Total bilirubin determination reagent capable of detecting heparin anticoagulant plasma

Abstract

The invention relates to the technical field of analysis and detection, in particular to a total bilirubin determination reagent capable of detecting heparin anticoagulant plasma. The zwitterionic surfactant, the cationic surfactant and the nonionic surfactant are added into the reagent, so that the interference of heparin with different concentrations on the detection result of total bilirubin in a plasma sample can be eliminated, and the accuracy of the detection result of total bilirubin in heparin anticoagulant plasma is improved. The reagent developed by the invention is convenient for clinical detection of a total bilirubin item on a heparin anticoagulant plasma sample in emergency treatment, and the variety of detection samples of the item is expanded; meanwhile, the reagent provided by the invention is simple in component, low in production and use cost, easy to produce and prepare, easy to commercialize and wide in application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of analysis and detection, in particular to a total bilirubin determination reagent capable of detecting heparin anticoagulated plasma. Background Art

[0002] Total bilirubin is the general term for conjugated and unconjugated bilirubin produced by heme metabolism in the human body. It mainly comes from aging red blood cells and is degraded by hemoglobin in the spleen, liver and bone marrow, accounting for 70% to 80% of the total bilirubin. The decomposition of hemoglobin in the liver such as P450 and B5 catalase and the decomposition of immature red blood cells in the spleen and bone marrow (also known as bypass bilirubin) account for 10% to 15% of the total bilirubin.

[0003] Bilirubin reflects the secretion and excretion function of the liver and is a routine liver function indicator. Under normal circumstances, aging red blood cells are destroyed by mononuclear macrophages, and the released hemoglobin is decomposed into bilirubin, iron and globin. Unconjugated bilirubin reaches the liver with the blood, and combines with glucuronic acid in the liver cells to form glucuronic acid bilirubin, which is called conjugated bilirubin. Conjugated bilirubin is water-soluble and can be excreted from the urine through glomerular filtration, while the conjugated bilirubin that enters the capillary bile duct enters the intestine with bile, and is reduced to colorless urobilinogen under the action of intestinal bacteria, and most of it is excreted from the feces. The determination of total bilirubin in human serum, plasma and urine is one of the most routine items in medical examinations. In practical applications, total bilirubin detection is often used in combination with direct bilirubin detection. Elevated total bilirubin is mainly seen in liver damage, liver disease, jaundice, etc.; decreased total bilirubin is mainly seen in diseases with reduced bilirubin production such as iron deficiency anemia.

[0004] Commonly used methods for clinical detection of total bilirubin indicators include diazonium salt method, vanadate oxidation method, bilirubin oxidation, enzyme method, etc. The diazo method reagent is unstable and must be temporarily prepared with sodium nitrite and aminosulfonic acid, and the storage time is short. The enzyme method has good specificity and is almost not affected by hemolysis, lipid turbidity, etc., but it is difficult to promote due to the source, purity, stability and price of the enzyme, and the bilirubin oxidase in it is also prone to decreased activity, affecting the accuracy of the measurement; therefore, the vanadate oxidation method is currently more widely used.

[0005] The basic principle of the vanadate oxidation method for determining total bilirubin is as follows: Under specific conditions (pH 3.0), bilirubin in serum reacts with vanadic acid to generate biliverdin. The decrease in its absorbance at 450nm is proportional to the total bilirubin content in serum. The change in absorbance at a wavelength of 450nm is detected using a biochemical analyzer to determine the total bilirubin concentration in the tested serum.

[0006] Compared with the diazo method, the vanadate oxidation method has better anti-hemolysis and lipid turbidity ability. This is related to the fact that vanadic acid can oxidize heme while oxidizing bilirubin. However, the traditional vanadate oxidation method can only detect serum samples. For heparin plasma samples, it will be interfered by heparin (heparin lithium or heparin sodium). Heparin is a linear anionic mucopolysaccharide containing different lengths, with a large amount of negative charge and strong acidity. It may react chemically with strongly alkaline surfactants with ammonium ions to form an insoluble white suspension, which increases the absorbance at 450nm. Therefore, the turbidity caused by heparin reduces the decrease in absorbance before and after oxidation, thereby reducing the determination result of total bilirubin. Different concentrations of heparin produce different interferences in plasma, and gradually increase with the increase of heparin content. Therefore, the commonly used amount of heparin anticoagulated plasma has a greater impact on the results of total bilirubin determination by the vanadate method.

[0007] Therefore, how to provide a detection reagent suitable for detecting total bilirubin in heparin anticoagulated plasma has become a technical problem that urgently needs to be solved in this field. Summary of the invention

[0008] In order to solve the problem of inaccurate results of total bilirubin determination using a vanadate method for heparin anticoagulated plasma, the present invention provides a total bilirubin determination reagent, comprising a reagent R1, a reagent R2 and a calibrator; the reagent R1 comprises a buffer, an EDTA salt and a surfactant; the surfactant comprises a zwitterionic surfactant, a cationic surfactant and a non-ionic surfactant; the reagent R2 comprises a buffer and sodium metavanadate.

[0009] The present invention finds that by adding three different surfactants (zwitterionic surfactant, cationic surfactant and non-ionic surfactant) to the reagent, especially the zwitterionic surfactant and the cationic surfactant must each be combined with at least one component to prepare the reagent R1, by utilizing the interaction between different surfactants, the interference of different concentrations of heparin on the total bilirubin detection result in the plasma sample can be eliminated, thereby improving the accuracy of the total bilirubin detection result of the heparin anticoagulated plasma sample.

[0010] In some embodiments, the zwitterionic surfactant includes at least one of Amphitol-24B, fatty acid amide alkyl betaine, and lauryl betaine.

[0011] In some embodiments, the cationic surfactant includes at least one of cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, tetradecyltrimethylammonium bromide, and tetradecyltrimethylammonium chloride.

[0012] In some embodiments, the nonionic surfactant includes at least one of Tween-80, fatty alcohol containing ethylene oxide with 8 EOs, fatty alcohol ethylene oxide-propylene oxide adduct, and polyoxyethylene lauryl ether.

[0013] Preferably, the fatty acid amide alkyl betaine is AMPHOTENSID B 4 F (Schirmach, Germany).

[0014] Preferably, the fatty alcohol containing ethylene oxide of 8 EOs is ZUSOLAT 1008 / 85 or OXETAL 800 / 85; and / or the fatty alcohol ethylene oxide-propylene oxide adduct is PROPETAL 100.

[0015] In some embodiments, the buffer in the reagent R1 is a citrate buffer; and / or, the EDTA salt in the reagent R1 is an EDTA potassium salt or an EDTA sodium salt; and / or, the buffer in the reagent R2 is a phosphate buffer; and / or, the calibrator includes bilirubin and a phosphate buffer.

[0016] Preferably, the EDTA sodium salt is disodium EDTA or tetrasodium EDTA.

[0017] Preferably, the EDTA potassium salt is dipotassium EDTA or tripotassium EDTA.

[0018] In some embodiments, in the reagent R1, the buffer is a citric acid buffer with a pH of 2.5-3.5 (preferably a pH of 2.8-3.2) and a concentration of 50-150 mmol / L (preferably 80-120 mmol / L), the EDTA salt is EDTA sodium salt with a concentration of 1-5 mmol / L, the volume percentage of the zwitterionic surfactant in the reagent R1 is 0.1%-25% (preferably 10%-15%), the concentration of the cationic surfactant is 1-20 g / L (preferably 5-15 g / L), and the volume percentage of the non-ionic surfactant in the reagent R1 is 0.1%-25% (preferably 1%-5%).

[0019] In some embodiments, in the reagent R2, the buffer is a phosphate buffer with a pH of 6.5-7.5 (preferably pH 6.8-7.2) and a concentration of 50-150 mmol / L (preferably 80-120 mmol / L), and the concentration of sodium metavanadate is 1-10 mmol / L (preferably 2-6 mmol / L).

[0020] In some embodiments, in the calibrator, the phosphate buffer is a phosphate buffer with a pH of 6.5-7.5 (preferably pH 6.8-7.2) and a concentration of 50-150 mmol / L (preferably 80-120 mmol / L), and the concentration of bilirubin is 150-200 μmol / L.

[0021] Furthermore, the present invention provides the use of the reagent in detecting total bilirubin.

[0022] Preferably, the sample detected in the application is heparin anticoagulated plasma.

[0023] Preferably, the heparin anticoagulated plasma is lithium heparin anticoagulated plasma or sodium heparin anticoagulated plasma.

[0024] Preferably, the heparin concentration in the heparin anticoagulated plasma ranges from 6.25 U / mL to 100 U / mL.

[0025] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a total bilirubin detection reagent that eliminates heparin interference, so as to eliminate the interference of different concentrations of heparin on the total bilirubin detection results in plasma samples, and improve the accuracy of the total bilirubin detection results in heparin anticoagulated plasma. The reagent developed by the present invention is convenient for clinical emergency department to detect the total bilirubin project of heparin anticoagulated plasma samples, and expands the types of samples detected for this project; at the same time, the reagent provided by the present invention has simple components, low production and use costs, and is easy to produce and prepare, easy to commercialize, and has broad application prospects. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0028] Example 1 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator, and the specific components are shown in Table 1.

[0029] Table 1 Composition of total bilirubin assay reagent

[0030] Example 2

[0031] This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: AMPHOTENSID B 4 F was replaced with an equal amount of Amphitol-24B.

[0032] Example 3 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: AMPHOTENSID B 4 F was replaced with an equal amount of lauryl betaine.

[0033] Example 4 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: The tetradecyltrimethylammonium bromide was replaced with an equal amount of hexadecyltrimethylammonium chloride.

[0034] Example 5 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: Replace OXETAL 800 / 85 with an equal amount of Tween-80.

[0035] Example 6 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: Mix OXETAL 800 / 85 and ZUSOLAT 1008 / 85 at 1% each.

[0036] Example 7 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: Replace OXETAL 800 / 85 with an equal amount of ZUSOLAT 1008 / 85.

[0037] Example 8 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: Replace OXETAL 800 / 85 with an equal amount of PROPETAL 100.

[0038] Example 9 This embodiment provides a total bilirubin determination reagent, which is composed of reagent R1, reagent R2 and a calibrator. The specific components are different from those in Example 1 only in that: Replace OXETAL 800 / 85 with an equal amount of polyoxyethylene lauryl ether.

[0039] Test example This test example uses sodium heparin anticoagulated plasma (the concentration of sodium heparin is 100 U / mL, which is 4 times the conventional clinical dosage) and the same human serum without sodium heparin anticoagulant, and uses the total bilirubin determination reagent in the above embodiment for detection, and uses a fully automatic biochemical analyzer with a dual reagent function for determination. The detection steps are shown in Table 2. Wavelength: 450nm (440nm~470nm), temperature: 37°C, colorimetric cup light path: 1cm.

[0040] Table 2 Automatic biochemical analyzer test steps

[0041] The test results are shown in Table 3.

[0042] Table 3 Results of the high and low concentration samples measured in each embodiment

[0043] The deviation of the total bilirubin determination reagents in the above embodiments for determining high and low concentration levels of heparin sodium anticoagulated plasma and serum samples is within ±5%, indicating that the total bilirubin determination reagents in the embodiments can be used to determine heparin anticoagulated plasma.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A total bilirubin assay reagent, characterized in that: The invention comprises reagent R1, reagent R2 and a calibrator; the reagent R1 comprises a buffer solution, EDTA salt and a surfactant; the surfactant comprises a zwitterionic surfactant, a cationic surfactant and a nonionic surfactant; the reagent R2 comprises a buffer solution and sodium metavanadate.

2. The reagent according to claim 1, characterized in that The zwitterionic surfactant includes at least one of Amphitol-24B, fatty acid amide alkyl betaine, and dodecyl betaine.

3. The reagent according to claim 1, characterized in that The cationic surfactant includes at least one of cetyltrimethylammonium bromide, cetyltrimethylammonium chloride, tetradecyltrimethylammonium bromide and tetradecyltrimethylammonium chloride.

4. The reagent according to claim 1, characterized in that The nonionic surfactant includes at least one of Tween-80, fatty alcohol containing ethylene oxide with 8 EOs, fatty alcohol ethylene oxide-propylene oxide adduct, and polyoxyethylene lauryl ether.

5. The reagent according to claim 2, characterized in that The fatty acid amide alkyl betaine is AMPHOTENSID B 4 F.

6. The reagent according to claim 4, characterized in that The fatty alcohol containing ethylene oxide of 8 EOs is ZUSOLAT 1008 / 85 or OXETAL 800 / 85; and / or, the fatty alcohol ethylene oxide-propylene oxide adduct is PROPETAL 100.

7. The reagent according to claim 1, characterized in that The buffer in the reagent R1 is a citric acid buffer; and / or, the EDTA salt in the reagent R1 is an EDTA potassium salt or an EDTA sodium salt; and / or, the buffer in the reagent R2 is a phosphate buffer; and / or, the calibrator includes bilirubin and a phosphate buffer.

8. The reagent according to claim 7, characterized in that In the reagent R1, the buffer is a citric acid buffer with a pH of 2.5 to 3.5 and a concentration of 50 to 150 mmol / L, the EDTA salt is EDTA sodium salt with a concentration of 1 to 5 mmol / L, the volume percentage of the zwitterionic surfactant in the reagent R1 is 0.1% to 25%, the concentration of the cationic surfactant is 1 to 20 g / L, and the volume percentage of the nonionic surfactant in the reagent R1 is 0.1% to 25%; And / or, in the reagent R2, the buffer is a phosphate buffer with a pH of 6.5-7.5 and a concentration of 50-150 mmol / L, and the concentration of sodium metavanadate is 1-10 mmol / L; And / or, in the calibrator, the phosphate buffer is a phosphate buffer with a pH of 6.5-7.5 and a concentration of 50-150 mmol / L, and the concentration of bilirubin is 150-200 μmol / L.

9. Use of the reagent according to any one of claims 1 to 8 in detecting total bilirubin.

10. The use according to claim 9, characterized in that: The samples tested were heparin-anticoagulated plasma.

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