A direct bilirubin detection kit and a preparation method thereof

Abstract

The application belongs to the field of in-vitro diagnostic reagents, and discloses a direct bilirubin detection kit and a preparation method thereof. The direct bilirubin detection kit is characterized by comprising R1 reagent and R2 reagent. The R1 reagent comprises the following components: tartaric acid-sodium hydroxide buffer, a surfactant, a reducing agent and a metal ion chelating agent. The R2 reagent comprises the following components: PB buffer, sodium metavanadate and NaCl. The direct bilirubin detection kit eliminates the influence of interference factors caused by hemolysis through a plurality of anti-interference substances, and then performs bilirubin detection, thereby improving the anti-interference capability for hemolytic samples and the accuracy of detection results.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagents, and more particularly to a direct bilirubin detection kit and its preparation method. Background Technology

[0002] Direct bilirubin is an important form of bilirubin, produced from the breakdown of hemoglobin in aging red blood cells. After processing by the liver, it conjugates with glucuronic acid. Bilirubin that is not conjugated with glucuronic acid is called indirect bilirubin. The sum of the two is total bilirubin. The detection of direct bilirubin is of great significance for assessing liver function, diagnosing hepatobiliary diseases, and monitoring the progression of certain systemic diseases.

[0003] Clinically, direct bilirubin is mainly used for the diagnosis of liver diseases and biliary obstruction. Under normal physiological conditions, the level of direct bilirubin in the blood is generally maintained between 1.7 and 7.0 μmol / L. When liver function is impaired or the biliary system is obstructed, the metabolism and excretion of direct bilirubin are affected, leading to an increase in its concentration in the blood. Elevated direct bilirubin levels are commonly seen in hepatocellular jaundice caused by hepatitis and cirrhosis, as well as in intrahepatic and extrahepatic obstructive jaundice.

[0004] Currently, the main methods for direct bilirubin detection include the bilirubin oxidase method, the diazo method, and the vanadate oxidation method. The bilirubin oxidase method utilizes bilirubin oxidase to oxidize bilirubin to biliverdin, simultaneously producing hydrogen peroxide. The concentration of bilirubin is indirectly determined by detecting changes in the amount of hydrogen peroxide produced or other related substances. The bilirubin oxidase method has advantages such as high specificity, good repeatability, and high sensitivity; however, due to enzyme stability and cost issues, this method has not been widely used. The diazo reagent method involves the reaction of direct bilirubin with a diazo reagent under acidic conditions to generate azobilirubin. The concentration of bilirubin is indirectly determined based on the light absorption intensity of azobilirubin at a specific wavelength. This method has high sensitivity, low cost, and simple operation, but its repeatability is poor. The vanadate oxidation method involves the oxidation of bilirubin to biliverdin under the action of vanadate. The concentration of bilirubin is determined by measuring the change in absorbance before and after oxidation. This method has high sensitivity, good specificity, and repeatability, and is gradually being promoted in clinical applications. In practical applications, it has been found that hemolyzed samples can negatively interfere with the vanadate oxidation method, affecting the accuracy of the test results.

[0005] Although existing direct bilirubin test kits have made some progress in terms of accuracy and sensitivity, in practical applications, the test results are often affected by hemolysis of the sample, resulting in insufficient accuracy.

[0006] Therefore, existing technologies need further improvement. Summary of the Invention

[0007] To address the interference of hemolyzed samples on direct bilirubin detection results in the prior art, this invention provides a novel direct bilirubin detection kit and its preparation method. This application improves the reagents in the kit by using anti-interference substances to eliminate the influence of interference factors caused by hemolysis before bilirubin detection, effectively improving the accuracy of direct bilirubin detection results and the anti-interference ability against hemolyzed samples.

[0008] To address the above problems, this application provides the following technical solution: In a first aspect, this application provides a direct bilirubin detection kit, comprising: reagent R1 and reagent R2; wherein: The R1 reagent consists of the following components: tartaric acid-sodium hydroxide buffer, surfactant Triton X-405, reducing agent, and metal ion chelating agent.

[0009] The R2 reagent consists of the following components: PB buffer, sodium metavanadate, and NaCl.

[0010] Optionally, the tartaric acid-sodium hydroxide buffer solution has a pH of 2.5 to 3.5, and the PB buffer solution has a pH of 6.6 to 7.5.

[0011] Optionally, the surfactant includes one or more of Triton X-405, lauryl polyoxyethylene ether (Brij-35), sodium dodecylbenzenesulfonate, Tween 20, and polyoxyethylene alkyl ether.

[0012] The polyoxyethylene alkyl ether used is EMULGEN 709, commonly known as Kao 709.

[0013] Optionally, in the direct bilirubin detection kit, the metal ion chelating agent is selected from one or both of ethylenediaminetetraacetic acid (EDTA) and hydroxyethylidene diphosphonic acid (HEDP).

[0014] Preferably, the metal ion chelating agent is composed of ethylenediaminetetraacetic acid (EDTA) and hydroxyethylidene diphosphonic acid (HEDP).

[0015] Optionally, the reducing agent includes one or more of potassium iodide, hydroxylamine hydrochloride, thiourea, and acetone oxime. Preferably, the reducing agent consists of potassium iodide and hydroxylamine hydrochloride.

[0016] In the R1 reagent of the aforementioned direct bilirubin assay kit, the selected surfactant can remove serum interfering substances, eliminate the influence of indirect bilirubin, and improve the solubility of direct bilirubin. The metal ion chelating agent, a combination of EDTA and HEDP, can not only shield the background signal generated by metavanadate in the R2 reagent by chelating vanadium ions, but also remove interference from other impurity ions on direct bilirubin detection. Furthermore, this application preferably uses a combination of potassium iodide and hydroxylamine hydrochloride as reducing agents to achieve multiple functions. Potassium iodide can react with oxidizing substances to prevent hemoglobin oxidation from interfering with bilirubin detection, and it can also react with free radicals to remove active substances and ensure the stability of the bilirubin detection reaction system. Hydroxylamine hydrochloride can act as a protective agent, working with potassium iodide to prevent premature bilirubin oxidation, and it can also consume excess metavanadate through a redox reaction with metavanadate, thereby reducing the background signal.

[0017] Based on the synergistic effect of the components in the above-mentioned kit, the anti-interference performance and stability of kit R1 are greatly improved, especially the anti-hemolytic interference effect, which significantly improves the accuracy and stability of the detection results of direct bilirubin.

[0018] Optionally, the ratio of reagent R1 to reagent R2 is 4:1.

[0019] Optionally, in the direct bilirubin detection kit, the R1 reagent comprises the following components at the following concentrations: Tartaric acid-sodium hydroxide buffer solution 20-150 mmol / L; EDTA 0.1-1.0 g / L; Triton X4051-5g / L; Hydroxylamine hydrochloride 1-10 g / L; HEDP 2-5mmol / L; KI 0.5-2g / L.

[0020] Optionally, in the direct bilirubin detection kit, the R2 reagent comprises the following components at the following concentrations: PB buffer 5-100 mmol / L pH 6.6~7.5; NaCl 5-15g / L; Sodium metavanadate 5-10 mmol / L.

[0021] Preferably, the direct bilirubin detection kit comprises: reagent R1 and reagent R2, wherein, Reagent R1 comprises the following components at the following concentrations: Tartaric acid-sodium hydroxide buffer solution, 100 mmol / L, pH 3.0; EDTA 0.1 g / L; Triton X4052g / L; Hydroxylamine hydrochloride 3g / L; HEDP 4 mmol / L; KI 0.8g / L.

[0022] The R2 reagent comprises the following components at the following concentrations: PB buffer 10 mmol / L pH 7.0; NaCl 10g / L; Sodium metavanadate 6 mmol / L.

[0023] Secondly, this application also provides a method for preparing a direct bilirubin detection kit, which includes the following steps: The preparation method for reagent R1 is as follows: use tartaric acid-sodium hydroxide buffer as a solvent, add and mix all other raw materials, and wait until all components are completely dissolved. The preparation method for R2 reagent is as follows: using PB buffer as solvent, add and mix all other raw materials as described above, and wait until all components are completely dissolved.

[0024] The present invention has the following beneficial effects: 1. This invention uses two reagents, R1 and R2, in combination to detect direct bilirubin. By modifying reagent R1, multiple components in R1 are used as anti-interference substances to eliminate various interfering factors in the sample, including hemolytic components. Then, R2 is added to detect direct bilirubin, which improves the accuracy of the direct bilirubin detection results.

[0025] 2. The HEDP and EDTA in reagent R1 shield the background signal generated by metavanadate itself, eliminating interference from impurity ions. Furthermore, the reaction of potassium iodide with oxidants and free radicals in the sample eliminates interference from hemoglobin oxidation and reactive substances, ensuring the stability of the bilirubin detection reaction system. Simultaneously, hydroxylamine hydrochloride acts as a protective agent, preventing premature bilirubin oxidation and consuming excess metavanadate, thus reducing background signal. Based on the synergistic effect of these components, the kit's anti-interference capability and stability are improved, especially its resistance to hemolytic interference is significantly enhanced. This kit exhibits high detection accuracy and good stability, fully meeting the needs of clinical diagnosis. Detailed Implementation

[0026] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. In the present invention, unless otherwise specified, the equipment and raw materials used can be purchased from the market or are commonly used in the art. Unless otherwise specified, the methods in the following embodiments are conventional methods in the art.

[0027] Example 1: Direct bilirubin detection kit and its preparation 1. This embodiment provides a direct bilirubin detection kit, which includes reagent R1 and reagent R2, with the following formulation: R1 reagent:

[0028] R2 reagent:

[0029] 2. This embodiment also provides a method for preparing the above-mentioned direct bilirubin detection kit, which includes the following steps: (1) Preparation of R1 reagent: Using tartaric acid-sodium hydroxide buffer as solvent, add and mix all the other raw materials mentioned above until all components are completely dissolved.

[0030] (2) Preparation of R2 reagent: Using PB buffer as solvent, add and mix all other raw materials mentioned above until all components are completely dissolved.

[0031] Example 2 1. This embodiment provides a direct bilirubin detection kit, which includes reagent R1 and reagent R2, with the following formulation (KI is omitted from this formulation compared to Example 1): R1 reagent:

[0032] R2 reagent:

[0033] 2. This embodiment also provides a method for preparing the above-mentioned direct bilirubin detection kit, which is described in Example 1.

[0034] Example 3 1. This embodiment provides a direct bilirubin detection kit, which includes reagent R1 and reagent R2, with the following formulation (HEDP is omitted from this formulation compared to Example 1): R1 reagent:

[0035] R2 reagent:

[0036] 2. This embodiment also provides a method for preparing the above-mentioned direct bilirubin detection kit, which is described in Example 1.

[0037] Example 4 1. This embodiment provides a direct bilirubin detection kit, which includes reagent R1 and reagent R2 (compared to Example 1, this formulation omits hydroxylamine hydrochloride), and the formulation is as follows: R1 reagent:

[0038] R2 reagent:

[0039] 2. This embodiment also provides a method for preparing the above-mentioned direct bilirubin detection kit, which is described in Example 1.

[0040] Example 5: Direct bilirubin detection kit and its preparation 1. This embodiment provides a direct bilirubin detection kit, which includes reagent R1 and reagent R2 (in reagent R1, the concentrations of HEDP and KI used are different from those in Example 1), and the formulation is as follows: R1 reagent:

[0041] R2 reagent:

[0042] 2. This embodiment also provides a method for preparing the above-mentioned direct bilirubin detection kit, which is described in Example 1.

[0043] Example 6: Application of the kit of this application in the detection of direct bilirubin content. The specific method of using the direct bilirubin detection kit provided in this application is as follows: Place R1 and R2 in the reagent tray of a fully automated biochemical analyzer at a 4:1 ratio, such as the Hitachi 7600 fully automated biochemical analyzer. Test parameters: main wavelength 450nm, secondary wavelength 546nm. Place the calibrator, quality control, and test sample in their corresponding positions on the sample rack, and perform sample determination using the two-point endpoint method.

[0044] The specific steps and data are shown in Table 1 below. First, add 280 μL of reagent R1 and the corresponding other components to each tube, mix well, and incubate at 37°C for 5 minutes. Read the absorbance A1. Then add 70 μL of reagent R2, mix well, incubate at 37°C for 5 minutes, and read the absorbance A2. Calculate ΔA = A2 - A1.

[0045] The formula for calculating direct bilirubin content is as follows:

[0046] Table 1. Procedure for Direct Bilirubin Detection

[0047] Example 7: Determination of the Reagent Kit's Effectiveness 1. Anti-hemolytic interference test (1) Detection method: A. Take normal serum and divide it into 4 portions, and 1 portion of severely hemolyzed sample. Do not treat one portion of normal serum, and add hemoglobin at different concentrations (3.0g / L, 6.0g / L, 9.0g / L) to the other 3 portions of normal serum to simulate hemolyzed samples.

[0048] The kits prepared in Examples 1-4 above and the direct bilirubin assay kit (vanadate oxidation method) produced by Shanghai Kehua Bioengineering Co., Ltd. were used to detect the direct bilirubin content of the above five different samples according to the method in Example 6.

[0049] (2) Test results and analysis As can be seen from the test results in Table 2: A. For Examples 2-4 without HEDP, hydroxylamine hydrochloride, and potassium iodide, the detection results of direct bilirubin decreased significantly, even showing negative values, as the concentration of hemoglobin in the serum increased. With the combined action of the three anti-interference substances, the detection results of the kit in Example 1 were significantly less affected by hemoglobin, and the detection results showed good accuracy regardless of the concentration of hemoglobin added to the sample.

[0050] B. For samples with severe hemolysis, the kit of Example 1 of this invention showed better resistance to hemolysis interference, and its effect was not only better than that of Examples 2-4, but also better than that of commercially available comparative kits.

[0051] Table 2. Detection results of five reagents on samples interfering with hemolysis (unit: μmol / L)

[0052] 2. Performance comparison of the kits in Example 1 and Example 5 (1) Experimental method: High and low value quality control samples from Landau were taken, and the kits prepared in Examples 1 and 5 above were used to directly detect the bilirubin content of the high and low value quality control samples according to the method in Example 6. The high and low value quality control samples were manufactured by Landau Laboratory Diagnostics Ltd., UK. The product instructions include the target value and range of the item for this batch. The target value is the target concentration reference value of each analyte in the quality control sample, and the range is used to evaluate the accuracy of the test results and ensure that the results are within the range.

[0053] (2) Experimental results and analysis: The test results are shown in Table 3. From these results, we can see that: For both high- and low-value quality control samples from Landau, the relative deviation of the test results from the kit in Example 1 was significantly smaller than that from Example 5. This result indicates that the kit in Example 1 has higher accuracy and is significantly better than that from Example 5, suggesting that the formulation of the kit in Example 1 is more optimized.

[0054] Table 3. Accuracy of two reagent tests on Landau quality control samples (unit: μmol / L)

[0055] 3. Determination of the linear range of the reagent kit The linear range of the kit in Example 1 was determined using the following method.

[0056] (1) Experimental method: At least five dilutions were prepared by mixing a high-concentration sample (close to the upper limit of the linear range) with distilled water (close to the lower limit of the linear range). The detection system was then calibrated on a Hitachi 7600 biochemical analyzer using calibrators provided with a certified reagent kit. The calibrated system was used to test the five dilutions, with each dilution tested three times, and the mean of the results was calculated. A linear regression equation was derived with dilution concentration as the independent variable and the mean of the results as the dependent variable. The correlation coefficient r ≥ 0.9900. Specifically, for direct bilirubin levels below 2 μmol / L, the absolute deviation of the linear regression should not exceed ±1 μmol / L; for levels between 2 and 342 μmol / L, the relative deviation should not exceed ±10%.

[0057] (2) Test results and analysis The test results are shown in Table 4. As can be seen from Table 4, the reagent of the present invention has good linearity, with a correlation r > 0.999, and the linear deviation meets the requirements, that is, the linear range can reach 342 μmol / L.

[0058] Table 4. Experimental results for linear range (μmol / L)

[0059] 4. Determination of the precision and accuracy of the reagent kit (1) Detection method The reagent kit was prepared according to Example 1. High-value and low-value direct bilirubin samples from the hospital were measured using the reagent kit and the control reagent of Example 1 of this invention. The measurements were repeated 10 times, and the CV value was calculated. The detailed detection steps were the same as in Example 1.

[0060] (2) Test results and analysis The test results are shown in Table 5. As can be seen from Table 5, the average value, standard deviation, and coefficient of variation of the high-concentration sample measured by the kit of Example 1 of this invention were 12.181 μmol / L, 0.0863, and 0.7088%, respectively; while the average value, standard deviation, and coefficient of variation of the high-concentration sample measured by the comparative kit were 13.184 μmol / L, 0.1286, and 0.9754%, respectively. The average value, standard deviation, and coefficient of variation of the low-concentration sample measured by the kit of Example 1 of this invention were 5.946 μmol / L, 0.056, and 0.9421%, respectively; while the average value, standard deviation, and coefficient of variation of the low-concentration sample measured by the comparative kit were 5.813 μmol / L, 0.1098, and 1.8880%, respectively.

[0061] Therefore, compared with the comparative kit, the kit of the present invention has smaller standard deviation and coefficient of variation of the measured samples. Thus, the kit of the present invention has better precision and accuracy than the comparative kit.

[0062] Table 5. Results of Precision and Accuracy Experiments

[0063] 5. Accelerated thermal stability test of the reagent kit (1) Experimental methods The experiment was conducted using the kit prepared in Example 1. Reagents R1 and R2 were placed in a 37°C water bath and removed from the water bath after heating for 1 day, 3 days, 5 days, 7 days, and 9 days, respectively, for testing. Reagents R1 and R2 without thermal acceleration were used as a comparison, and the relative deviation between the K value and the quality control measurement value was calculated.

[0064] (2) Experimental results and analysis The experimental results are shown in Table 6. As can be seen from the table, the quality control measurements of the kit of the present invention remained stable with small relative deviations after 9 days of heat degradation, and the reagent performance showed almost no decline. Therefore, the kit of the present invention has excellent thermal stability.

[0065] Table 6 Results of accelerated thermal stability experiments

[0066] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A direct bilirubin detection kit, characterized in that, include: Reagents R1 and R2; where: Reagent R1 comprises the following components at the following concentrations: Tartaric acid-sodium hydroxide buffer solution 20-150 mmol / L pH 2.5-3.5; Ethylenediaminetetraacetic acid (EDTA) 0.1-1.0 g / L; Triton X 4051-5g / L; Hydroxylamine hydrochloride 1-10 g / L; Hydroxyethylidene diphosphonic acid 2-5 mmol / L; KI 0.5-2g / L; The R2 reagent comprises the following components at the following concentrations: PB buffer 5-100 mmol / L pH 6.6~7.5; NaCl 5-15g / L; Sodium metavanadate 5-10 mmol / L.

2. The direct bilirubin detection kit according to claim 1, characterized in that, The direct bilirubin detection kit includes: reagent R1 and reagent R2. Reagent R1 comprises the following components at the following concentrations: Tartaric acid-sodium hydroxide buffer solution, 100 mmol / L, pH 3.0; Ethylenediaminetetraacetic acid 0.1 g / L; Triton X 4052g / L; Hydroxylamine hydrochloride 3g / L; Hydroxyethylidene diphosphonic acid 4 mmol / L; KI 0.8g / L; The R2 reagent comprises the following components at the following concentrations: PB buffer 10 mmol / L pH 7.0; NaCl 10g / L; Sodium metavanadate 6 mmol / L.

3. The method for preparing the direct bilirubin detection kit according to any one of claims 1 to 2, characterized in that, Includes the following steps: The preparation method for reagent R1 is as follows: use tartaric acid-sodium hydroxide buffer as a solvent, add all other components and mix them until all components are completely dissolved. The preparation method for R2 reagent is as follows: use PB buffer as solvent, add and mix all other components mentioned above, and wait until all components are completely dissolved.

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