay kits after opening. The cerebrospinal fluid or urine total protein detection kit, by adding Tris to reagent R1, can maintain the stability of small amounts of reagent after opening, which is beneficial for further promotion and use in the market, ensuring the long-term reliability of test results and the economy of reagent use.

Description

Technical Field

[0001] This invention relates to the field of detection kit technology, specifically to a detection kit for total protein in cerebrospinal fluid or urine. Background Technology

[0002] Cerebrospinal fluid (CSF) total protein refers to the total content of all proteins in CSF. CSF is a fluid found in the ventricles and subarachnoid space, primarily secreted by the choroid plexus of the ventricles. The total CSF protein content can reflect the physiological state of the central nervous system (CNS), and its changes may be associated with damage, inflammation, or degenerative diseases of the nervous system. Normal CSF total protein content is low, typically between 150-450 mg / L. Urine total protein refers to the total content of all proteins in urine. Urine protein testing is mainly used to assess kidney function, especially glomerular filtration and tubular reabsorption. In normal individuals, the total 24-hour urinary protein content generally does not exceed 150 mg. Random urinary protein concentration is usually below 150 mg / L. Clinically, it is mainly used as an adjunct to the diagnosis of central nervous system (e.g., meningitis, brain tumors) / kidney diseases.

[0003] US Patent 4485176A discloses a turbidimetric method for determining proteins in urine and cerebrospinal fluid. This method involves stepwise addition of reagents: first, alkali, EDTA, and a nonionic surfactant, followed by incubation, and then the addition of a high concentration of cationic surfactant. This significantly shortens the reaction time and improves sensitivity and interference resistance. However, commercially available total protein assay kits are prone to pH drops when the reagents are opened and stored, especially when the reagent volume is approximately 50 test samples, leading to reagent instability.

[0004] Therefore, this application is hereby submitted. Summary of the Invention

[0005] The technical problem this invention aims to solve is that existing turbidimetric total protein assay kits suffer from poor stability and pH drop after opening. The objective is to provide a kit for detecting total protein in cerebrospinal fluid or urine. The inventors discovered in experiments that adding the alkaline buffer Tris to reagent R1 significantly improves the stability of small amounts of reagent after opening.

[0006] This invention is achieved through the following technical solution: In a first aspect, the present invention provides a cerebrospinal fluid or urine total protein detection kit, the kit comprising reagent R1 and reagent R2, wherein reagent R1 comprises a buffer stabilizer, the buffer stabilizer being Tris, for maintaining the stability of reagent R1 during the opening and use process.

[0007] As one of the optional implementation methods, the concentration of Tris in reagent R1 is 10-25 mM.

[0008] As one of the optional embodiments, reagent R1 further includes EDTA·Na2, sodium hydroxide and purified water.

[0009] This invention uses sodium hydroxide to provide an alkaline environment, which can adjust the pH and disrupt the protein hydration layer, reducing the hydration of protein molecules and promoting precipitation formation.

[0010] This invention uses EDTA·Na2 as a chelating agent to complex metal ions such as magnesium, calcium, and iron, thereby removing interference and making the reaction mixture clear.

[0011] The inventors discovered through experiments that using Tris can prevent the pH of the reaction system from decreasing, thereby maintaining the stability of the reagents.

[0012] As one of the optional embodiments, the concentration of EDTA·Na2 in reagent R1 is 25mM, and the concentration of sodium hydroxide is 0.4-1.2M.

[0013] As one of the optional implementation methods, the reagent R2 includes benzyl chloride and purified water.

[0014] The reaction mechanism of turbidimetric detection of total protein: (1) The role of alkaline environment: pH adjustment (usually pH 9-12): Under alkaline conditions, the carboxyl group (-COO) of protein... - ) and amino (-NH3) + (1) Dissociation of the group makes the protein surface carry a net negative charge, making it easier to bind with the cationic surfactant (benzyl chloride). In addition, the alkaline environment will reduce the hydration of protein molecules and promote precipitation. (2) Neutralization of the binding charge of benzyl chloride with protein: The quaternary ammonium cation of benzyl chloride binds with the negatively charged group of protein to form an insoluble complex. Hydrophobic interaction: The long-chain alkyl and benzene ring of benzyl chloride bind with the hydrophobic region of protein, enhancing precipitation stability. Formation of suspension: The protein-benzyl chloride complex forms fine particles in the solution, making the reaction solution turbid. The turbidity is proportional to the protein concentration in the sample. (3) Quantitative analysis: The turbidity of the reaction solution is measured at a wavelength of 505 nm using a specific protein analyzer, and the concentration of total protein in the sample is calculated according to the standard curve.

[0015] As one of the optional implementation methods, the concentration of benzyl chloride in reagent R2 is 10-25 g / L.

[0016] As one optional implementation, reagent R1 in the kit is configured as follows: EDTA·Na2 25mM Sodium hydroxide 0.7M Tris 15mM The rest is purified water. The preparation of reagent R2 is as follows: Benzyl chloride 20g / L The rest is purified water. As one of the optional implementation methods, the preparation methods of reagent R1 and reagent R2 are as follows: first add 80% of the prepared volume of purified water to the preparation container, then accurately weigh the corresponding materials in sequence, place them in the preparation container and stir. After the materials are completely dissolved, make up the volume with purified water to the prepared volume, and finally filter with a filter membrane with a pore size of 0.45µm.

[0017] Secondly, the present invention provides a method for detecting total protein in a sample, comprising the following steps: The sample to be tested is mixed with reagent R1 of the detection kit according to any one of claims 1-8 and incubated, and then reagent R2 is added and mixed for detection; Quantitative analysis involves measuring the turbidity of the reaction solution using a detection instrument and calculating the total protein concentration in the sample based on the calibration curve.

[0018] As one of the optional implementation methods, the detection instrument is a fully automated specific protein analyzer.

[0019] Compared with the prior art, the present invention has the following advantages and beneficial effects: 1. The cerebrospinal fluid or urine total protein detection kit disclosed in this invention, by adding Tris to reagent R1, can maintain the stability of the reagent when opening a small amount of reagent, which is conducive to further promotion and use in the market, and ensures the long-term reliability of the test results and the economy of reagent use.

[0020] 2. This invention conducted comparative experiments with other buffer systems (Na2CO3, CAPS, and Na2HPO4). The results showed that adding the buffer system did not affect the detection results. However, in the open-bottle stability verification test, the detection kits with added buffer systems (Na2CO3, CAPS, and Na2HPO4) showed poorer stability compared to the kits without added buffer systems. The addition of the Tris buffer system in this invention significantly improved the open-bottle stability of small amounts of reagents. This may be because the pKa of Tris perfectly matches the pH range of reagent R1 in this invention. Adding Tris can directly form a highly efficient conjugate buffer pair, and a small amount of Tris can quickly construct a strong buffer pair to stabilize the pH. In contrast, the pKa of Na2CO3, CAPS, and Na2HPO4 deviates significantly, resulting in extremely weak buffering capacity or even a counterproductive effect. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of the present invention and should not be considered as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort. In the drawings: Figure 1 This is a graph showing the results of the accuracy verification experiment in Embodiment 1 of the present invention. Figure 2 This is a graph showing the results of the accuracy verification experiment for Comparative Example 1 of this invention. Figure 3 This is a graph showing the results of the accuracy verification experiment for Comparative Example 3 of this invention. Figure 4 The graph shows the results of the accuracy verification experiment of Comparative Example 5 of this invention. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments and accompanying drawings. The illustrative embodiments and descriptions of the present invention are only used to explain the present invention and are not intended to limit the present invention.

[0023] Throughout this specification, references to "an embodiment," "an example," or "an example" mean that a particular feature, structure, or characteristic described in connection with that embodiment or example is included in at least one embodiment of the invention. Therefore, the phrases "an embodiment," "an example," "an example," or "an example" appearing in various places throughout the specification do not necessarily refer to the same embodiment or example. Furthermore, specific features, structures, or characteristics can be combined in one or more embodiments or examples in any suitable combination and / or sub-combination. Moreover, those skilled in the art will understand that the illustrations provided herein are for illustrative purposes and are not necessarily drawn to scale. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0024] Currently available total protein assay kits using turbidimetric methods operate on the principle that proteins react with benzyl chloride in an alkaline matrix (NaOH aqueous solution) to form a precipitate, with the turbidity directly proportional to the protein content. However, in practical applications, excessively low NaOH concentrations can cause the pH to drop due to the absorption of carbon dioxide from the air, or the reagent pH can easily decrease after opening, leading to reagent instability, especially when the reagent volume is approximately 50 test samples. A review of mainstream manufacturer A showed that, with a reagent volume of approximately 5 mL, the relative deviations between the two quality control levels reached 25.1% and 16.0% on day 7 after opening.

[0025] Example 1: The total protein detection kit includes reagent R1 and reagent R2. Preparation of reagent R1: Add 80% of the prepared volume of purified water to the preparation container, then accurately weigh EDTA·Na2, sodium hydroxide and tris(hydroxymethyl)aminomethane (Tris) in sequence, and place them in the preparation container and stir. After the materials are completely dissolved, make up the volume with purified water to the prepared volume, and then filter it through a filter membrane with a pore size of 0.45 μm.

[0026] Preparation of reagent R2: The procedure is the same as for reagent R1.

[0027] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M Tris 15mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Regarding the detection method: The detection protocol is provided in the operation manual or instrument software of the BioSystems BA400, Chongqing Bosideng BA200, Chongqing Bosideng BST300, Chongqing Bosideng BST320, and Chongqing Bosideng BA400 fully automated specific protein analyzers. The operation is as follows: Open the total protein assay reagent bottle cap and place it in the corresponding reagent slot on the instrument. Before first use, gently shake to avoid air bubbles, which could cause sampling errors. After removing the calibrator from 2-8℃, allow it to equilibrate at room temperature (10-30℃) for 10-15 minutes. Before sampling, gently rotate to mix; do not shake vigorously to avoid foam formation. Carefully open the bottle cap and proceed as with routine samples. After sampling, wipe the bottle opening clean and tighten the cap as soon as possible. Store immediately at 2-8℃.

[0028] Collect cerebrospinal fluid / urine samples using standard methods and place them in the corresponding sample slots for testing. When testing, please select the correct sample type; otherwise, incorrect results will occur. Click "Start" on the instrument. The sampling needle will aspirate 170 µL of reagent R1 into the reaction dish; then aspirate 10 µL of sample, mix well, incubate at 37°C for 5 minutes, and then add 30 µL of reagent R2. The instrument will automatically detect and calculate the results.

[0029] Example 2 is based on Example 1. The kit preparation in this example is basically the same as that in Example 1, except that the concentration of Tris in reagent R1 is 10mM.

[0030] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M Tris 10mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 1, based on Example 1, is basically the same as the kit prepared in Example 1, except that Na2CO3 is used instead of Tris in reagent R1 to maintain the stability of a small amount of reagent after opening, wherein the concentration of Na2CO3 is 20mM.

[0031] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M Na2CO3 20mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 2, based on Example 1, is basically the same as the kit preparation of Example 1, except that Na2CO3 is used instead of Tris in reagent R1 to maintain the opening stability of a small amount of reagent, wherein the concentration of Na2CO3 is 10mM.

[0032] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M Na2CO3 10mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 3, based on Example 1, is basically the same as the kit prepared in Example 1, except that 3-(cyclohexylamine)-1-propanesulfonic acid (CAPS) is used instead of Tris in reagent R1 to maintain the stability of a small amount of reagent after opening, wherein the CAPS concentration is 25mM.

[0033] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M CAPS 25mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 4, based on Example 1, is basically the same as the kit prepared in Example 1, except that 3-(cyclohexylamine)-1-propanesulfonic acid (CAPS) is used instead of Tris in reagent R1 to maintain the stability of a small amount of reagent after opening, wherein the CAPS concentration is 15mM.

[0034] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M CAPS 15mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 5, based on Example 1, is basically the same as the kit prepared in Example 1, except that Na2HPO4 is used instead of Tris in reagent R1 to maintain the stability of a small amount of reagent after opening, wherein the concentration of Na2HPO4 is 20mM.

[0035] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M Na2HPO4 20mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 6, based on Example 1, is basically the same as the kit prepared in Example 1, except that Na2HPO4 is used instead of Tris in reagent R1 to maintain the stability of a small amount of reagent after opening, wherein the concentration of Na2HPO4 is 10mM.

[0036] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M Na2HPO4 10mM Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 7, based on Example 1, is basically the same as the kit prepared in Example 1, except that Tris is not added to reagent R1 and the sodium hydroxide concentration is 1.0M.

[0037] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 1.0M Purified water Reagent R2: Benzyl chloride 20g / L Purified water Comparative Example 8, based on Example 1, is basically the same as the kit prepared in Example 1, except that Tris is not added to reagent R1 and the concentration of benzyl chloride is 25 g / L.

[0038] Reagent R1: EDTA·Na2 25mM Sodium hydroxide 0.7M Purified water Reagent R2: Benzyl chloride 25g / L Purified water Comparative Example 9: This comparative example uses the detection kit from existing manufacturer A.

[0039] Detailed operation steps: 1) Calibration procedure: Place the calibrator at the designated position on the instrument for multi-point calibration to obtain the calibration curve.

[0040] 2) Sample testing process: Place reagent kits R1 and R2 into the instrument's fixed reagent slots, and place the cerebrospinal fluid / urine sample into the fixed sample slots. After clicking the start button, the instrument will automatically detect the sample and calculate the total protein concentration in the cerebrospinal fluid / urine based on the calibration curve.

[0041] Accuracy verification experiment: The accuracy of the reagent kits prepared in Example 1, Comparative Example 1, Comparative Example 3, and Comparative Example 5 was experimentally verified: The reagent kits from Examples 1, 1, 3, and 5 were used as the experimental group, while a commercially recognized high-performance total protein reagent kit (manufacturer A) was used as the control group for comparison. Forty samples were tested, and the results are as follows: Figure 1-4 .

[0042] pass Figure 1-4The detection data show that the regression equation for the detection results of the test kit in Example 1 and the control test kit is y = 1.0452x - 0.1646, with a correlation of 0.9990; the regression equation for the detection results of the test kit in Comparative Example 1 and the control test kit is y = 1.0146x + 4.3164, with a correlation of 0.9987; the regression equation for the detection results of the test kit in Comparative Example 3 and the control test kit is y = 1.0869x - 3.9144, with a correlation of 0.9975; and the regression equation for the detection results of the test kit in Comparative Example 5 and the control test kit is y = 1.0495x + 2.093, with a correlation of 0.9980. All these correlations are good, indicating that the test kit of the present invention is highly consistent with commercially available total protein detection kits with excellent accuracy. This proves that the addition of Tris, CAPS, and Na2HPO4 to the test kit of the present invention does not affect its accuracy, and the test kit still maintains good accuracy.

[0043] Linear correlation verification experiment: A sample with a high total protein concentration of 2089 mg / L was identified. Seven samples of different concentrations were prepared by serial dilution with physiological saline. Each sample at each concentration level was measured three times, and the average value was taken. The samples were then tested using reagents from Examples 1, 1, 3, 5, 7, and 8. The results are shown in Table 1.

[0044]

[0045] Using theoretical total protein concentration as the independent variable X on the x-axis and actual measured value as the dependent variable Y on the y-axis, a linear regression equation was derived, and the correlation coefficient r was calculated. The results showed that the linear regression equation for Example 1 was y = 0.9983x + 10.649, with a correlation coefficient r = 0.9998; the linear regression equation for Comparative Example 1 was y = 0.9945x - 18.878, with a correlation coefficient r = 0.9998; the linear regression equation for Comparative Example 3 was y = 1.0062x - 10.954, with a correlation coefficient r = 0.9999; the linear regression equation for Comparative Example 5 was y = 1.0119x - 29.423, with a correlation coefficient r = 0.9995; the linear regression equation for Comparative Example 7 was y = 1.0083x + 5.8819, with a correlation coefficient r = 0.9998; and the linear regression equation for Comparative Example 8 was y = 0.9998. =1.0078x + 4.4376, with a correlation coefficient r=0.9999, indicating that the present invention has good correlation within the linear range of 40mg / L-2000mg / L.

[0046] The bottle-opening stability verification experiments are shown in Table 2:

[0047] Each of the reagent kits of this invention and manufacturer A (approximately 50 test samples each) was calibrated on the testing instrument. The stability of the reagent kits was tested using two levels of quality control samples after being opened and stored at 2-8℃ for 7 days. Each concentration level was measured three times, and the average value was taken. The deviation of the test results at each test time point was calculated. The results are shown in Table 3.

[0048]

[0049] Table 3 shows that 7 days after opening, the relative deviations of the two levels of quality control samples for reagent kit A (Comparative Example 9) were 25.06% and 16.04%, respectively. The relative deviation of the low-level quality control sample was greater than 20%, and the relative deviation of the high-level quality control sample was greater than 15%. Compared with the control kit from Manufacturer A, the reagent kit of this invention showed better stability after opening with a small amount of reagent. The relative deviations of the quality control samples for reagent kits in Examples 1 and 2 were: 3.67% and 6.18% for level 1, and 2.19% and 5.02% for level 2. The relative deviations of both the low-level and high-level quality control samples in Example 1 were less than 5%. The relative deviations of the two levels of quality control samples for reagent kits in Comparative Examples 1-6 ranged from 8% to 20%. The relative deviations of the quality control samples for reagent kits in Comparative Examples 7 and 8 were: 8.91% and 9.19% for level 1, and 5.46% and 5.93% for level 2. The relative deviations of both the low-level and high-level quality control samples ranged from 5% to 10%. Compared with Manufacturer A and Comparative Examples 1-8, the addition of Tris to Reagent R1 significantly improves the open-bottle stability of small amounts of reagents in the kit.

[0050] In summary, the total protein detection kit provided by this invention significantly improves the open-bottle stability of small quantities of reagents by adding a certain amount of Tris to reagent R1. Therefore, the total protein detection kit provided by this invention is beneficial for further promotion and use in the market.

[0051] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A kit for detecting total protein in cerebrospinal fluid or urine, characterized in that, The kit includes reagent R1 and reagent R2. Reagent R1 includes a buffer stabilizer, which is Tris, to maintain the stability of reagent R1 during the opening and use process.

2. The cerebrospinal fluid or urine total protein detection kit according to claim 1, characterized in that, In the reagent R1, the concentration of Tris is 10-25 mM.

3. The cerebrospinal fluid or urine total protein detection kit according to claim 1, characterized in that, The reagent R1 also includes disodium ethylenediaminetetraacetate, sodium hydroxide, and purified water.

4. The cerebrospinal fluid or urine total protein detection kit according to claim 3, characterized in that, The concentration of disodium ethylenediaminetetraacetate in reagent R1 is 25 mM, and the concentration of sodium hydroxide is 0.4-1.2 M.

5. The cerebrospinal fluid or urine total protein detection kit according to claim 1, characterized in that, The reagent R2 comprises benzyl chloride and purified water.

6. The cerebrospinal fluid or urine total protein detection kit according to claim 5, characterized in that, The concentration of benzyl chloride in reagent R2 is 10-25 g / L.

7. The cerebrospinal fluid or urine total protein detection kit according to any one of claims 1-6, characterized in that, The reagent R1 in the kit is prepared as follows: EDTA·Na2 25mM Sodium hydroxide 0.7M Tris 15mM The rest is purified water. The preparation of reagent R2 is as follows: Benzyl chloride 20g / L The rest is purified water.

8. The cerebrospinal fluid or urine total protein detection kit according to claim 1, characterized in that, The preparation methods for both reagent R1 and reagent R2 are as follows: first, add 80% of the prepared volume of purified water to the preparation container, then accurately weigh the corresponding materials in sequence, place them in the preparation container and stir. After the materials are completely dissolved, adjust the volume to the prepared volume with purified water, and finally filter with a filter membrane with a pore size of 0.45µm.

9. A method for detecting total protein in a sample, characterized in that, Includes the following steps: The sample to be tested is mixed with reagent R1 of the detection kit according to any one of claims 1-8 and incubated, and then reagent R2 is added and mixed for detection; Quantitative analysis involves measuring the turbidity of the reaction solution using a detection instrument and calculating the total protein concentration in the sample based on a standard curve.

10. The method for detecting total protein in a sample according to claim 9, characterized in that, The detection instrument is a fully automated specific protein analyzer.

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