Total bilirubin detection and concentration reagent as well as preparation method and detection method thereof

Abstract

The invention relates to a total bilirubin detection and concentration reagent. The detection concentration reagent comprises a first concentration reagent, and the first concentration reagent comprises a first buffer solution and at least one quaternary ammonium salt cationic surfactant; and the second concentration reagent comprises a second buffer solution and metavanadate with the content of 7 mmol / L or above. According to the concentration reagent, by optimizing a high-concentration formula, the clinical detection number is effectively increased, good stability is achieved, and the accuracy of a detection result is effectively improved.

Description

Technical Field

[0001] The present disclosure relates to the field of blood analysis, and particularly to a total bilirubin detection concentrated reagent, a preparation method thereof, and a detection method thereof. Background Art

[0002] Bilirubin in blood is divided into direct bilirubin and indirect bilirubin, and the two are collectively referred to as total bilirubin. The content of total bilirubin in blood is an important basis for the determination of jaundice clinically and is also an important indicator for judging the liver metabolism of the human body.

[0003] With the continuous improvement of the medical system and the maturity of the medical consumption concept, the demand for in vitro diagnosis has increased rapidly. As a result, the number of hospital test samples has been increasing continuously, and patients have higher requirements for the timeliness of test results. Due to the large demand for reagents, the workload of reagent loading has been greatly increased, and at the same time, more stringent requirements have been put forward for the reagent refrigeration space.

[0004] In addition, the vanadate oxidation method is a commonly used biochemical detection method, and its detection principle is to use sodium metavanadate as an oxidant to oxidize total bilirubin in blood into biliverdin. The concentration of total bilirubin in blood is detected by the absorbance method.

[0005] Clinical samples may undergo hemolysis during the pre-sample analysis process. In addition, there are various reasons for in vivo hemolysis. When hemolysis occurs, the sample will release free hemoglobin, and the free hemoglobin may cause optical interference or chemical interference in the determination of the analyte.

[0006] Therefore, it is necessary to provide a concentrated reagent for total bilirubin detection with high detection efficiency and accurate detection results. Summary of the Invention

[0007] In view of this, the present disclosure aims to provide a total bilirubin detection concentrated reagent using the vanadate method, which can improve the number of tests in clinical detection and improve the detection efficiency; and further aims to provide a total bilirubin detection reagent with anti-hemolysis interference, which can effectively improve the accuracy of clinical detection.

[0008] The first aspect of the present disclosure provides a total bilirubin detection concentrated reagent, including: a first concentrated reagent, the first concentrated reagent includes a first buffer solution and at least one quaternary ammonium salt cationic surfactant; and a second concentrated reagent, the second concentrated reagent includes a second buffer solution and sodium metavanadate with a content of more than 7 mmol / L.

[0009] In some embodiments, the content of the quaternary ammonium salt cationic surfactant is 15 g / L or more.

[0010] In some embodiments, the content of the quaternary ammonium salt cationic surfactant in the first concentrated reagent is 15-40 g / L; the content of the metavanadate in the second concentrated reagent is 7-16 mmol / L.

[0011] In some embodiments, the quaternary ammonium salt cationic surfactant in the first concentrated reagent is CTAB, and the content is 20-30 g / L; the content of the metavanadate in the second concentrated reagent is 8-12 mmol / L.

[0012] In some embodiments, the first concentrated reagent further comprises at least one non-ionic surfactant.

[0013] In some embodiments, the non-ionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, polyoxyethylene lauryl ether, and polyoxyethylene castor oil ether.

[0014] In some embodiments, in the first concentrated reagent, the content of the non-ionic surfactant is 30 g / L - 80 g / L, preferably 40 g / L - 60 g / L.

[0015] In some embodiments, the pH of the first concentrated reagent is 2.5 - 3.5, and the pH of the second concentrated reagent is 6.0 - 8.0.

[0016] The second aspect of the present disclosure provides a method for detecting total bilirubin in blood. This method uses the concentrated reagent as described above for detection, and includes:

[0017] Dilute the first concentrated reagent with the first dilution water and mix it with the sample to be tested to obtain a first mixture, and obtain the first absorbance A1 after incubation;

[0018] Dilute the second concentrated reagent with the second dilution water and add it to the first mixture to obtain a second mixture, and obtain the second absorbance A2 after incubation; and

[0019] Obtain the content of total bilirubin in the sample to be tested according to the first absorbance A1 and the second absorbance A2.

[0020] The third aspect of the present disclosure provides a preparation method for a concentrated reagent for total bilirubin detection, including:

[0021] Prepare the first concentrated reagent by dissolving at least one quaternary ammonium salt cationic surfactant in a first buffer solution;

[0022] Prepare the second concentrated reagent by dissolving metavanadate in a second buffer solution, wherein the content of the metavanadate in the second concentrated reagent is 7 mmol / L or more.

[0023] In some embodiments, in the first concentrated reagent, the content of the quaternary ammonium salt surfactant is 15 g / L or more.

[0024] In some embodiments, in the first concentrated reagent, the content of the quaternary ammonium salt cationic surfactant is 15 - 40 g / L; in the second concentrated reagent, the content of the metavanadate is 7 - 16 mmol / L.

[0025] In some embodiments, the quaternary ammonium salt cationic surfactant in the first concentrated reagent is CTAB, and the content is 20 - 30 g / L; in the second concentrated reagent, the content of the metavanadate is 8 - 12 mmol / L.

[0026] In some embodiments, preparing the first concentrated reagent further includes:

[0027] Adding at least one non-ionic surfactant to the first buffer solution. Preferably, the non-ionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, polyoxyethylene lauryl ether, and polyoxyethylene castor oil ether.

[0028] More preferably, in the first concentrated reagent, the content of the non-ionic surfactant is 30 - 80 g / L, preferably 40 - 60 g / L.

[0029] In some embodiments, the pH of the first concentrated reagent is 2.5 - 3.5, and the pH of the second concentrated reagent is 6.0 - 8.0.

[0030] The fourth aspect of the present disclosure further provides a total bilirubin detection reagent, including a first reagent, the first reagent includes a first buffer solution, at least one quaternary ammonium salt cationic surfactant, and at least one non-ionic surfactant; and a second reagent, the second reagent includes a second buffer solution and metavanadate; wherein, the non-ionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, polyoxyethylene lauryl ether, and polyoxyethylene castor oil ether.

[0031] In some embodiments, in the first reagent, the content of the quaternary ammonium salt cationic surfactant is 5 - 15 g / L, and the content of the non-ionic surfactant is 10 g / L - 30 g / L; the content of the metavanadate in the second reagent is less than 7 mmol / L;

[0032] Preferably, in the first reagent, the content of the quaternary ammonium salt cationic surfactant is 5 - 10 g / L, and the content of the non-ionic surfactant is 10 - 20 g / L; the content of the metavanadate in the second reagent is 2 - 6.5 mmol / L.

[0033] In some embodiments, the pH of the first reagent is 2.5 - 3.5, and the pH of the second reagent is 6.0 - 8.0.

[0034] The total bilirubin detection concentrated reagent provided by the present disclosure effectively increases the number of test samples per bottle of reagent, improving the detection efficiency; at the same time, it improves the utilization rate of the reagent positions of the instrument and the cold storage space for the stored reagents, saving costs. Through a specific combination of surfactants, it is possible to reduce or even offset the physical and chemical interferences of hemolysis, improve the anti-interference ability of the detection reagent, and thus improve the detection accuracy of the detection reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is the sample reaction curve obtained by the detection reagents of the examples and comparative examples of the present application for detecting high-value clinical serum samples.

[0036] Figure 2 It is a photo of the concentrated detection reagents of the examples and comparative examples of the present application stored at refrigerated temperature for 48 hours. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0037] The technical solutions of the present disclosure will be clearly and completely described below in conjunction with the embodiments and examples. Obviously, the specific embodiments described are only some embodiments, rather than all embodiments. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

[0038] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as having the meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meaning as the general understanding of those skilled in the art to which the present invention pertains. In case of contradiction, this specification shall prevail.

[0039] In this text, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, such that a method or product comprising a series of elements not only includes those elements expressly recited, but also includes other elements not expressly listed, or further includes elements inherent to the implementation of the method or product.

[0040] Unless otherwise specified, the singular forms "a / an" and "the" as used herein include the plural of the nouns referred to.

[0041] Unless otherwise specified, the % used herein refers to mass percentage.

[0042] In the vanadate oxidation method, using metavanadate as an oxidant, in the presence of a surfactant at around pH = 3, total bilirubin in blood is oxidized to biliverdin. Due to the decrease in the specific absorbance of bilirubin's yellow color, a change in the detection signal is generated, and the biochemical analyzer converts this signal into the concentration of total bilirubin in the blood. Automatic detection equipment, such as a fully automatic biochemical analyzer, has been used to detect bilirubin in blood samples. However, for the currently used detection reagents, the number of test samples per bottle of reagent is limited, and the bottle needs to be frequently replaced midway, which seriously reduces the clinical detection efficiency.

[0043] Total bilirubin detection concentrated reagent

[0044] To solve the above problems, the present disclosure provides a total bilirubin detection concentrated reagent. The concentrated reagent includes a first concentrated reagent, the first concentrated reagent includes a first buffer solution and at least one quaternary ammonium salt cationic surfactant; and a second concentrated reagent, the second concentrated reagent includes a second buffer solution and metavanadate with a content of 7 mmol / L or more.

[0045] Compared with conventional reagents, on the basis of not changing the packaging amount of the reagent, the concentrated reagent of the present disclosure controls the amount of reagent to be aspirated for a single test through reasonable reagent design, effectively increasing the number of tests per bottle of concentrated reagent, reducing the bottle replacement frequency of the concentrated reagent, and thus improving the detection efficiency.

[0046] In the detection of total bilirubin, due to the action of intramolecular hydrogen bonds, the reactive groups of free bilirubin cannot be oxidized by the oxidant sodium metavanadate. During the reaction process, a quaternary ammonium salt cationic surfactant needs to be added as an accelerator for the reaction to open the molecular structure of bilirubin, expose its reactive groups, and enable it to be oxidized by sodium metavanadate. To further increase the number of detections, the first reagent also adopts the form of a concentrated reagent. In one embodiment, in the first concentrated reagent, the content of the quaternary ammonium salt cationic surfactant is 15 g / L or more.

[0047] The above first concentrated reagent and second concentrated reagent, in cooperation with the corresponding analyzer, increase the number of test samples and reduce the reagent loading workload. At the same time, the concentrated reagents stored in the same cold storage space can be used to detect more samples, saving storage space and thus reducing the detection cost.

[0048] In one embodiment, the content of the quaternary ammonium cationic surfactant in the first concentrated reagent is 15 - 40 g / L; the content of the metavanadate in the second concentrated reagent is 7 - 16 mmol / L. In the second concentrated reagent, when the content of metavanadate in the diluted detection reagent used for each detection is too low, part of the bilirubin cannot react fully, thus affecting the accuracy of the detection; or in order to ensure that the content of metavanadate in the diluted detection reagent is sufficient, the number of the diluted detection reagents obtained is limited, which in turn leads to a limited increase in the number of sample detections. When the content of metavanadate is too high, there is partial overlap between the absorption peaks of metavanadate and bilirubin, increasing the background absorbance of the reaction endpoint reagent and reducing the sample reactivity, thereby affecting the accuracy of the detection result. In addition, high-concentration metavanadate is difficult to dissolve, which may increase the difficulty of preparing the concentrated reagent, making it inconvenient for operation and mass production of the product. The content of the quaternary ammonium cationic surfactant in the first concentrated reagent is coordinated with the content of metavanadate. If the content of the quaternary ammonium cationic surfactant is too low, the hydrogen bonds in the free bilirubin in the sample cannot be completely opened, which will affect the reactivity of the detection reagent with the total bilirubin in the sample, and in turn lead to poor accuracy of the detection results for clinically high-value samples. If the content is too high, it will also increase the actual production difficulty.

[0049] Exemplarily, in the first concentrated reagent, the content of the quaternary ammonium cationic surfactant can be 15 g / L, 20 g / L, 25 g / L, 30 g / L, 35 g / L, 40 g / L, or the range between any two of these values. The content of metavanadate in the second concentrated reagent can be 7 mmol / L, 7.5 mmol / L, 8 mmol / L, 9 mmol / L, 10 mmol / L, 11 mmol / L, 12 mmol / L, 13 mmol / L, 14 mmol / L, 15 mmol / L, 16 mmol / L, or the range between any two of these values.

[0050] The present disclosure does not make a special limitation on the selection of the quaternary ammonium cationic surfactant, as long as it can be used as a reaction accelerator for the total bilirubin detection. For example, the quaternary ammonium cationic surfactant can be selected from one or more of dodecyl trimethyl ammonium bromide, tetradecyl trimethyl ammonium bromide, and cetyl trimethyl ammonium bromide (CTAB).

[0051] In a preferred embodiment, the quaternary ammonium cationic surfactant in the first concentrated reagent is CTAB, and the content is 20 - 30 g / L; the content of metavanadate in the second concentrated reagent is 8 - 12 mmol / L.

[0052] Total bilirubin detection reagents usually need to be stored refrigerated. Due to the existence of the Krafft Point, the water solubility of quaternary ammonium cationic surfactants decreases at refrigerated temperatures (2 - 8°C), which is not conducive to the formation of micelles and easily aggregates to form nuclei and precipitate crystals. And reagent concentration requires a higher concentration of quaternary ammonium cationic surfactants, which further increases the risk of crystal precipitation during low-temperature storage of the reagents.

[0053] To solve the above problems, in some embodiments, the first concentrated reagent in the total bilirubin detection concentrated reagent provided by the present disclosure further includes at least one non-ionic surfactant. Suitable non-ionic surfactants act as co-solvents and form mixed micelles with quaternary ammonium cationic surfactants, thereby increasing the water solubility of the cationic surfactant at refrigerated temperatures, and further reducing crystal precipitation during low-temperature storage of the reagent.

[0054] In addition, the interaction between the non-ionic surfactant and hemoglobin will cause changes in the conformation of hemoglobin, resulting in the exposure of heme or changes in the secondary and tertiary structures of hemoglobin, leading to changes in the spectral properties and electrochemical behavior of hemoglobin. Thus, the non-ionic surfactant provided by the present disclosure can also improve the anti-hemolysis interference ability of the detection concentrated reagent.

[0055] In some specific embodiments, the non-ionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, lauryl alcohol polyoxyethylene ethers, and castor oil polyoxyethylene ethers. The polyoxyethylene sorbitan fatty acid ester series of surfactants includes, for example, TWEEN-20, TWEEN-40, TWEEN-60, TWEEN-80, etc. The lauryl alcohol polyoxyethylene ether series of surfactants includes, for example, lauryl polyether-9, lauryl polyether-20, lauryl polyether-23 (Brij L23), etc. The castor oil polyoxyethylene ether series of surfactants includes, for example, EL-20, EL-35, EL-40, etc.

[0056] In the first concentrated reagent, the content of the non-ionic surfactant is 30 - 80 g / L. An appropriate amount of non-ionic surfactant can effectively form composite micelles with quaternary ammonium cationic surfactants, thereby increasing the water solubility of the quaternary ammonium cationic surfactant. If the content of the non-ionic surfactant is too high, it will cause an increase in the viscosity of the reagent and affect the precision of the detection results; if the content of the non-ionic surfactant is too low, the quaternary ammonium cationic surfactant will precipitate at the refrigerated temperature (2 - 8°C) of the reagent. Exemplarily, in the first concentrated reagent, the content of the non-ionic surfactant can be 30 g / L, 40 g / L, 50 g / L, 60 g / L, 70 g / L, 80 g / L, or the range between any two of these values. In a preferred embodiment, the content of the non-ionic surfactant is 40 g / L - 60 g / L.

[0057] In the above detection and concentration reagent, the buffer solution is used to provide a suitable detection pH value for the reaction system. The pH of the first concentrated reagent is 2.5 - 3.5, and can be, for example, 2.5, 3.0, 3.5. The pH of the second concentrated reagent is 6.0 - 8.0, and can be, for example, 6.0, 7.0, 8.0. The present disclosure has no particular limitation on the selection of the buffer solution. As long as it can provide a biochemical environment suitable for detection. The first buffer solution optional for the first concentrated reagent includes one or more selected from phosphate buffer solution, malate buffer solution, and citrate buffer solution. Preferably, the pH of the first buffer solution is 3.0. The second buffer solution optional for the second concentrated reagent includes Tris buffer solution, phosphate buffer solution, malate buffer solution, and glycine buffer solution. Preferably, the pH of the second buffer solution is 7.0.

[0058] Detection method for total bilirubin in blood

[0059] The second aspect of the present disclosure provides a detection method for total bilirubin in blood, which uses the total bilirubin detection and concentration reagent as described above for detection. The detection method includes diluting the first concentrated reagent with the first dilution water and then mixing it with the sample to be tested to obtain a first mixture, incubating and then obtaining the first absorbance A1; diluting the second concentrated reagent with the second dilution water and then adding it to the first mixture to obtain a second mixture, incubating and then obtaining the second absorbance A2; and obtaining the content of total bilirubin in the sample to be tested according to the first absorbance A1 and the second absorbance A2.

[0060] The sample to be tested can be a direct blood sample from clinical sampling.

[0061] In one embodiment, the dosage of the sample to be tested can be 3.5 - 7 μL, the dosage of the first concentrated reagent is 73 - 146 μL, the dosage of the first dilution water is 25 - 50 μL, the dosage of the second concentrated reagent is 14.5 - 29 μL, and the dosage of the second dilution water is 10 - 20 μL. The dosages of the concentrated reagent and the dilution water can be adjusted according to the required volume of the reaction system.

[0062] In this detection method, the absorbance of the mixture is measured using a bilirubin detector, with the main wavelength set at 450 nm and the secondary wavelength set at 546 nm.

[0063] In order to obtain the content of total bilirubin, this detection method further includes treating a calibration product with the same dosage in the same method as the sample to be tested, measuring the first absorbance A1' and the second absorbance A2' of the calibration product, and calculating the content of total bilirubin using the following formula:

[0064]

[0065] Preparation method of total bilirubin detection and concentration reagent

[0066] The present disclosure also provides a method for preparing a total bilirubin detection concentrated reagent, including:

[0067] Preparing a first concentrated reagent by dissolving at least one quaternary ammonium salt cationic surfactant in a first buffer solution;

[0068] Preparing a second concentrated reagent by dissolving vanadate in a second buffer solution, wherein the content of vanadate in the second concentrated reagent is above 7 mmol / L.

[0069] In the above preparation method, the buffer solution is used to dissolve the surfactant and vanadate, and provides a suitable pH for the reaction system. In the first concentrated reagent, optional buffer solutions include phosphate buffer solution, malate buffer solution, citrate buffer solution. In the second concentrated reagent, optional buffer solutions include Tris buffer solution, phosphate buffer solution, malate buffer solution, glycine buffer solution. In one embodiment, the pH of the first concentrated reagent is 2.5 - 3.5, preferably 3.0; the pH of the second concentrated reagent is 6.0 - 8.0, preferably 7.0.

[0070] In the above preparation method, by reasonable reagent design to control the amount of reagent aspirated for a single detection, the number of tests per bottle of concentrated reagent is increased, the reagent replacement frequency is reduced, and thus the detection efficiency is improved.

[0071] In one embodiment, the content of the quaternary ammonium salt cationic surfactant in the first concentrated reagent is above 15 g / L. The above first concentrated reagent and second concentrated reagent cooperate with the corresponding analyzer, increasing the number of test samples and reducing the reagent loading workload. At the same time, the concentrated reagents stored in the same refrigerated space can be used to detect more samples, saving the reagent refrigerated storage space, and thus reducing the detection cost.

[0072] In some embodiments, as described above, the first concentrated reagent further includes a nonionic surfactant to improve the water solubility of the quaternary ammonium salt cationic surfactant and prevent the crystallization and precipitation of the quaternary ammonium salt cationic surfactant during low-temperature storage of the reagent. At the same time, the nonionic surfactant also helps to eliminate hemolysis interference.

[0073] In the preparation method, a predetermined amount of the nonionic surfactant and the quaternary ammonium salt cationic surfactant can be dissolved in the first buffer solution to obtain the first concentrated reagent.

[0074] The components and contents of the first concentrated reagent and the second concentrated reagent are the same as those described above and will not be elaborated here.

[0075] Total bilirubin detection reagent

[0076] The present disclosure also provides a total bilirubin detection reagent, which includes a first reagent. The first reagent includes a first buffer, at least one quaternary ammonium salt cationic surfactant, and at least one nonionic surfactant; and a second reagent, which includes a second buffer and metavanadate.

[0077] Among the above surfactants, the quaternary ammonium salt cationic surfactant serves as a reaction accelerator to open the molecular structure of free bilirubin, expose its reactive groups, enabling it to be oxidized by sodium metavanadate, thereby ensuring the accuracy of the test results. The nonionic surfactant interacts with hemoglobin, causing a conformational change in hemoglobin, and thus can improve the anti-hemolysis interference ability of the detection reagent.

[0078] In one embodiment, the nonionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, polyoxyethylene lauryl ether, and polyoxyethylene castor oil ether. The polyoxyethylene sorbitan fatty acid ester series surfactants include, for example, TWEEN-20, TWEEN-40, TWEEN-60, TWEEN-80, etc. The polyoxyethylene lauryl ether series surfactants include, for example, laureth-9, laureth-20, laureth-23 (Brij L23), etc. The polyoxyethylene castor oil ether series surfactants include, for example, EL-20, EL-35, EL-40, etc.

[0079] In some embodiments, in the first reagent, the content of the quaternary ammonium salt cationic surfactant is 5-15 g / L, and the content of the nonionic surfactant is 10-30 g / L; in the second reagent, the content of the metavanadate is less than 7 mmol / L. In the above detection reagent, when the contents of the quaternary ammonium salt cationic surfactant, the nonionic surfactant, and the metavanadate are within the above ranges, the accuracy and anti-hemolysis interference ability of the detection reagent can be improved.

[0080] Exemplarily, in the first reagent, the content of the quaternary ammonium salt cationic surfactant can be 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L or the range between any of these values. The content of the non-ionic surfactant can be 10 g / L, 15 g / L, 20 g / L, 25 g / L, 30 g / L or the range between any of these values. The content of the vanadate can be 6.5 mmol / L, 6 mmol / L, 5.5 mmol / L, 5 mmol / L, 4.5 mmol / L, 4 mmol / L, 3.5 mmol / L, 3 mmol / L, 2.5 mmol / L, 2 mmol / L or the range between any of these values. In a preferred embodiment, in the first reagent, the content of the quaternary ammonium salt cationic surfactant is 5 - 10 g / L, and the content of the non-ionic surfactant is 10 - 20 g / L; in the second reagent, the content of the metavanadate is 2 - 6.5 mmol / L.

[0081] In the above detection reagent, the buffer solution is used to provide a suitable detection pH value for the reaction system. In the first reagent, the first buffer solution includes one or more selected from phosphate buffer solution, malate buffer solution, and citrate buffer solution. In one embodiment, the pH of the first reagent is 2.5 - 3.5, preferably 3.0. In the second reagent, the second buffer solution includes one or more selected from Tris buffer solution, phosphate buffer solution, malate buffer solution, and glycine buffer solution. In one embodiment, the pH of the second reagent is 6.0 - 8.0, preferably 7.0.

[0082] The present invention is further illustrated by the following specific examples. Unless otherwise specified, the reagents are all commercially available conventional reagents.

[0083] Examples

[0084] Preparation of the sample to be tested: A self-made interference solution containing a high concentration (200 g / L) of hemoglobin (200 μL) was used as the hemoglobin interference mother liquor and added to random serum (4 mL) to prepare serum with a hemoglobin content of 1000 mg / dL.

[0085] Pure water of the same volume was added to the same volume of the same random serum as the control group serum.

[0086] Subsequently, the two parts of serum were mixed with each other in different proportions to obtain a series of serum samples with different hemoglobin contents.

[0087] Detection method:

[0088] Concentrated reagent: Add 3.5 μL of calibrator to 73 μL of the first concentrated reagent and 25 μL of the first concentrated reagent diluent water, mix well. After incubating at 37 °C for 5 min, read the absorbance A1' at the 15th measurement point. Then add 14.5 μL of the second concentrated reagent and 10 μL of the second concentrated reagent diluent water, mix well. After incubating at 37 °C for 5 min, read the absorbance A2' at the 32nd measurement point. Among them, the reading period of the measurement points is 18 s.

[0089] Then, use the same method to detect the first absorbance A1 and the second absorbance A2 of the test samples with different hemoglobin contents respectively. According to the following formula:

[0090]

[0091] Calculate the total bilirubin content in each test sample. Among them:

[0092] ΔA 样品 =(A2 - A1);

[0093] ΔA 校准品 =(A2' - A1');

[0094] ΔA is converted and calculated into the corresponding result through the calibration curve.

[0095] The test uses an automatic biochemical analyzer BS - 2800M, with the main wavelength set at 405 nm and the secondary wavelength at 540 nm.

[0096] Each group of reagents is tested in parallel three times, and the average value is taken. Taking the test average value of the test sample with the lowest hemoglobin concentration as the standard, calculate the relative deviation of the test average value of other test samples from the test average value of the test sample with the lowest hemoglobin concentration.

[0097] Non - concentrated reagent: Add 7 μL of calibrator to 196 μL of the first reagent, mix well. After incubating at 37 °C for 5 min, read the absorbance A1' at the 15th measurement point. Then add 49 μL of the second reagent, mix well. After incubating at 37 °C for 5 min, read the absorbance A2' at the 32nd measurement point. Among them, the reading period of the measurement points is 18 s.

[0098] Then, use the same method to detect the first absorbance A1 and the second absorbance A2 of the test samples with different hemoglobin contents respectively.

[0099] Use the calculation method in the above - mentioned concentrated reagent to calculate the total bilirubin content in each test sample, and the relative deviation of each test sample from the test average value of the test sample with the lowest hemoglobin concentration. The test instrument and parameters used are the same as those for the concentrated reagent.

[0100] Examples 1 - 4

[0101] Prepare the total bilirubin detection concentrated reagent according to Table 1 below.

[0102] Comparative Example 1

[0103] Prepare the total bilirubin detection reagent according to Table 1 below.

[0104] Table 1:

[0105]

[0106] Use the detection reagents of the above Examples 1-4 and Comparative Example 1 to test high-value clinical serum samples respectively according to the detection method of the above concentrated reagent, record the absorbance value of each detection point, and draw the reaction curve graph of the sample, as shown in Figure 1 . From Figure 1 It can be seen that the reagents of Examples 1-4 tend to be flat at the end of the reaction curve, indicating that the reaction is complete, while the reagent of Comparative Example 1 does not react completely at the test end, and the finally measured total bilirubin content value is low.

[0107] Example 5

[0108] Prepare the total bilirubin concentrated reagent according to Table 2 below.

[0109] Table 2:

[0110]

[0111] Examples 6-10

[0112] Prepare the total bilirubin concentrated reagent according to the method of Example 5 above, except that in Examples 6-10, the non-ionic surfactants in Table 3 below are selected respectively.

[0113] Comparative Examples 2-3

[0114] Prepare the total bilirubin concentrated reagent according to the method of Example 5 above, except that in Comparative Examples 2-3, the non-ionic surfactants in Table 3 below are selected respectively.

[0115] Table 3:

[0116]

[0117] According to the above test method of the concentrated reagent, use the above concentrated reagent to detect the samples to be tested with different hemoglobin concentrations respectively, and obtain the total bilirubin content in each sample to be tested. And finally calculate the relative deviation of the content between each sample to be tested and the sample to be tested with the lowest hemoglobin concentration. The smaller the relative deviation, the stronger the anti-hemolysis interference ability of the detection concentrated reagent. The test results are shown in Table 4.

[0118] Table 4:

[0119]

[0120] Examples 11 - 13

[0121] Prepare the total bilirubin detection concentrated reagent according to Table 5 below.

[0122] Comparative Examples 4 - 6

[0123] Prepare the total bilirubin detection concentrated reagent according to Table 5 below.

[0124] Table 5:

[0125]

[0126] Place the total bilirubin detection concentrated reagents of the above Examples 11 - 13 and Comparative Examples 4 - 6 at the refrigerated storage temperature (2 - 8 °C). After storing for 48 hours, visually observe the changes in the concentrated reagents. The results are shown in Figure 2 .

[0127] From Figure 2 it can be observed that when the content of the non - ionic surfactant in the concentrated reagent is greater than or equal to 30 g / L, no crystals precipitate in the concentrated reagent during storage at refrigerated temperature. While when the content of the non - ionic surfactant is small, crystals precipitate in the concentrated reagent.

[0128] Examples 14 - 16

[0129] Prepare the total bilirubin detection reagent according to Table 6 below.

[0130] Comparative Examples 7 - 8

[0131] Prepare the total bilirubin detection reagent according to Table 6 below.

[0132] Table 6:

[0133]

[0134] According to the above - mentioned test method for non - concentrated reagents, use the above - mentioned total bilirubin detection reagents to detect the test samples with different hemoglobin concentrations respectively, and obtain the content of total bilirubin in each test sample. And finally calculate the relative deviation of the content between each test sample and the test sample with the lowest hemoglobin concentration. The smaller the relative deviation, the stronger the anti - hemolysis interference ability of the detection reagent. The test results are shown in Table 7.

[0135] Table 7:

[0136]

[0137]

[0138] Control Example

[0139] A commercially available total bilirubin kit is used. The reagent formulations in the instruction manual include: First reagent R1: 0.1 mol / L citric acid buffer solution; Second reagent R2: 10 mmol / L phosphate buffer solution and 4 mmol / L sodium metavanadate. According to the above test method for non-concentrated reagents, the test samples with different hemoglobin concentrations are respectively detected to obtain the content of total bilirubin in each test sample. Each test sample is tested in parallel three times, and its average value is taken. Then, the absolute deviation of the average value of the total bilirubin content between each test sample and the test sample with the lowest hemoglobin concentration is calculated. According to the formula: relative deviation = (absolute deviation / average value of the content of the test sample with the lowest hemoglobin concentration) × 100%, the relative deviation of the test result is calculated. The smaller the relative deviation, the stronger the anti-hemolysis interference ability of the detected concentrated reagent. The test results are shown in Table 8.

[0140] Table 8:

[0141]

[0142] Test Example

[0143] Test Example 1

[0144] The concentrated reagents of manufacturers A, B, C and Example 1 are respectively loaded onto an automated biochemical analyzer for detection, and the volume of the test sample required for a single test and the volumes of the detection reagents R1 and R2 required are recorded. Among them, in the concentrated reagent according to Example 1 of the present application, 25 μL of dilution water is added to the first concentrated reagent for dilution before use, and 10 μL of dilution water is added to the second concentrated reagent R2 before use. The results are shown in Table 10 below.

[0145] Table 10

[0146] Concentrated reagent of Example 1 Manufacturer - A Manufacturer - B Manufacturer - C Sample volume required for a single test (μL) 3.5 10 10 10 Volume of reagent R1 required for a single test (μL) 73 280 280 280 Volume of reagent R2 required for a single test (μL) 14.5 70 70 70

[0147] The above are only examples of some embodiments of the present invention, and thus do not limit the patent scope of the present invention. Any equivalent structural transformation made under the inventive concept of the present invention by using the content of the specification and drawings of the present invention, or any direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A total bilirubin detection concentrated reagent, comprising: A first concentrated reagent, which includes a first buffer and at least one quaternary ammonium salt cationic surfactant; And A second concentrated reagent, which includes a second buffer and metavanadate with a content of more than 7 mmol / L.

2. The concentrated reagent according to claim 1, wherein The content of the quaternary ammonium salt cationic surfactant is 15 g / L or more.

3. The concentrated reagent according to claim 1 or 2, wherein In the first concentrated reagent, the content of the quaternary ammonium salt cationic surfactant is 15 - 40 g / L; in the second concentrated reagent, the content of the metavanadate is 7 - 16 mmol / L.

4. The concentrated reagent according to any one of claims 1-3, wherein, In the first concentrated reagent, the quaternary ammonium salt cationic surfactant is CTAB, and the content is 20 - 30 g / L; in the second concentrated reagent, the content of the metavanadate is 8 - 12 mmol / L.

5. The concentrated reagent according to any one of claims 1-4, wherein, The first concentrated reagent further includes at least one nonionic surfactant.

6. The concentrated reagent according to claim 5, wherein, The nonionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, polyoxyethylene lauryl ether, and polyoxyethylene castor oil ether.

7. The concentrated reagent according to claim 5 or 6, wherein, In the first concentrated reagent, the content of the nonionic surfactant is 30 g / L - 80 g / L, preferably 40 g / L - 60 g / L.

8. The concentrated reagent according to any one of claims 1-7, wherein, The pH of the first concentrated reagent is 2.5 - 3.5, and the pH of the second concentrated reagent is 6.0 - 8.

0.

9. A method for detecting total bilirubin in blood, characterized in that, Using the concentrated reagent according to any one of claims 1 - 8 for detection, including: Diluting the first concentrated reagent with a first dilution water and mixing it with a sample to be tested to obtain a first mixture, and obtaining a first absorbance A1 after incubation; Diluting the second concentrated reagent with a second dilution water and adding it to the first mixture to obtain a second mixture, and obtaining a second absorbance A2 after incubation; and Obtaining the content of total bilirubin in the sample to be tested according to the first absorbance A1 and the second absorbance A2.

10. A preparation method of a total bilirubin detection concentrated reagent, comprising: Preparing the first concentrated reagent by dissolving at least one quaternary ammonium salt cationic surfactant in a first buffer; Preparing the second concentrated reagent by dissolving metavanadate in a second buffer, wherein the content of the metavanadate in the second concentrated reagent is more than 7 mmol / L.

11. The preparation method according to claim 10, wherein, In the first concentrated reagent, the content of the quaternary ammonium salt surfactant is 15 g / L or more.

12. The preparation method according to claim 10 or 11, wherein In the first concentrated reagent, the content of the quaternary ammonium salt cationic surfactant is 15 - 40 g / L; in the second concentrated reagent, the content of the metavanadate is 7 - 16 mmol / L.

13. The preparation method according to any one of claims 10 - 12, wherein, In the first concentrated reagent, the quaternary ammonium salt cationic surfactant is CTAB, and the content is 20 - 30 g / L; in the second concentrated reagent, the content of the metavanadate is 8 - 12 mmol / L.

14. The preparation method according to any one of claims 11-13, wherein, Preparing the first concentrated reagent further includes: Adding at least one nonionic surfactant to the first buffer, preferably, the nonionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, polyoxyethylene lauryl ether, and polyoxyethylene castor oil ether; More preferably, in the first concentrated reagent, the content of the nonionic surfactant is 30-80 g / L, preferably 40-60 g / L.

15. The preparation method according to any one of claims 11-14, wherein, The pH of the first concentrated reagent is 2.5-3.5, and the pH of the second concentrated reagent is 6.0-8.

0.

16. A total bilirubin detection reagent, comprising: A first reagent, the first reagent comprising a first buffer, at least one quaternary ammonium salt cationic surfactant and at least one nonionic surfactant; And A second reagent, the second reagent comprising a second buffer and metavanadate; Wherein, the nonionic surfactant is selected from polyoxyethylene sorbitan fatty acid esters, polyoxyethylene lauryl ether and polyoxyethylene castor oil.

17. The reagent according to claim 16, wherein, In the first reagent, the content of the quaternary ammonium salt cationic surfactant is 5-15 g / L, and the content of the nonionic surfactant is 10 g / L - 30 g / L; the content of metavanadate in the second reagent is less than 7 mmol / L; Preferably, in the first reagent, the content of the quaternary ammonium salt cationic surfactant is 5-10 g / L, and the content of the nonionic surfactant is 10-20 g / L; the content of metavanadate in the second reagent is 2-6.5 mmol / L.

18. The reagent according to claim 16 or 17, wherein, The pH of the first reagent is 2.5-3.5, and the pH of the second reagent is 6.0-8.0.

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