A test kit for total bile acids

Abstract

The application relates to the technical field of medical detection, in particular to a detection kit for total bile acid and a detection kit for total bile acid. The application adds polyoxyethylene-polyoxypropylene copolymer, Span 40 and oxide in R1, and adds EDTA-Na2 and sodium bicarbonate in R2, so that the stability and precision of reagents are effectively improved, and the situation that a negative value is generated in the detection process is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical detection, in particular to a total bile acid detection kit. BACKGROUND

[0002] TBA is metabolized by the liver and can reflect liver parenchymal injury. Once liver cells are diseased, the concentration of bile acid in blood is prone to increase. The TBA result of acute hepatitis, chronic active hepatitis and cirrhosis is significantly increased. Compared with ALT, both acute hepatitis and chronic active hepatitis are more sensitive, but the result of cirrhosis is significantly different: the positive rate of TBA is as high as 95%. Therefore, TBA determination has great value for monitoring chronic liver disease, and its determination method is simple and is a very practical liver function diagnostic index.

[0003] In 1974, the first generation of enzyme bile acid reagent was successfully produced by using 3α-hydroxysteroid by the Norwegian company KabiNordic, which made the determination of bile acid truly enter the clinical field, which is of great significance. Today, the detection of bile acid has developed to the 5th generation of circulating enzyme method. The prior art: under the catalysis of 3α-hydroxysteroid (3α-HSD), bile acid is specifically oxidized by β-thio-nicotinamide adenine dinucleotide oxidized form (Thio-NAD+) to generate 3-ketosteroid, and Thio-NAD+ is reduced to β-thio-nicotinamide adenine dinucleotide reduced form (Thio-NADH). The newly generated 3-ketosteroid is reduced to bile acid and NAD+ in the presence of 3α-HSD and NADH. In this way, trace amounts of bile acid in the sample are amplified in multiple cycles, and Thio-NADH generated is continuously expanded. The change of absorbance at 405 nm is positively correlated with the concentration of TBA.

[0004] TBA+Thio-NAD+(thio-oxidized coenzyme)+3α-HSD——3-ketosteroid+Thio-NADH

[0005] 3-ketosteroid+NADH+3α-HSD——TBA+NAD + SUMMARY

[0006] Therefore, the present application provides a total bile acid detection kit, which can effectively detect the content of serum total bile acid, has good stability and precision, can solve the problem of negative value of low-value samples, has low false negative, and the detection result is more accurate.

[0007] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions:

[0008] A total bile acid detection kit is composed of R1 reagent and R2 reagent.

[0009] The R1 reagent comprises a buffer, Thio-NAD+, oxamate sodium, mannitol, polyoxyethylene-polyoxypropylene copolymer, Span 40, an oxidant, and a preservative;

[0010] The R2 reagent comprises a buffer, NADH, BSA, 3a-HSD, EDTA-Na2, sodium bicarbonate, and a preservative.

[0011] In some embodiments, the R1 reagent comprises:

[0012]

[0013] In some embodiments, the R2 reagent comprises:

[0014]

[0015] In some embodiments, in the R1 reagent, the buffer is at least one of MES buffer, Tris buffer, HEPES buffer, PBS buffer, CAPS buffer, and the pH of the buffer is 8-10. In some specific embodiments, the buffer is MES buffer.

[0016] In some embodiments, in the R1 reagent, the oxidant is at least one of tert-butyl hydroperoxide, tert-butyl perbenzoate, potassium ferrocyanide, and sodium nitrite. In some specific embodiments, the oxidant is tert-butyl hydroperoxide.

[0017] In some embodiments, in the R1 reagent and the R2 reagent, the preservative is at least one of NaN3, PC300, PC950, IZU, CAA, and BND. In some specific embodiments, the preservative is NaN3.

[0018] In some embodiments, in the R2 reagent, the buffer is at least one of TAPS buffer, citric acid buffer, and glycine buffer, and the pH of the buffer is 3-5. In some specific embodiments, the buffer is TAPS buffer.

[0019] In some embodiments, the kit is composed of the R1 reagent and the R2 reagent;

[0020] The R1 reagent comprises:

[0021]

[0022] The R2 reagent comprises:

[0023]

[0024] The application effectively improves reagent stability and precision by adding polyoxyethylene-polyoxypropylene copolymer, Span 40 and oxide in R1 and adding EDTA-Na2 and sodium bicarbonate in R2, thereby solving the problem of negative value in the detection process. DETAILED DESCRIPTION

[0025] The application provides a detection kit for bile acids. Those skilled in the art can improve the process parameters according to the content herein. It is particularly pointed out that all similar replacements and changes are obvious to those skilled in the art and are considered to be included in the application. The method and application of the application have been described by the preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the application, to realize and apply the technology of the application.

[0026] The test materials used in the application are all ordinary commercially available products and can be purchased in the market.

[0027] The application is further described below in combination with examples:

[0028] Example 1

[0029] The R1 and R2 reagents comprise the following components:

[0030] The R1 reagent comprises the following components:

[0031]

[0032] The R2 reagent comprises the following components:

[0033]

[0034] Example 2

[0035] The R1 and R2 reagents comprise the following components:

[0036] The R1 reagent comprises the following components:

[0037]

[0038] The R2 reagent comprises the following components:

[0039]

[0040]

[0041] Example 3

[0042] The R1 and R2 reagents comprise the following components:

[0043] The R1 reagent comprises the following components:

[0044]

[0045] R2 reagent:

[0046]

[0047] Comparative Example 1

[0048] Compared with Example 1, R1 does not contain tert-butyl hydroperoxide, polyoxyethylene-polyoxypropylene copolymer and Span 40, R2 does not contain EDTA-Na2 and sodium bicarbonate. The composition of R1 and R2 is:

[0049] R1 reagent:

[0050]

[0051] R2 reagent:

[0052]

[0053] Comparative Example 2

[0054] Compared with Example 1, R1 does not contain tert-butyl hydroperoxide and Span 40, R2 does not contain EDTA-Na2 and sodium bicarbonate. The composition of R1 and R2 is:

[0055] R1 reagent:

[0056]

[0057] R2 reagent:

[0058]

[0059] Comparative Example 3

[0060] Compared with Example 1, R1 does not contain tert-butyl hydroperoxide, polyoxyethylene-polyoxypropylene copolymer, R2 does not contain EDTA-Na2 and sodium bicarbonate. The composition of R1 and R2 is:

[0061] R1 reagent:

[0062]

[0063] R2 reagent:

[0064]

[0065] Comparative Example 4

[0066] Compared with Example 1, R1 does not contain tert-butyl hydroperoxide, R2 does not contain EDTA-Na2 and sodium bicarbonate. The composition of R1 and R2 is:

[0067] R1 reagent:

[0068]

[0069] R2 reagent:

[0070]

[0071] Comparative Example 5

[0072] Compared with Example 1, R1 does not contain tert-butyl hydroperoxide, polyoxyethylene-polyoxypropylene copolymer and Span 40, and R2 does not contain sodium bicarbonate. The composition of R1 and R2 is:

[0073] R1 reagent:

[0074]

[0075] R2 reagent:

[0076]

[0077] Comparative Example 6

[0078] Compared with Example 1, R1 does not contain tert-butyl hydroperoxide, polyoxyethylene-polyoxypropylene copolymer and Span 40, and R2 does not contain EDTA-Na2. The composition of R1 and R2 is:

[0079] R1 reagent:

[0080]

[0081] R2 reagent:

[0082]

[0083] Comparative Example 7

[0084] Compared with Example 1, R1 does not contain tert-butyl hydroperoxide, polyoxyethylene-polyoxypropylene copolymer and Span 40. The composition of R1 and R2 is:

[0085] R1 reagent:

[0086]

[0087] R2 reagent:

[0088]

[0089] Comparative Example 8

[0090] Compared with Example 1, the polyoxyethylene-polyoxypropylene copolymer and Span 40 are not contained in R1, and EDTA-Na2 and sodium bicarbonate are not contained in R2. The composition of R1 and R2 is:

[0091] R1 reagent:

[0092]

[0093] R2 reagent:

[0094]

[0095]

[0096] Comparative Example 9

[0097] Compared with Example 1, the polyoxyethylene-polyoxypropylene copolymer and Span 40 are not contained in R1. The composition of R1 and R2 is:

[0098] R1 reagent:

[0099]

[0100] R2 reagent:

[0101]

[0102] Test kit performance test

[0103] The kits of Examples 1- and Comparative Examples 1- were detected according to the following table of detection parameters by using a full-automatic biochemical analyzer:

[0104] Table 1. Detection parameters of the kit according to the present application on a full-automatic biochemical analyzer

[0105]

[0106]

[0107] The above kits were used to detect samples according to the above parameters, and were compared with reagents on the market, and the related reagent stability, precision and negative value were detected.

[0108] (1) Precision

[0109] The average value, standard deviation and coefficient of variation CV of the measured values were calculated by measuring 10 random sera, which were the measurement precision of the kit, and the precision of the formula was compared.

[0110] Table 2

[0111]

[0112] It can be seen that the comparative example 4 can obviously improve the low value precision of the reagent by adding double active addition in the R1 reagent.

[0113] (2) Stability

[0114] The set value serum was tracked for 30 days of open bottle stability, and the bias was calculated for 240 days of real-time stability to compare the stability effect of the formula.

[0115] Table 3 Detection values at different open bottle days

[0116]

[0117]

[0118] Table 4 Deviation of detection values at different open bottle days from open bottle 0 days

[0119]

[0120] Table 5 Detection values at different real-time days

[0121]

[0122] Table 6 Deviation of detection values at different real-time days from real-time 0 days

[0123]

[0124]

[0125] From the data in Tables 3-6, it can be seen that the addition of EDTA-Na2 and sodium bicarbonate in R2 can effectively improve the stability of the reagent. Stability and precision, thereby solving the negative value situation.

[0126] (3) Negative value

[0127] The negative value situation of the low value sample and the negative value situation after the open bottle for 30 days without re-calibration and re-measurement were compared using the reagent kits of Comparative Example 1 and Examples 1-3:

[0128] Table 7

[0129] Comparative Example 1 Example 1 Example 2 Example 3 1 1.6 1.8 1.7 1.7 2 -0.1 0.2 0.1 0.1 3 6.8 6.8 6.7 6.9 4 0.5 0.5 0.4 0.6 5 2.8 2.8 2.7 2.9 6 0 0.4 0.3 0.3 7 2.8 2.5 2.4 2.9 8 1.1 1.1 1 1.2 9 0.5 0.5 0.4 0.6 10 -0.2 0.1 0.2 0.1 11 1.4 1.5 1.4 1.5 12 2.7 2.9 2.8 2.8 13 4.6 4.7 4.6 4.7 14 0.7 0.8 0.7 0.8 15 0.5 0.6 0.5 0.6 16 3.4 3.5 3.4 3.5 17 0.8 0.9 0.8 0.9 18 5.8 6 5.9 5.9 19 0.3 0.5 0.4 0.4 20 0.6 0.7 0.6 0.7

[0130] The results show that compared with Comparative Example 1, the reagent kits of Examples 1-3 of the present application have obvious improvement in negative value situation by improving precision and stability.

[0131] The above is only a preferred embodiment of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. A test kit for total bile acids, characterized by, consists of R1 reagent and R2 reagent; the R1 reagent consists of: buffer 50-100 mmol / L; Thio-NAD + 1-10 mmol / L; sodium oxamate 1-100 mmol / L; mannitol 1-100 g / L; polyoxyethylene-polyoxypropylene copolymer 0.1-10 g / L; Span 40 0.1-10 g / L; oxidant 1-10 g / L; preservative 1-2 g / L; the R2 reagent consists of: buffer 100-200 mmol / L; NADH 1-10 mmol / L; BSA 1-10 g / L; 3α-HSD 1-10 KU / L; EDTA-Na2 1-20 g / L; sodium bicarbonate 1-20 mmol / L; preservative 1-2 g / L; In the R1 reagent, the oxidant is at least one of tert-butyl hydroperoxide, tert-butyl perbenzoate, potassium ferrocyanide, and sodium nitrite.

2. The test kit according to claim 1, characterized in that, In the R1 reagent, the buffer is at least one of MES buffer, Tris buffer, HEPES buffer, PBS buffer, and CAPS buffer, and the pH of the buffer is 8-10.

3. The test kit according to claim 1, characterized in that, In the R1 reagent and the R2 reagent, the preservative is at least one of NaN3, PC300, PC950, IZU, CAA, and BND.

4. The test kit according to claim 1, characterized in that, In the R2 reagent, the buffer is at least one of TAPS buffer, citric acid buffer, and glycine buffer, and the pH of the buffer is 3-5.

5. The test kit according to any one of claims 1 to 4, characterized in that, the R1 reagent consists of: MES buffer with pH 8-10 50-100 mmol / L; Thio-NAD + 1-10 mmol / L; sodium oxamate 1-100 mmol / L; mannitol 1-100 g / L; polyoxyethylene-polyoxypropylene copolymer 1-10 g / L; Span 40 1-10 g / L; oxidant 1-10 g / L; NaN3 1-2 g / L; the R2 reagent consists of: TAPS buffer with pH 3-5 100-200 mmol / L; NADH 1-10 mmol / L; BSA 1-10 g / L; 3α-HSD 1-10 KU / L; EDTA-Na2 1-20 g / L; sodium bicarbonate 1-20 mmol / L; NaN3 1-2 g / L.

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