A direct bilirubin assay kit and its usage method

Abstract

This invention discloses a direct bilirubin assay kit and its usage method. The kit includes reagent A, reagent B, and reagent C. Reagent A contains sulfuric acid; reagent B contains 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and sulfuric acid; and reagent C contains N,N-diisopropylethylamine and n-hexane. Using this kit, the upper limit of linearity reaches 500 μmol / L, making it suitable for the determination of higher concentrations of direct bilirubin. When measuring high-concentration samples, sample dilution is not required, reducing operational steps and minimizing the influence of human factors on the results. This kit shows no significant interference in DBIL determination when hemoglobin concentration is ≤4.5 g / L, and no significant interference in DBIL determination when triglyceride concentration is ≤40 mmol / L (P>0.05). This indicates that the kit is more suitable for the detection of lipemia and hemolyzed samples.

Description

Technical Field

[0001] This invention relates to the field of medical testing technology, specifically to a direct bilirubin assay kit and its usage method. Background Technology

[0002] Direct bilirubin (DBil) is formed when indirect bilirubin enters the liver and combines with glucuronic acid under the action of enzymes. Direct bilirubin is soluble in water and reacts directly with diazo reagent. The determination of direct bilirubin is helpful in the diagnosis and differentiation of jaundice types.

[0003] Currently, the main methods for determining serum direct bilirubin include enzymatic methods, chemical oxidation methods, and diazo methods. Enzymatic methods have good specificity, but enzyme reagents are expensive, and bilirubin oxidase has poor thermal stability, easily leading to a decrease in activity and affecting the accuracy of the determination. Chemical oxidation methods mainly involve vanadate oxidation, where reagents are relatively stable but have certain toxicity, and false positives are common in clinical practice, affecting the accuracy of the test results. Furthermore, when measured on an automated biochemical analyzer, it seriously interferes with the determination of other items. Diazo methods are widely used in clinical laboratories, but conventional diazo methods are subject to interference from lipemia and hemolysis, thus affecting the accuracy of the determination. The research results of Li Xiaomei et al. (Li Xiaomei. Methodological evaluation of bilirubin detection by vanadate oxidation method and diazo method [J]. China Medical Guide, 2009(20):2.) showed that when the hemoglobin concentration was above 2.0 g / L, the diazo method had extremely significant interference in the determination of DBIL. When the triglyceride concentration was 8.0 mmol / L, the diazo method had significant interference in the determination of DBIL. To date, the specific mechanisms by which hemoglobin and triglycerides interfere with diazo determination have not been clearly revealed, and no diazo method has been found that can effectively overcome the interference of lipemia and hemolysis. Therefore, it is of great significance to provide a diazo method that can effectively avoid the interference of hemoglobin and triglycerides. Summary of the Invention

[0004] The purpose of this invention is to provide a direct bilirubin determination method based on the diazo method that is resistant to interference from hemoglobin and triglycerides.

[0005] This invention includes the following key aspects:

[0006] A direct bilirubin assay kit includes reagent A, reagent B and reagent C, wherein reagent A contains sulfuric acid; reagent B contains 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and sulfuric acid; and reagent C contains N,N-diisopropylethylamine and n-hexane.

[0007] Preferably, the concentration of the sulfuric acid is 20-50 mM.

[0008] Preferably, the concentration of 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate (CAS No. 32178-39-5) is 3.5 to 7.0 mM.

[0009] Preferably, the concentration of N,N-diisopropylethylamine is 3–8 μM.

[0010] Preferably, the concentration of n-hexane is 3–10 mM.

[0011] Preferably, the concentration of the sulfuric acid is 30 mM.

[0012] Preferably, the concentration of 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate is 6.0 mM.

[0013] Preferably, the concentration of N,N-diisopropylethylamine is 5 μM, and the concentration of n-hexane is 5 mM.

[0014] The present invention also provides a method for using the kit, the method comprising the following steps: during the determination, reagent A, reagent B and reagent C are first mixed, and then the sample to be tested is added; the volume ratio of the sample to be tested, reagent A, reagent B and reagent C is 200:1600~2300:300~700:30~60; after reacting for 3~5 minutes, the absorbance value is measured.

[0015] On the other hand, the present invention provides the application of the direct bilirubin assay kit in the determination of bilirubin content.

[0016] Compared with the prior art, the beneficial effects of the present invention are:

[0017] ① Using the kit of this invention, the upper limit of linearity reaches 500 μmol / L, which is higher than that of conventional products, making it suitable for the determination of higher concentrations of direct bilirubin. When measuring high-concentration samples, sample dilution is not required, reducing operational steps and minimizing the impact of human factors on the results.

[0018] ② The kit of the present invention showed no significant interference in the determination of DBIL when the hemoglobin concentration was ≤4.5g / L and when the triglyceride concentration was ≤40mmol / L, indicating that the kit of the present invention is more suitable for the detection of lipemia and hemolysis samples.

[0019] ③The reagent kit of this invention is relatively simple to use and easy to promote and use. Detailed Implementation

[0020] To better understand the technical content of this invention, specific embodiments are provided below to further illustrate the invention.

[0021] Example 1:

[0022] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0023] Reagent A: 30mM sulfuric acid;

[0024] Reagent B contains 6.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 30 mM sulfuric acid;

[0025] Reagent C contains 5 μM N,N-diisopropylethylamine and 5 mM n-hexane.

[0026] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0027] Example 2:

[0028] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0029] Reagent A: 50mM sulfuric acid;

[0030] Reagent B contains 7.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 50 mM sulfuric acid;

[0031] Reagent C contains 5 μM N,N-diisopropylethylamine and 5 mM n-hexane.

[0032] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0033] Example 3:

[0034] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0035] Reagent A: 50mM sulfuric acid;

[0036] Reagent B contains 7.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 50 mM sulfuric acid;

[0037] Reagent C contains 8 μM N,N-diisopropylethylamine and 10 mM n-hexane.

[0038] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0039] Example 4:

[0040] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0041] Reagent A: 20mM sulfuric acid;

[0042] Reagent B: 7.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 20 mM sulfuric acid;

[0043] Reagent C: 8 μM N,N-diisopropylethylamine and 10 mM n-hexane.

[0044] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0045] Example 5:

[0046] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0047] Reagent A: 40mM sulfuric acid;

[0048] Reagent B: 3.5 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 40 mM sulfuric acid;

[0049] Reagent C: 8 μM N,N-diisopropylethylamine and 10 mM n-hexane.

[0050] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0051] Example 6:

[0052] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0053] Reagent A: 50mM sulfuric acid;

[0054] Reagent B contains 7.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 50 mM sulfuric acid;

[0055] Reagent C: 3 μM N,N-diisopropylethylamine and 10 mM n-hexane.

[0056] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0057] Example 7:

[0058] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0059] Reagent A: 50mM sulfuric acid;

[0060] Reagent B: 5 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 50 mM sulfuric acid;

[0061] Reagent C: 5 μM N,N-diisopropylethylamine and 5 mM n-hexane.

[0062] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0063] Example 8:

[0064] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0065] Reagent A: 50mM sulfuric acid;

[0066] Reagent B: 5 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 50 mM sulfuric acid;

[0067] Reagent C: 5 μM N,N-diisopropylethylamine and 3 mM n-hexane.

[0068] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 2 to 5 minutes, measure the absorbance value.

[0069] Comparative Example 9

[0070] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0071] Reagent A: 30mM sulfuric acid;

[0072] Reagent B: 6.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 30 mM sulfuric acid;

[0073] Reagent C: 1 μM N,N-diisopropylethylamine and 5 mM n-hexane.

[0074] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0075] Comparative Example 10

[0076] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0077] Reagent A: 30mM sulfuric acid;

[0078] Reagent B: 6.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 30 mM sulfuric acid;

[0079] Reagent C: 5 μM N,N-diisopropylethylamine and 0.5 mM n-hexane.

[0080] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0081] Comparative Example 11

[0082] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0083] Reagent A: 30mM sulfuric acid;

[0084] Reagent B: 6.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 30 mM sulfuric acid;

[0085] Reagent C: 0.1 mM N,N-diisopropylethylamine and 5 mM n-hexane.

[0086] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0087] Comparative Example 12

[0088] A direct bilirubin assay kit, comprising reagent A, reagent B and reagent C.

[0089] Reagent A: 30mM sulfuric acid;

[0090] Reagent B: 6.0 mM 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and 30 mM sulfuric acid;

[0091] Reagent C: 5 μM N,N-diisopropylethylamine and 25 mM n-hexane.

[0092] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2000 μL: 500 μL: 50 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0093] Comparative Example 13 (Conventional Product)

[0094] A direct bilirubin assay kit, comprising reagent A and reagent B.

[0095] Reagent A: 30mM hydrochloric acid;

[0096] Reagent B: 6.0 mM 2,4-dichloroaniline diazonium salt and 30 mM hydrochloric acid.

[0097] The method of using the kit includes the following steps: When performing the assay, first mix all the reagents, then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, and reagent B is 200 μL: 2000 μL: 500 μL; after reacting at 37℃ for 3 to 5 minutes, measure the absorbance value.

[0098] Application Example 1:

[0099] The method of using the reagent kits in Examples 1-12 above can also be as follows: During the determination, mix the reagents first, and then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 1600 μL: 300 μL: 30 μL; after reacting at 37°C for 3-5 min, measure the absorbance value.

[0100] Application Example 2:

[0101] The method of using the reagent kits in Examples 1-12 above can also be as follows: During the determination, mix the reagents first, and then add the sample to be tested; the volume ratio of the sample to be tested, reagent A, reagent B, and reagent C is 200 μL: 2300 μL: 70 μL: 60 μL; after reacting at 37°C for 3-5 min, measure the absorbance value.

[0102] Detection parameters: wavelength 600nm, temperature 37℃, reagent blank zeroing, absorbance values ​​of standard and unknown sample to be tested were measured using a biochemical analyzer.

[0103] Direct bilirubin (μmol / L) = (Absorbance of sample / Absorbance of standard) × Concentration of standard

[0104] Linearity tests showed that the upper limit of linearity for DBIL determination using the kits from Examples 1-12 was 500 μmol / L, while the upper limit of linearity using the kit from Example 13 was 300 μmol / L. It is understood that the highest upper limit of linearity for currently commercially available direct bilirubin detection kits is 430 μmol / L. Therefore, the kit provided by this invention has a higher upper limit of linearity and is suitable for the determination of higher concentrations of direct bilirubin.

[0105] Experiment: Interference Experiment

[0106] 1.1 Hemoglobin interference

[0107] Serum samples (direct bilirubin 12–18 μmol / L) from jaundice patients were collected and divided into two portions. The first portion was mixed with physiological saline at a 1:1 ratio (sample 1). The second portion was mixed with hemoglobin standard solution at a 1:1 ratio to achieve a hemoglobin concentration of 6.0 g / L (sample 5). Samples 1 and 5 were mixed at volume ratios of 3:1, 2:2, and 1:3, respectively, to obtain samples 2–4. DBIL was measured four times in each of the five samples using the kits and methods described in Examples 1–13. The mean and standard deviation were calculated. The mean values ​​of samples 2–5 were compared with those of sample 1 to determine if significant interference existed.

[0108] The results showed that in Examples 1-8, DBIL determination was not significantly affected when the hemoglobin concentration was ≤4.5 g / L (P>0.05), but was significantly affected when the hemoglobin concentration was 6.0 g / L (P<0.05); in Examples 9-13, DBIL determination was significantly affected when the hemoglobin concentration was above 3.0 g / L (P<0.01).

[0109] 1.2 Triglyceride interference

[0110] The method was the same as in 1.1, but hemoglobin was replaced with triglycerides. The triglyceride content in sample 5 was 40 mmol / L.

[0111] Examples 1-8 showed no significant interference in DBIL determination when triglyceride concentration was ≤40 mmol / L (P>0.05); Examples 9-12 showed significant interference in DBIL determination when triglyceride concentration was above 20 mmol / L (P<0.05), indicating that the anti-interference effect of N,N-diisopropylethylamine and n-hexane was significantly weakened when the concentrations were too high or too low; Example 13 showed extremely significant interference in DBIL determination when triglyceride concentration was 10 mmol / L (P<0.01).

[0112] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A direct bilirubin assay kit, characterized in that, The reagents include reagent A, reagent B, and reagent C. Reagent A contains sulfuric acid; reagent B contains 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate and sulfuric acid; and reagent C contains N,N-diisopropylethylamine and n-hexane. The concentration of the sulfuric acid is 20~50mM; The concentration of the 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate is 3.5~7.0 mM; The concentration of the N,N-diisopropylethylamine is 3~8 μM; The concentration of the n-hexane is 3~10 mM.

2. The direct bilirubin assay kit according to claim 1, characterized in that, The concentration of the sulfuric acid is 30 mM.

3. The direct bilirubin assay kit according to claim 1, characterized in that, The concentration of the 2,5-diethoxy-4-(4-morpholino)diazobenzene sulfate is 6.0 mM.

4. The direct bilirubin assay kit according to claim 1, characterized in that, The concentration of N,N-diisopropylethylamine is 5 μM, and the concentration of n-hexane is 5 mM.

5. The method of using the direct bilirubin assay kit according to claim 1, characterized in that, Includes the following steps: During the determination, reagents A, B, and C are mixed first, and then the sample to be tested is added. The volume ratio of the sample to be tested, reagents A, B, and C is 200:1600~2300:300~700:30~60. After reacting for 3~5 minutes, the absorbance value is measured.

6. The application of the direct bilirubin assay kit according to any one of claims 1 to 4 in the determination of bilirubin content.