Direct bilirubin detection reagent and detection method

Abstract

The invention relates to a direct bilirubin detection and concentration reagent. The concentration reagent comprises a first concentration reagent, and the first concentration reagent comprises a first buffer solution, a surfactant, hydroxylamine hydrochloride with the content being 1.5 g / L or above and hydroxyethylidene diphosphoric acid with the content being 4 g / L or above; the second concentration reagent comprises a second buffer solution and metavanadate with the content of 0.5 g / L or above. According to the concentration reagent, by optimizing a high-concentration formula, the clinical detection number is effectively increased, the reagent cost is reduced, 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 reagent for detecting direct bilirubin and a method for detecting direct bilirubin. Background Art

[0002] Direct bilirubin in blood is mainly formed by the conjugation of indirect bilirubin entering the liver with glucuronic acid in hepatocytes (monoglucuronide and diglucuronide bilirubin). The content of direct bilirubin is an important basis for judging jaundice clinically and is also an important indicator of liver function. An increase in the content of direct bilirubin in blood is mainly seen in obstructive jaundice, hepatocellular jaundice, liver cancer, pancreatic head cancer, cholelithiasis, cholangiocarcinoma, etc. Existing methods for detecting direct bilirubin in blood include enzymatic method, diazonium salt method, and chemical oxidation method, such as vanadate method. Among them, the diazonium salt method and vanadate method are relatively commonly used biochemical detection methods.

[0003] With the increasing demand for sample testing in hospitals day by day, conventional biochemical reagents have gradually been unable to meet the testing volume requirements of hospitals. In order to increase the testing volume, it is often necessary to change bottles and pour bottles midway, which seriously reduces the testing efficiency of clinical detection and increases the clinical risk of biochemical detection.

[0004] In addition, the vanadate method acts on direct bilirubin through the oxidizing property of metavanadate, oxidizes bilirubin to biliverdin, and detects it by absorbance method. If red blood cells are broken for various reasons in the sample and hemoglobin therein enters the detection solution, it will interfere with the detection result due to the oxidizing property of hemoglobin. Therefore, it is necessary to provide a direct bilirubin detection reagent with high detection efficiency and accurate detection results. Summary of the Invention

[0005] In view of this, the present disclosure aims to provide a direct bilirubin detection concentrated reagent using the vanadate method, which can improve the number of tests in clinical detection and enhance the detection efficiency; and further aims to provide a direct bilirubin detection reagent with good anti-hemoglobin interference performance, which can effectively improve the accuracy of clinical detection.

[0006] A first aspect of the present disclosure provides a direct bilirubin detection concentrated reagent, including: a first concentrated reagent, the first concentrated reagent includes a first buffer solution, a surfactant, hydroxylamine hydrochloride with a content of more than 1.5 g / L, and 1-hydroxyethylidene-1,1-diphosphonic acid with a content of more than 4 g / L; and a second concentrated reagent, the second concentrated reagent includes a second buffer solution and metavanadate with a content of more than 0.5 g / L.

[0007] In some embodiments, the content of hydroxylamine hydrochloride in the first concentrated reagent is 1.5 - 20 g / L, and the content of hydroxyethane diphosphonic acid is 4 - 50 g / L; the content of the metavanadate in the second concentrated reagent is 0.5 - 1.2 g / L; preferably, the content of hydroxylamine hydrochloride in the first concentrated reagent is 2.0 - 5.0 g / L, and the content of hydroxyethane diphosphonic acid is 5.0 - 10.0 g / L; the content of the metavanadate in the second concentrated reagent is 0.5 - 1.0 g / L.

[0008] In some embodiments, the first concentrated reagent further comprises a reducing agent.

[0009] In some embodiments, the reducing agent comprises at least one of tris(2 - carboxyethyl)phosphine hydrochloride and a mercapto - containing compound; preferably, the mercapto - containing compound comprises at least one selected from mercaptoethanol and dithiothreitol.

[0010] In some embodiments, the content of the reducing agent is 0.1 - 0.5 g / L, preferably 0.2 - 0.4 g / L, and more preferably 0.25 - 0.35 g / L.

[0011] In some embodiments, the second concentrated reagent further comprises an inorganic salt; preferably, the inorganic salt is selected from at least one of the alkali metal salts of hydrochloric acid, sulfuric acid, and nitric acid; more preferably, the inorganic salt is selected from one of NaCl, Na2SO4, and NaNO3.

[0012] In some embodiments, in the second concentrated reagent, the content of the inorganic salt is 1 - 8 g / L, preferably 2 - 6 g / L, and more preferably 3 - 5 g / L.

[0013] In some embodiments, the content of the surfactant in the first concentrated reagent is 1 - 15 g / L, preferably 5 - 15 g / L.

[0014] In some embodiments, the surfactant is a non - ionic surfactant; preferably, the surfactant comprises at least one selected from polyoxyethylene surfactants Triton - 405, Brij - 35, Tween - 80, and Tween - 20.

[0015] In some embodiments, the pH of the first concentrated reagent is 2.5 - 3.0, and the pH of the second concentrated reagent is 6.2 - 7.0.

[0016] The second aspect of the present disclosure provides a method for detecting direct bilirubin in blood, which uses the concentrated reagent in the above - mentioned embodiments for detection. The detection method includes:

[0017] The first concentrated reagent is diluted with a first diluent and then mixed with a sample to be tested to obtain a first mixture. After incubation, a first absorbance A1 is obtained.

[0018] The second concentrated reagent is diluted with a second diluent and then added to the first mixture to obtain a second mixture. After incubation, a second absorbance A2 is obtained; and

[0019] The content of direct bilirubin in the sample to be tested is obtained based on the first absorbance A1 and the second absorbance A2.

[0020] Preferably, the dosage of the sample to be tested is 3 - 10 μL, the dosage of the first concentrated reagent is 10 - 70 μL, the dosage of the first diluent is 10 - 70 μL, the dosage of the second concentrated reagent is 10 - 50 μL, and the dosage of the second diluent is 5 - 50 μL.

[0021] The third aspect of the present disclosure further provides a direct bilirubin detection reagent, including: a first reagent, the first reagent includes a first buffer, a surfactant, a reducing agent, hydroxylamine hydrochloride, and hydroxyethylidene diphosphonic acid; and a second reagent, the second reagent includes a second buffer and vanadate.

[0022] In some embodiments, the reducing agent includes at least one of tris(2 - carboxyethyl)phosphine hydrochloride and a thiol - containing compound; preferably, the reducing agent includes at least one selected from mercaptoethanol and dithiothreitol.

[0023] In some embodiments, the content of the reducing agent in the first reagent is 0.1 - 0.5 g / L, preferably 0.2 - 0.4 g / L, and more preferably 0.25 - 0.35 g / L.

[0024] In some embodiments, the second reagent further includes an inorganic salt; preferably, the inorganic salt is at least one selected from alkali metal salts of hydrochloric acid, sulfuric acid, and nitric acid; more preferably, the inorganic salt is selected from NaCl, Na2SO4, and NaNO3.

[0025] In some embodiments, in the second reagent, the content of the inorganic salt is 1 - 4 g / L, preferably 2 - 3 g / L.

[0026] In some embodiments, the content of hydroxylamine hydrochloride in the first reagent is 0.1 - 1 g / L, and the content of hydroxyethylidene diphosphonic acid is 0.1 - 3 g / L; the content of vanadate in the second reagent is 0.05 - 0.45 g / L.

[0027] In some embodiments, the surfactant is a nonionic surfactant. Preferably, the surfactant includes at least one selected from polyoxyethylene surfactants. More preferably, the surfactant includes Brij-35. More preferably, the content of the surfactant in the first reagent is 5-15 g / L.

[0028] The fourth aspect of the present disclosure also provides a use of a reducing agent as an anti-hemolysis interference agent in a direct bilirubin detection reagent.

[0029] The fifth aspect of the present disclosure also provides a use of a reducing agent in the preparation of a direct bilirubin detection reagent for anti-hemolysis interference.

[0030] In some embodiments, the direct bilirubin detection reagent includes a first reagent containing the reducing agent and a second reagent containing vanadate. The reducing agent includes at least one of tris(2-carboxyethyl)phosphine hydrochloride and a thiol-containing compound. Preferably, the thiol-containing compound includes at least one selected from mercaptoethanol and dithiothreitol. More preferably, the content of the reducing agent in the first reagent is 0.1-0.5 g / L, preferably 0.2-0.4 g / L.

[0031] In some embodiments, the second reagent further includes an inorganic salt selected from at least one of alkali metals of hydrochloric acid, sulfuric acid, and nitric acid. Preferably, the inorganic salt is selected from one of NaCl and Na2SO4. Preferably, in the second reagent, the content of the inorganic salt is 1-8 g / L, preferably 2-6 g / L.

[0032] The present disclosure provides a direct bilirubin detection concentrated reagent, which effectively increases the number of clinical test samples per set of reagents, improves the detection efficiency, and reduces the reagent cost. Further, the reducing agent in the concentrated reagent enhances its reducing ability, so that direct bilirubin is not affected by hemoglobin entering the detection solution due to red blood cell hemolysis in the sample. In combination with the inorganic salt and surfactant in the reagent, it can effectively reduce or even eliminate the interference of hemoglobin on the biochemical reagent to be detected. The detection method for direct bilirubin in blood samples provided by the present disclosure is simple to operate, and the use of the concentrated reagent can effectively increase the number of tests in clinical detection. In addition, the present disclosure also provides a direct bilirubin detection reagent, which can effectively reduce or eliminate the interference of hemoglobin in the biochemical reagent to be detected by the reducing agent, and improve the accuracy of the detection result. Detailed Embodiments

[0033] 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 a part of the embodiments, rather than all the embodiments. Based on these embodiments, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0034] 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.

[0035] 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 includes not only those elements expressly recited, but also other elements not expressly listed, or elements inherent to the implementation of the method or product.

[0036] Unless otherwise specified, the singular forms "a / an" and "the" used herein include the plural of the nouns referred to.

[0037] Unless otherwise specified, the % used herein refers to mass percentage.

[0038] The method for detecting direct bilirubin in blood by the vanadate method is to act vanadate on a blood sample. Due to the oxidizing property of vanadate, direct bilirubin is oxidized to biliverdin, and the total bilirubin content in the blood is calculated by detecting the change in absorbance before and after the oxidation of vanadate. Automatic detection equipment, such as biochemical analyzers, has been used to detect bilirubin in blood samples. However, for the currently used detection reagents, the number of test samples per set of reagents is limited, and it is necessary to frequently change and pour the reagent bottles midway, which seriously reduces the clinical detection efficiency and increases the clinical risk.

[0039] Direct bilirubin detection concentrated reagent

[0040] To solve the above problems, the present disclosure provides a direct bilirubin detection concentrated reagent. The concentrated reagent includes a first concentrated reagent, and the first concentrated reagent includes a first buffer, a surfactant, hydroxylamine hydrochloride with a content of more than 1.5 g / L, and 1-hydroxyethylidene-1,1-diphosphonic acid (also known as hydroxyethane diphosphonic acid) with a content of more than 4 g / L; and a second concentrated reagent, and the second concentrated reagent includes a second buffer and vanadate with a content of more than 0.5 g / L.

[0041] In a conventional bilirubin detection reagent, the concentration of metavanadate in the second reagent is usually 4 mmol / L (i.e., about 0.488 g / L) or lower. In the second concentrated reagent of the present disclosure, the content of metavanadate is above 0.5 g / L. When combined with the first concentrated reagent, the content of hydroxylamine hydrochloride is above 1.5 g / L and the content of hydroxyethylidene diphosphonic acid is above 4 g / L. Thus, when combined with an analyzer, without changing the reagent packaging volume, the number of tests with a set of reagents can be effectively increased, the liquid change frequency can be reduced, thereby improving the detection efficiency, reducing the detection risk, and reducing the detection cost.

[0042] In one embodiment, the content of hydroxylamine hydrochloride in the first concentrated reagent is 1.5 - 20 g / L, and the content of hydroxyethylidene diphosphonic acid is 4 - 50 g / L; the content of metavanadate in the second concentrated reagent is 0.5 - 1.2 g / L. In the second concentrated reagent, if the content of metavanadate is too low, the effect of the concentrated reagent cannot be achieved. After dilution of the concentrated reagent, the content of metavanadate in the solvent may be insufficient, and some direct bilirubin may not react fully, thus affecting the accuracy of the detection. If the content of metavanadate is too high, it will exceed the upper limit of the instrument absorbance, restricting the use of the vanadate concentration. The contents of hydroxylamine hydrochloride and hydroxyethylidene diphosphonic acid in the first concentrated reagent are coordinated with the content of metavanadate. If the contents of hydroxylamine hydrochloride and hydroxyethylidene diphosphonic acid are too low, the reactivity of the detection reagent with direct bilirubin will be affected, and further the accuracy of the detection result will be poor. If the contents are too high, it will also increase the actual production difficulty.

[0043] Exemplarily, in the first concentrated reagent, the content of hydroxylamine hydrochloride can be 1.5 g / L, 2 g / L, 3 g / L, 4 g / L, 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, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, and values within the range composed of any two of these values. In a preferred embodiment, the content of hydroxylamine hydrochloride in the first concentrated reagent is 2 - 10 g / L, and more preferably, 2 - 5 g / L.

[0044] Exemplarily, in the first concentrated reagent, the content of hydroxyethane diphosphonic acid can be 4 g / L, 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, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L, 30 g / L, 40 g / L, 50 g / L, and values between the ranges formed by any two values. In a preferred embodiment, the content of hydroxyethane diphosphonic acid is 4.0 - 20 g / L, more preferably 5.0 - 10.0 g / L; the content of the vanadate in the second concentrated reagent is 0.5 - 1.0 g / L, more preferably 0.5 - 0.8 g / L.

[0045] In some embodiments, the first concentrated reagent provided by the present disclosure further includes a reducing agent. Since hemoglobin in the blood sample to be tested has oxidizing properties, hemoglobin will consume a part of the reducing direct bilirubin, resulting in deviation of the results, thus reducing the accuracy of the detection results. The reducing agent in the first concentrated reagent increases the reducing ability of the detection system. When the first concentrated reagent is diluted and mixed with the blood sample, the reducing agent preferentially binds to the oxidizing hemoglobin and acts as a protector for direct bilirubin, so that direct bilirubin is not affected by hemoglobin, enabling the concentrated reagent to have the ability to resist hemoglobin interference.

[0046] In some specific embodiments, the reducing agent includes at least one selected from tris(2 - carboxyethyl)phosphine hydrochloride (TCEP) and sulfhydryl - containing compounds. In some preferred embodiments, the sulfhydryl - containing compound includes at least one of mercaptoethanol and dithiothreitol (DTT). The above - mentioned reducing agents have high safety, are friendly to production and the environment, and are suitable for the direct bilirubin detection reagent of this application, having good temperature stability and system compatibility. In a preferred embodiment, the reducing agent is TCEP, which has excellent anti - interference performance.

[0047] In the first concentrated reagent, the content of the above - mentioned reducing agent is 0.1 - 0.5 g / L. When the concentration of the reducing agent is within the above range, it shows a good effect of resisting hemoglobin interference. In a preferred embodiment, the content of the reducing agent is 0.2 - 0.4 g / L, more preferably 0.25 - 0.35 g / L. Exemplarily, the content of the reducing agent can be 0.15 g / L, 0.20 g / L, 0.25 g / L, 0.28 g / L, 0.30 g / L, 0.32 g / L, 0.35 g / L, 0.40 g / L, 0.45 g / L, etc., and values between the ranges formed by any two values.

[0048] In some embodiments, the second concentrated reagent further comprises an inorganic salt. Adding an inorganic salt to the second concentrated reagent can increase the ion concentration in the detection system. Due to the primary salt effect in solution reactions, the higher the ion concentration in the solution, the faster the reaction rate, thereby accelerating the reaction rate between vanadate and direct bilirubin. It can be understood that the first concentrated reagent may also include an inorganic salt, and the present invention does not make a special limitation on this.

[0049] Research has found that the addition of an inorganic salt affects the anti-hemoglobin interference effect of the detection system. A suitable inorganic salt combined with the above reducing agent can further enhance the anti-hemoglobin interference ability of the detection system.

[0050] In some embodiments, the inorganic salt is selected from alkali metal or alkaline earth metal salts of hydrochloric acid, sulfuric acid, and nitric acid. In some specific embodiments, the inorganic salt is selected from one or more of sodium chloride (NaCl), sodium sulfate (Na2SO4), and sodium nitrate (NaNO3). In a preferred embodiment, the inorganic salt is NaCl.

[0051] According to one embodiment, in the second concentrated reagent, the content of the inorganic salt can be 1 - 8 g / L. The content of the inorganic salt within the above range can cooperate with the reducing agent to further enhance the anti-hemoglobin interference effect of the detection system, and the detection system has good detection accuracy for both high-concentration bilirubin samples to be tested and low-concentration bilirubin samples to be tested.

[0052] Exemplarily, the content of the inorganic salt can be 2 g / L, 3 g / L, 3.5 g / L, 4 g / L, 4.5 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, or the range between any of their values. In some preferred embodiments, the content of the inorganic salt is 2 - 6 g / L, more preferably, the content of the inorganic salt is 3 - 5 g / L, and most preferably, the content of the inorganic salt is 4 g / L.

[0053] In a preferred embodiment, in the above concentrated reagent, the reducing agent in the first concentrated reagent is tris(2-carboxyethyl)phosphine hydrochloride, and its content is 0.35 g / L. The inorganic salt in the second concentrated reagent is NaCl, and its content is 4 g / L. The combination of the above reducing agent and inorganic salt effectively enhances the anti-hemoglobin interference ability of the above concentrated reagent and increases the accuracy of the detection result.

[0054] In the first concentrated reagent, the surfactant helps the dissolution and dispersion of each component in the reagent, and resists the adverse effects on the homogeneous stability caused by a series of changes such as external temperature and pH value. According to some embodiments, the content of the surfactant in the first concentrated reagent is 1 - 15 g / L, preferably 5 - 15 g / L.

[0055] The present disclosure places no particular restrictions on the type of surfactant, as long as it can contribute to the dissolution and dispersion of the components in the reagent. In some embodiments, the surfactant is a non-ionic surfactant. More preferably, the surfactant includes at least one selected from polyoxyethylene surfactants. Examples include, but are not limited to, Triton series such as Triton X-405, Triton X-100, etc.; Tween series such as Tween 80, Tween 20, etc.; and Brij35. Preferably, Brij35 and Tween-20.

[0056] In the first concentrated 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 - 3.0, and the pH of the second concentrated reagent is 6.2 - 7.0. The present disclosure places no particular restrictions on the selection of the buffer solution, as long as it can provide a biochemical environment suitable for detection. Optional buffer solutions include tartaric acid buffer solution, citric acid buffer solution, phosphate buffer solution, etc.

[0057] Detection method for direct bilirubin in blood

[0058] The second aspect of the present disclosure provides a method for detecting direct bilirubin in blood, which uses the direct bilirubin detection concentrated 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 direct bilirubin in the sample to be tested based on the first absorbance A1 and the second absorbance A2.

[0059] The sample to be tested can be a direct blood sample from clinical sampling.

[0060] In some embodiments, the dosage of the sample to be tested can be 3 - 10 μL, the dosage of the first concentrated reagent is 10 - 70 μL, the dosage of the first dilution water is 10 - 70 μL, the dosage of the second concentrated reagent is 10 - 50 μL, and the dosage of the second dilution water is 5 - 50 μL. The dosages of the concentrated reagent and the dilution water can be adjusted according to the final concentration of vanadate in the detection reagent required.

[0061] 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.

[0062] To obtain the content of direct bilirubin, the detection method further includes treating the calibrator in the same way as the test sample to be processed, measuring the first absorbance A1' and the second absorbance A2' of the calibrator, and calculating the content of direct bilirubin using the following formula:

[0063]

[0064] Direct bilirubin detection reagent

[0065] The present disclosure also provides a direct bilirubin detection reagent, which includes a first reagent and a second reagent. The first reagent includes a first buffer, a surfactant, a reducing agent, hydroxylamine hydrochloride, and hydroxyethylidene diphosphonic acid; the second reagent includes a second buffer and vanadate.

[0066] In the first reagent, the reducing agent is used to enhance the reduction ability of the detection system. When the first reagent is mixed with a blood sample, the reducing agent preferentially binds to hemoglobin in the blood sample, so that direct bilirubin is not affected by hemoglobin, thereby improving the ability of the detection reagent to resist hemoglobin interference. In some embodiments, the reducing agent includes at least one selected from tris(2-carboxyethyl)phosphine hydrochloride and sulfhydryl-containing compounds. In a preferred embodiment, the sulfhydryl-containing compound includes at least one selected from mercaptoethanol and dithiothreitol. The above reducing agents are highly safe and friendly to production and the environment. In a preferred embodiment, the reducing agent is TCEP, which has excellent ability to resist hemoglobin interference.

[0067] In the first reagent, the content of the reducing agent is 0.1 - 0.5 g / L. Exemplarily, the content of the reducing agent can be 0.25 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, and any range between the values. In a preferred embodiment, the content of the reducing agent is 0.2 - 0.4 g / L, more preferably 0.25 - 0.35 g / L. In a preferred embodiment, the content of the reducing agent is 0.35 g / L.

[0068] In some embodiments, the second reagent further includes inorganic salts. Adding inorganic salts to the second reagent can increase the ion concentration in the detection system. Since there is a salt effect in the solution reaction, the higher the ion concentration in the solution, the faster the reaction rate, thereby accelerating the reaction rate between vanadate and direct bilirubin. In addition, appropriate inorganic salts cooperate with the above reducing agent to further increase the ability of the bilirubin detection reagent to resist hemoglobin interference. It can be understood that the first reagent can also include inorganic salts, and the present application does not make special limitations on this.

[0069] In some embodiments, the inorganic salt is selected from alkali metal or alkaline earth metal salts of hydrochloric acid, sulfuric acid and nitric acid. In a specific embodiment, the inorganic salt is selected from one or more of sodium chloride (NaCl), sodium sulfate (Na2SO4), and sodium nitrate (NaNO3). In a preferred embodiment, the inorganic salt is selected from NaCl and Na2SO4, and more preferably NaCl.

[0070] According to some embodiments, in the second reagent, the content of the inorganic salt can be the same or different, and is 1-4 g / L respectively. Exemplarily, the content of the inorganic salt can be 1 g / L, 2 g / L, 3 g / L, 4 g / L or the range between any values thereof. In a preferred embodiment, the content of the inorganic salt is 2-3 g / L, and more preferably, the content of the inorganic salt is 2 g / L.

[0071] In a preferred embodiment, in the above-mentioned concentrated reagent, the reducing agent in the first concentrated reagent is tris(2-carboxyethyl)phosphine hydrochloride, and its content is 0.35 g / L, and the inorganic salt in the second concentrated reagent is NaCl, and its content is 2 g / L.

[0072] In one embodiment, in the first reagent, the content of hydroxylamine hydrochloride is 0.1-1 g / L, and the content of hydroxyethylidene diphosphonic acid is 0.1-3 g / L; the content of the metavanadate in the second reagent is 0.05-0.45 g / L. The concentration of each reagent in the detection system is coordinated to achieve the best detection ability. Exemplarily, in the first reagent, the content of hydroxylamine hydrochloride can be 0.1 g / L, 0.3 g / L, 0.5 g / L, 0.7 g / L, 0.9 g / L, 1.0 g / L, and the values between the ranges formed by any two values. Preferably, the content of hydroxylamine hydrochloride can be 0.5-1.0 g / L. Exemplarily, in the first reagent, the content of hydroxyethylidene diphosphonic acid can be 0.1 g / L, 0.5 g / L, 1.0 g / L, 1.4 g / L, 1.8 g / L, 2.0 g / L, 2.2 g / L, 2.4 g / L, 2.6 g / L, 2.8 g / L, 3.0 g / L, and the values between the ranges formed by any two values. Preferably, the content of hydroxyethylidene diphosphonic acid can be 1-3 g / L. Exemplarily, in the second reagent, the content of the metavanadate can be 0.05 g / L, 0.1 g / L, 0.2 g / L, 0.3 g / L, 0.35 g / L, 0.4 g / L, 0.45 g / L and the values between the ranges formed by any two values. Preferably, the content of the metavanadate can be 0.2-0.45 g / L.

[0073] In the first reagent, a surfactant is used to promote the dissolution and dispersion among the components to resist the adverse effects on the homogeneous stability caused by a series of changes such as external temperature, pH value, etc. The present disclosure has no particular limitation on the type of the surfactant, as long as it can contribute to the dissolution and dispersion of the components in the reagent. In some embodiments, the surfactant is a nonionic surfactant. More preferably, the surfactant includes at least one selected from polyoxyethylene surfactants. Exemplarily, the surfactant is, for example, the Triton series, such as Triton X-405, Triton X-100; the Tween series, such as Tween 80, Tween 20, etc.; and Brij35, but not limited thereto. Preferably, Brij35 and Tween80. According to some embodiments, the content of the surfactant in the first reagent is 1-15 g / L, preferably 5-15 g / L.

[0074] The present disclosure also provides the use of a reducing agent as an anti-hemolysis interference agent in a direct bilirubin detection reagent.

[0075] The present disclosure also provides the use of a reducing agent in the preparation of a direct bilirubin detection reagent for anti-hemolysis interference.

[0076] In some embodiments, the direct bilirubin detection reagent includes a first reagent containing the reducing agent and a second reagent containing metavanadate, and the reducing agent includes a thiol-containing compound; preferably, the reducing agent includes at least one selected from tris(2-carboxyethyl)phosphine hydrochloride, mercaptoethanol, and dithiothreitol; more preferably, the content of the reducing agent in the first reagent is 0.1-0.5 g / L, preferably 0.2-0.4 g / L.

[0077] In some embodiments, the second reagent further includes an inorganic salt, and the inorganic salt is selected from at least one of alkali metals of hydrochloric acid, sulfuric acid, and nitric acid; preferably, the inorganic salt is selected from one of NaCl and Na2SO4; preferably, in the second reagent, the content of the inorganic salt is 1-8 g / L, preferably 2-6 g / L.

[0078] The present invention is further illustrated by the following specific examples. Unless otherwise specified, the reagents are all commercially available conventional reagents.

[0079] Examples

[0080] Preparation of the sample to be tested:Use the composite calibrator (including the direct bilirubin item), composite high-value quality control material (including the direct bilirubin item), and composite low-value quality control material (including the direct bilirubin item) from Mindray. According to the instructions, add the corresponding volume of deionized water to dissolve the calibrator and quality control material respectively, mix well to obtain the samples to be tested, and aliquot them at 300 μL per portion. The bilirubin concentrations in the samples to be tested of the calibrator, low-value quality control material, and high-value quality control material are 38.3 μmol / L, 10 ± 2.4 μmol / L, and 31.2 ± 6.9 μmol / L respectively. Store the aliquoted samples in a -80 °C refrigerator. When taking samples, the samples must be thawed at 2 - 8 °C and mixed well before use.

[0081] Interference sample preparation: Add different masses of hemoglobin to the samples to be tested of the above high-value quality control material and low-value quality control material respectively to prepare the samples to be tested with different hemoglobin concentrations (0 - 500 mg / dL) for interference.

[0082] Absorbance measurement:

[0083] Concentrated reagent: Mix 50 μL of the first concentrated reagent R1 and 50 μL of the first concentrated reagent diluent water, then add 5 μL of the calibrator. After incubating at 37 °C for 5 min, read the first absorbance A1' at the 15th measurement point. Then, add 20 μL of the second concentrated reagent R2 and 20 μL of the second concentrated reagent diluent water, mix well, and read the second absorbance A2' at the 32nd measurement point after incubating at 37 °C for 5 min.

[0084] Then, use the same method to detect the first absorbance A1 and the second absorbance A2 of the high-value quality control material, low-value quality control material, and the samples to be tested with different hemoglobin concentrations for interference respectively. According to the following formula:

[0085]

[0086] Calculate the content of direct bilirubin in each sample to be tested and interference sample.

[0087] The above test uses the Mindray biochemical analyzer BS-2800M, sets the main wavelength at 450 nm and the secondary wavelength at 546 nm to measure the absorbance of the reagent, and records the absorbance value.

[0088] Using the above test method, use the above concentrated reagent to detect each sample to be tested and interference sample respectively. Each sample is tested in parallel three times to obtain the average value of the content of direct bilirubin in each sample to be tested and interference sample. Finally, calculate the relative deviation between the interference sample and the non-interference sample without adding hemoglobin interference substance. Relative deviation = (interference sample - non-interference sample) / non-interference sample. The smaller the relative deviation, the stronger the anti-interference ability of the detection concentrated reagent.

[0089] Example 1-2

[0090] Prepare the direct bilirubin detection concentrated reagent according to the formula in Table 1 below. The difference between Example 1 and Example 2 lies in the types of inorganic salts in the second concentrated reagent. Then, use the same method to detect the direct bilirubin concentration in the test sample and the interference sample, and the test results are shown in Table 2.

[0091] Comparative Example 1

[0092] Prepare the direct bilirubin detection concentrated reagent according to the formula in Table 1 below. The difference from Example 1 is that thiourea is used as the reducing agent, and the direct bilirubin concentration in the test sample and the interference sample is detected by the same method, and the results are shown in Table 2.

[0093] Table 1

[0094]

[0095] Table 2

[0096]

[0097]

[0098] Example 3-6

[0099] Prepare the direct bilirubin detection concentrated reagent according to the formula in Table 3 below. The difference between Example 3-6 is only the concentration of the reducing agent TCEP used. Then, use the same method to detect the direct bilirubin concentration in the test sample and the interference sample, and the test results are shown in Table 4.

[0100] Table 3.

[0101]

[0102] Table 4.

[0103]

[0104] Example 7-12

[0105] Prepare the direct bilirubin detection concentrated reagent according to the formula in Table 5 below. The difference between Example 7-12 is only the concentration of the inorganic salt NaCl used. Then, use the same method to detect the direct bilirubin concentration in the test sample and the interference sample, and the test results are shown in Table 6.

[0106] Table 5:

[0107]

[0108] Table 6:

[0109]

[0110] Examples 13 - 16

[0111] Prepare direct bilirubin detection reagents according to the formulation in Table 7 below. The differences among Examples 13 - 16 lie in the different concentrations of the reducing agent TECP used.

[0112] Then, use the same method to detect the direct bilirubin concentration in the sample to be tested and the interfering sample.

[0113] Absorbance test:

[0114] Add 7 μL of the calibrator to 196 μL of the first reagent R1. After incubating at 37 °C for 5 min, read the first absorbance A1’ at the 15th measurement point. Then, add 49 μL of the second reagent R2 and mix well. After incubating at 37 °C for 5 min, read the second absorbance A2’ at the 32nd measurement point.

[0115] Then, use the same method to detect the first absorbance A1 and the second absorbance A2 of the high - value quality control product, the low - value quality control product, and the sample to be tested with interfering samples of different hemoglobin concentrations respectively. According to the following formula:

[0116]

[0117] Calculate the content of direct bilirubin in each sample to be tested and interfering sample.

[0118] The test is carried out using Mindray biochemical analyzer BS2800M. Set the main wavelength to 450 nm and the secondary wavelength to 546 nm to measure the absorbance of the reagent, and record the absorbance value.

[0119] According to the above test method, use the above detection reagents to detect each sample to be tested and interfering sample respectively. Each sample is tested in parallel 3 times, and calculate the average value of the content of direct bilirubin in each sample to be tested and interfering sample. Finally, calculate the relative deviation between the interfering sample and the non - interfering sample without adding hemoglobin interfering substance. Relative deviation = (interfering sample - non - interfering sample) / non - interfering sample. The smaller the relative deviation, the stronger the anti - interference ability of the detection reagent. The test results are shown in Table 8.

[0120] Table 7.

[0121]

[0122] Table 8.

[0123]

[0124] Examples 17 - 18

[0125] Prepare a direct bilirubin detection reagent according to the formulation in Table 9. The only difference between Examples 17-18 is the concentration of the inorganic salt NaCl used. Then, detect the direct bilirubin concentration in the test sample and interference sample in the same manner as in Examples 13-16. The test results are shown in Table 10.

[0126] Table 9.

[0127]

[0128] Table 10

[0129]

[0130] The above are only examples of some embodiments of the present invention, and do not limit the patent scope of the present invention. Any equivalent structural transformation made using the content of the specification of the present invention under the inventive concept of the present invention, or direct / indirect application in other related technical fields, is included in the patent protection scope of the present invention.

Claims

1. A direct bilirubin detection concentrated reagent, comprising: A first concentrated reagent, the first concentrated reagent comprising a first buffer, a surfactant, hydroxylamine hydrochloride with a content of 1.5 g / L or more, and 1-hydroxyethylidene-1,1-diphosphonic acid with a content of 4 g / L or more; A second concentrated reagent, the second concentrated reagent comprising a second buffer and metavanadate with a content of 0.5 g / L or more.

2. The concentrated reagent according to claim 1, wherein In the first concentrated reagent, the content of hydroxylamine hydrochloride is 1.5 - 20 g / L, and the content of 1-hydroxyethylidene-1,1-diphosphonic acid is 4 - 50 g / L; in the second concentrated reagent, the content of the metavanadate is 0.5 - 1.2 g / L; Preferably, in the first concentrated reagent, the content of hydroxylamine hydrochloride is 2.0 - 5.0 g / L, and the content of 1-hydroxyethylidene-1,1-diphosphonic acid is 5.0 - 10.0 g / L; in the second concentrated reagent, the content of the metavanadate is 0.5 - 1.0 g / L.

3. The concentrated reagent according to claim 1 or 2, wherein The first concentrated reagent further comprises a reducing agent.

4. The concentrated reagent according to claim 3, wherein, The reducing agent comprises at least one of tris(2-carboxyethyl)phosphine hydrochloride and a sulfhydryl group-containing compound; preferably, the sulfhydryl group-containing compound comprises at least one selected from mercaptoethanol and dithiothreitol.

5. The concentrated reagent according to claim 4, wherein The content of the reducing agent is 0.1 - 0.5 g / L, preferably 0.2 - 0.4 g / L, more preferably 0.25 - 0.35 g / L.

6. The concentrated reagent according to any one of claims 1 to 5, wherein, The second concentrated reagent further comprises an inorganic salt; preferably, the inorganic salt is selected from at least one of alkali metal salts of hydrochloric acid, sulfuric acid, and nitric acid; more preferably, the inorganic salt is selected from one of NaCl, Na2SO4, and NaNO3.

7. The concentrated reagent according to claim 6, wherein, In the second concentrated reagent, the content of the inorganic salt is 1 - 8 g / L, preferably 2 - 6 g / L, more preferably 3 - 5 g / L.

8. The concentrated reagent according to any one of claims 1 to 7, wherein, The content of the surfactant in the first concentrated reagent is 1 - 15 g / L, preferably 5 - 15 g / L.

9. The concentrated reagent according to any one of claims 1 to 8, wherein, The surfactant is a non-ionic surfactant, preferably, the surfactant comprises at least one selected from polyoxyethylene surfactants Triton-405, Brij-35, Tween-80, and Tween-20.

10. The concentrated reagent according to any one of claims 1 to 9, wherein, The pH of the first concentrated reagent is 2.5 - 3.0, and the pH of the second concentrated reagent is 6.2 - 7.

0.

11. A method for detecting direct bilirubin in blood, characterized in that, Using the concentrated reagent according to any one of claims 1 - 10 for detection, comprising: 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 direct bilirubin in the sample to be tested according to the first absorbance A1 and the second absorbance A2.

12. A direct bilirubin detection reagent, comprising: A first reagent, the first reagent comprising a first buffer, a surfactant, a reducing agent, hydroxylamine hydrochloride, and 1-hydroxyethylidene-1,1-diphosphonic acid; A second reagent, the second reagent comprising a second buffer and metavanadate.

13. The reagent according to claim 12, wherein, The reducing agent includes at least one of tris(2-carboxyethyl)phosphine hydrochloride and a mercapto group-containing compound; preferably, the reducing agent includes at least one selected from mercaptoethanol and dithiothreitol.

14. The reagent according to claim 13, wherein, The content of the reducing agent in the first reagent is 0.1 - 0.5 g / L, preferably 0.2 - 0.4 g / L, more preferably 0.25 - 0.35 g / L.

15. The reagent according to any one of claims 12 to 14, wherein, The second reagent further includes an inorganic salt; preferably, the inorganic salt is at least one selected from the alkali metal salts of hydrochloric acid, sulfuric acid, and nitric acid; more preferably, the inorganic salt is selected from NaCl, Na2SO4, and NaNO3.

16. The reagent according to claim 6, wherein, In the second reagent, the content of the inorganic salt is 1 - 4 g / L, preferably 2 - 3 g / L.

17. For the reagent according to any one of claims 12 - 16, the content of hydroxylamine hydrochloride in the first reagent is 0.1 - 1 g / L, and the content of hydroxyethylidene diphosphonic acid is 0.1 - 3 g / L; the content of the vanadate in the second reagent is 0.05 - 0.45 g / L.

18. The reagent according to any one of claims 12 to 17, wherein The surfactant is a non-ionic surfactant. Preferably, the surfactant includes at least one selected from polyoxyethylene surfactants. Preferably, the surfactant includes Brij-35; more preferably, the content of the surfactant in the first reagent is 5 - 15 g / L.

19. Use of a reducing agent as an anti-hemolysis interference agent in a direct bilirubin detection reagent.

20. Use of a reducing agent in the preparation of a direct bilirubin detection reagent for anti-hemolysis interference.

21. The use according to claim 19 or 20, wherein The direct bilirubin detection reagent includes a first reagent containing the reducing agent and a second reagent containing vanadate. The reducing agent includes at least one of tris(2-carboxyethyl)phosphine hydrochloride and a mercapto group-containing compound; preferably, the mercapto group-containing compound includes at least one selected from mercaptoethanol and dithiothreitol; more preferably, the content of the reducing agent in the first reagent is 0.1 - 0.5 g / L, preferably 0.2 - 0.4 g / L.

22. The use according to claim 21, wherein, The second reagent further includes an inorganic salt, and the inorganic salt is at least one selected from the alkali metals of hydrochloric acid, sulfuric acid, and nitric acid. Preferably, the inorganic salt is one selected from NaCl and Na2SO4. Preferably, in the second reagent, the content of the inorganic salt is 1 - 8 g / L, preferably 2 - 6 g / L.

Images