Total bile acid detection kit and method

Abstract

The present invention relates to a TBA detection kit, which includes a first reagent, wherein the first reagent contains Thio-NAD; and a second reagent, wherein the second reagent contains NADH, 3α-HSD, a nonionic surfactant, and an antibiotic preservative. Through the combination of the nonionic surfactant and the antibiotic surfactant, the present invention prolongs the storage time of the TBA cyclic enzyme detection reagent and is not prone to causing a decrease in the stability of the reagent. In addition, the present invention also relates to a method for detecting TBA.

Description

Technical Field

[0001] The invention relates to the field of total bile acid detection, and in particular to a reagent for detecting bile acid based on a cyclic enzyme method. Background Art

[0002] Total bile acids (TBA) is the general term for the metabolites of cholesterol in the liver and in the enterohepatic circulation. It is the main end product of cholesterol metabolism in the body and an important component of bile, playing an important role in fat metabolism. In normal people's blood, the level of TBA is extremely low, but when TBA metabolism in liver cells is abnormal, TBA flows back into the blood, resulting in an increase in serum TBA. Therefore, the serum TBA level can be combined with a number of liver enzyme indicators to assist in the judgment of the occurrence, progression and prognosis of chronic liver disease, cirrhosis, alcoholic liver disease and cholestasis.

[0003] The TBA detection kits currently used in clinical practice include the enzyme colorimetric method and the cyclic enzyme method. The reaction principle of the enzyme colorimetric method is that 3α-hydroxybile acid is converted into NAD + In the presence of , 3-keto bile acid and NADH are generated by 3α-hydroxysteroid dehydrogenase, and NADH reacts with nitro blue tetrazolium (NBT) to generate a stable blue dye under the action of diaphorase, and then the total bile acid concentration is determined by colorimetric determination. However, on the one hand, the enzyme colorimetric method is susceptible to interference from substances such as lactate dehydrogenase in serum; on the other hand, the interference of sample blank cannot be removed on most automatic analyzers.

[0004] The cyclic enzyme method is another commonly used TBA detection method, which avoids the above-mentioned defects of the enzyme colorimetric method and can achieve convenient and highly accurate detection. The reaction principle of the cyclic enzyme method is that bile acid will be oxidized by 3-hydroxysterol dehydrogenation (3-HSD) and β-thionicotinamide adenine dinucleotide oxidized form (Thio-NAD) to generate 3-ketosteroids and β-thionicotinamide adenine dinucleotide reduced form (Thio-NADH). The generated 3-ketosteroids will generate bile acid and NAD in the presence of 3-HSD and NADH. According to the above cycle, the trace amount of bile acid is gradually amplified, and the concentration of bile acid can be calculated by measuring the absorbance change of Thio-NADH generated per unit time at 405nm.

[0005] The TBA reagent of the cyclic enzyme method contains a large amount of NADH. In order to maintain the stability of NADH, the pH needs to be adjusted to alkaline. Under this pH condition, the preservative effect of the preservatives commonly used in biochemical reagents will be weakened or even lost, thereby greatly shortening the shelf life of the reagents. Even if a few preservatives have a good preservative effect under this pH condition, they have a greater impact on enzyme activity, which will cause the stability of the reagent to decrease, and are not suitable for use in the TBA detection system based on the cyclic enzyme method.

[0006] Accordingly, in the field of TBA detection, there is a strong demand for extending the storage time of NADH-containing reagents while maintaining the enzyme activity of the reaction system. Summary of the invention

[0007] As mentioned above, the cyclic enzyme method is a commonly used method that can accurately detect TBA in serum, in which NADH is a necessary reaction component in the cyclic enzyme method. Since NADH needs to be under alkaline conditions, most common preservatives cannot work well. Although some preservatives still have a preservative effect under alkaline conditions, they will affect the activity of the enzyme in the cyclic enzyme reaction system, causing a decrease in the stability of the reagent. In other words, in the current TBA detection reagents based on the cyclic enzyme method, on the one hand, it is difficult to obtain excellent reagent preservation effects, and on the other hand, excellent preservation effects and reagent stability cannot be taken into account at the same time.

[0008] In order to solve the above problems, the present invention studied the TBA detection reagent of the cyclic enzyme method, and unexpectedly found that adding a non-ionic surfactant and an antibiotic preservative simultaneously to the TBA detection reagent containing NADH can produce a kit that takes into account both the preservation effect and the system stability, thereby completing the present invention.

[0009] Accordingly, in a first aspect, the present invention provides a kit comprising:

[0010] A first reagent comprising Thio-NAD, a pH adjuster and a preservative, wherein the pH adjuster optionally adjusts the pH to 3 to 4.5;

[0011] The second reagent contains NADH, 3α-HSD, a buffer, a nonionic surfactant and an antibiotic preservative. Optionally, the pH of the buffer is 8-10.

[0012] In some embodiments, the nonionic surfactant is a Tween series, such as one or more of Tween 20, Tween 60, and Tween 80.

[0013] In some embodiments, the nonionic surfactant is a Triton series surfactant, such as one or more of Triton X-100 and Triton X-114.

[0014] In some embodiments, the nonionic surfactant is Tertigol.

[0015] In some embodiments, the antibiotic preservative is selected from one or more of erythromycin, penicillin, natamycin and cefotaxime sodium salt.

[0016] In a specific embodiment, the antibiotic preservative is erythromycin.

[0017] In a specific embodiment, the antibiotic preservative is penicillin.

[0018] In a specific embodiment, the antibiotic preservative is cefotaxime sodium salt.

[0019] In some embodiments, the concentration of the Tween series is about 1 to 8 g / L, such as about 3 to 8 g / L.

[0020] In some embodiments, the concentration of the Triton series is about 2-8 g / L, such as about 3-5 g / L.

[0021] In some embodiments, the concentration of Tertigol is about 0.5-2.5 g / L, preferably about 0.5-1.5 g / L.

[0022] In some embodiments, the concentration of the antibiotic preservative is about 0.1 to 1.5 g / L, preferably about 0.2 to 1 g / L.

[0023] In a second aspect, the present invention also provides a method for detecting TBA, comprising the following steps:

[0024] 1) mixing the sample to be tested with the first reagent of the present invention and incubating;

[0025] 2) adding the second reagent of the present invention, mixing and incubating;

[0026] 3) measuring a first absorbance;

[0027] 4) After 2 to 4 minutes, measuring a second absorbance; and

[0028] 5) Calculating TBA concentration according to the first absorbance and the second absorbance.

[0029] In a third aspect, there is also provided use of the second reagent of the present invention in preparing a kit for detecting TBA.

[0030] The present invention improves the TBA detection reagent of the cyclic enzyme method, and simultaneously adds a nonionic surfactant and an antibiotic preservative into the TBA detection reagent containing NADH, so that the obtained reagent has the advantages of being able to take into account both the preservation effect and the system stability. DETAILED DESCRIPTION

[0031] The present invention will be described in detail below in conjunction with specific implementations and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific implementations and examples are used to illustrate the present invention, rather than to limit the present invention.

[0032] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0033] The TBA test reagent has a high content of organic matter and is particularly susceptible to the influence of microorganisms. For example, microorganisms decompose enzymes, causing the enzyme activity to decrease or even inactivate, thus causing the reagent to be unstable; the specific storage conditions of the reactant NADH in the reagent make it difficult for most preservatives to work well. Some microorganisms will inevitably be introduced during the production of raw materials or the preparation of reagents. Therefore, it is particularly important to find a substance or combination that can effectively play a preservative role without causing a decrease in the stability of the reagent.

[0034] Based on the detection system of TBA by cyclic enzyme method and aiming at the special conditions for the storage of reagents containing NADH, the present invention provides a TBA kit, which includes a first reagent containing Thio-NAD and a second reagent containing NADH and 3α-HSD, wherein a nonionic surfactant and an antibiotic preservative are also added to the second reagent.

[0035] In a preferred embodiment, the nonionic surfactant of the present invention is selected from the group consisting of Tween series, Triton series and Tertigol.

[0036] It should be noted that the Tween series, Triton series and Tertigol have the same hydrophilic group, namely the polyoxyethylene hydrophilic group, and only the hydrophobic group is different. The hydrophilic group enables them to play a similar role when used as a surfactant. I do not want to be bound by theory. For example, such a surfactant combines with the 3α-HSD molecule in the reagent, making its three-dimensional structure less susceptible to damage and maintaining its stability.

[0037] As used in the present invention, the term "preservative" refers to a substance used to extend the shelf life of a reagent, and "antibiotic preservative" refers to a substance belonging to the antibiotic class used to extend the shelf life of a reagent.

[0038] Specifically, the nonionic surfactant may be Tertigol. In the present invention, Tertigol refers to a substance having the following structure:

[0039]

[0040] Among them, n=6~17.

[0041] As demonstrated in the examples below, Tertigol can be added to the second agent of the present invention together with the antibiotic preservative erythromycin.

[0042] For example, the concentration of Tertigol can be about 0.5-2.5 g / L, such as about 1-2 g / L; and the concentration of erythromycin can be about 0.2-2.5 g / L, such as about 0.5-2 g / L. Whether compared with a control group without adding a surfactant and a preservative, or a control group with only one of the two added, the combination of Tertigol and erythromycin significantly enhances the preservative effect of the reagent; basic performance tests have confirmed that such a concentration also takes into account detection accuracy and analytical sensitivity.

[0043] Preferably, the concentration of Tertigol can be about 0.5-2.5 g / L, such as about 1-2 g / L, and the concentration of erythromycin can be about 0.8-1.2 g / L, such as about 1 g / L. By selecting such concentrations, the present invention further improves the antiseptic effect of the TBA cycle enzyme detection reagent.

[0044] Preferably, the concentration of Tertigol can be about 0.5-1.5 g / L, such as about 1 g / L, and the concentration of erythromycin can be about 0.2-1.2 g / L, such as about 0.5-1 g / L. By selecting such concentrations, the analytical sensitivity during the test is further maintained at a good level.

[0045] More preferably, the concentration of Tertigol can be about 0.5-1.5 g / L, such as about 1 g / L, and the concentration of erythromycin can be about 0.8-1.2 g / L, such as about 1 g / L. By selecting such concentrations, the present invention further improves the antiseptic effect of the TBA cycle enzyme detection reagent while maintaining the analytical sensitivity at a good level during the test.

[0046] As demonstrated in the examples below, Tertigol can also be added to the second agent of the present invention together with the antibiotic preservative penicillin.

[0047] For example, the concentration of Tertigol can be about 0.5-1.5 g / L, such as about 1 g / L; and the concentration of penicillin can be about 0.2-1.5 g / L, such as about 0.5-1 g / L. Whether compared with a control group without adding a surfactant and a preservative, or a control group with only one of the two added, the combination of Tertigol and penicillin significantly enhances the preservative effect of the reagent; basic performance tests have confirmed that such a concentration also takes into account detection accuracy and analytical sensitivity.

[0048] Preferably, the concentration of Tertigol can be about 0.5-1.5 g / L, such as about 1 g / L, and the concentration of penicillin can be about 0.8-1.2 g / L, such as about 1 g / L. By selecting such concentrations, the present invention further improves the antiseptic effect of the TBA cycle enzyme detection reagent.

[0049] Specifically, the nonionic surfactant may be selected from the Tween class. As demonstrated in the following examples, Tween 20 may be added to the second agent of the present invention together with the antibiotic preservative erythromycin.

[0050] For example, the concentration of Tween20 can be about 1-8 g / L, such as about 2-7 g / L; and the concentration of erythromycin can be about 0.5-1.5 g / L, such as about 1 g / L. Whether compared with the control group without adding surfactant or the control group with only adding erythromycin, the combination of Tween20 and erythromycin significantly enhances the antiseptic effect of the reagent; basic performance tests confirm that such a concentration also takes into account detection accuracy and analytical sensitivity.

[0051] Preferably, the concentration of Tween20 can be about 6-8 g / L, such as about 7 g / L, and the concentration of erythromycin can be about 0.5-1.5 g / L, such as about 1 g / L. By selecting such concentrations, the present invention further improves the antiseptic effect of the TBA cycle enzyme detection reagent.

[0052] Preferably, the concentration of Tween20 can be about 1-5 g / L, such as about 2-4 g / L, and the concentration of erythromycin can be about 0.5-1.5 g / L, such as about 1 g / L. By selecting such concentrations, the present invention further maintains the analytical sensitivity during the test at a good level.

[0053] As demonstrated in the examples below, Tween 20 can also be added to the second agent of the present invention together with the antibiotic preservative ceftriaxone sodium salt.

[0054] For example, the concentration of Tween20 can be about 6-8 g / L, such as about 7 g / L; and the concentration of cefotaxime sodium can be about 0.1-1.5 g / L, such as about 0.2-1 g / L. Whether compared with the control group without adding surfactant and preservative, or the control group with only adding cefotaxime sodium, the combination of Tween20 and cefotaxime sodium significantly enhances the antiseptic effect of the reagent; basic performance tests have confirmed that such a concentration also maintains the detection accuracy and analytical sensitivity at a better level.

[0055] In the first reagent of the present invention, Thio-NAD can be present in a concentration of 0.8 to 1.5 g / L, for example; the pH adjuster can be selected from HCl, formic acid and acetic acid, and can be present in a concentration of 0.35 to 0.45 g / L, and the pH is optionally adjusted to 3 to 4.5; the preservative can be selected from sodium benzoate, potassium sorbate, etc., and can be present in a concentration of 0.5 to 2 g / L.

[0056] In addition, the first reagent of the present invention may further include one or more of a chelating agent and a substance for protecting Thio-NAD.

[0057] The chelating agent may be selected from EDTA, HEDTANa3, ferrozine, 2,4,6-tris(2-pyridyl)triazine (TPTZ), etc., and may be present in a concentration of 0.8 to 4 g / L.

[0058] The substance used to protect Thio-NAD may be sodium sulfite.

[0059] In the second reagent of the present invention, NADH may be present at a concentration of 1 to 10 g / L; 3α-HSD may be present at a concentration of 5 to 12 KU / L; the buffer may be selected from carbonate buffer, Tris buffer, TAPS buffer, AMP buffer and Goods buffer, etc., wherein the pH of the buffer is 8 to 10 and the ion concentration is 0.08 to 0.2 M.

[0060] In addition, the second reagent of the present invention may further include one or more of an enzyme protectant and a substance for maintaining the stability of reactants.

[0061] The enzyme protectant may be selected from dextran, PEG, cyclodextrin, etc., and may be present at a concentration of 5 to 15 g / L.

[0062] The substance used to maintain the stability of the reactants may be selected from L-cysteine ​​and β-mercaptoethanol.

[0063] In a specific embodiment, the first reagent of the present invention includes 0.35-0.45 g / L HCl, 0.9-1.0 g / L Thio-NAD, and 0.8-1.2 g / L potassium sorbate.

[0064] In a specific embodiment, in addition to non-ionic surfactants and antibiotic preservatives, the second reagent of the present invention includes 1.5-3.5 g / L NADH, 10-12 KU / L 3α-HSD, 8-12 g / L dextran, and 4.2-16.8 g / L buffer, such as carbonate buffer.

[0065] The present invention will be described in more detail below by way of examples, but the present invention is not limited to these examples.

[0066] Example 1 Preparation of Reagent 1 and Reagent 2

[0067] According to Table 1 and Table 2, prepare Reagent 1 and Reagent 2 respectively.

[0068] Table 1

[0069]

[0070] Table 2

[0071]

[0072] Example 2 Preparation of Tertigol-based reagent combination

[0073] The reagent combination of the experimental group and the reagent combination of the control group for detecting TBA were prepared according to Table 1 and Table 2, wherein the type and content of the surfactant and the type and content of the antibiotic preservative are shown in Table 3 below.

[0074] Table 3

[0075]

[0076] Example 3 Microbial Challenge Experiment

[0077] In order to verify the antiseptic effect of the reagent, a microbial challenge experiment was conducted. The specific steps are as follows:

[0078] Take a portion of reagent 2 from the intact experimental group or control group, measure 20 mL of each in different sterilized reagent bottles under sterile conditions, and set aside.

[0079] Inoculate Acremonium truncatum on a modified Martin slant medium and culture for 24-48 hours. Rinse the bacterial moss with 5 mL of sterile saline and dilute it. Dilute and adjust its concentration and measure its absorbance with a spectrophotometer. Take the bacterial solution with absorbance A560nm = 0.09-0.11, which is 10 8 cfu / mL Acremonium suspension for later use.

[0080] Take 20 mL of the weighed reagent, add 0.2 ml of Acremonium suspension, mix well, store the sample at room temperature, detect the number of colonies in the reagent on days 0, 7, 14, and 28, and count the results to determine the antiseptic ability of the reagent.

[0081] Example 4 Results of microbial challenge experiments with Tertigol-based reagent combinations

[0082] According to the method described in Example 3, the antiseptic ability of the reagent 2 in the experimental group and the control group prepared in Example 2 was measured. The results are shown in Table 4 below.

[0083] Table 4

[0084]

[0085] As shown in Table 4, after inoculation with Acremonium spp., the colony count of control group A1 without adding surfactant and antibiotic preservatives once reached 3211.

[0086] For the control group A2 with 1g / L erythromycin added but no surfactant, the colony count once reached 559; for the control group A3 with 1g / L Tertigol added but no preservative, the colony count once reached 600. In the case of adding Tertigol and erythromycin at the same time, the colony count remained at a low level throughout the experimental period after inoculation, among which the experimental group A1 with 1g / L Tertigol and 1g / L erythromycin added and the experimental group A2 with 2g / L Tertigol and 1g / L erythromycin added had better preservative effects.

[0087] In addition, the colony count of the control group A4, which added 1g / L penicillin but no surfactant, once reached 512; and the colony count of the control group A3, which added 1g / L Tertigol but no preservative, once reached 600. When Tertigol and penicillin were added at the same time, the colony count remained at a low level, and the preservative effect of the experimental group A5, which added 1g / L Tertigol and 1g / L penicillin, was better.

[0088] Example 5 Basic Performance Test

[0089] In order to investigate the performance of each group of reagents when used to detect TBA, accuracy and analytical sensitivity tests were performed.

[0090] The specific steps of the accuracy test are as follows:

[0091] 1) Add 3 μl of sample (calibrator: 50 μmol / L sodium glycochenodeoxycholate + 1 g / L sodium azide; quality control: commercial quality control product from Mindray, catalog number: 105-003695-00) to 203 μl of reagent 1, mix well, and incubate at 37°C for 5-10 min; 2) Add 68 μl of reagent 2 to the incubated solution, mix well, and react at 37°C for 1 min;

[0092] 3) Use Mindray BS800 XE-78001209 to measure the absorbance A1 (main wavelength 412nm; secondary wavelength 660nm);

[0093] 4) After standing for 3 minutes, measure the absorbance A2 (main wavelength 412nm; secondary wavelength 660nm);

[0094] 5) Take the target values ​​of the calibrator and quality control product (recorded in the instructions of the kit) as the true value, compare the measured results with the true value, and calculate the relative deviation.

[0095] The specific steps of analytical sensitivity testing are as follows:

[0096] 1) Add 3 μl of sample (serum with TBA concentration of 50 mmol / L) to 203 μl of reagent 1, mix well, and incubate at 37°C for 5-10 min; 2) Add 68 μl of reagent 2 to the incubated solution, mix well, and react at 37°C for 1 min;

[0097] 3) Use Mindray BS800 XE-78001209 to measure the absorbance Am (main wavelength 412nm; secondary wavelength 660nm);

[0098] 4) After standing for 3 minutes, measure the absorbance An (main wavelength 412nm; secondary wavelength 660nm);

[0099] 5) Read the absorbance at points m and n in the reaction time (mn) on the reaction curve, and record them as Am and An respectively; calculate the absolute value of the absorbance change rate according to the following formula and record it as △A1 / min:

[0100] △A1 / min=abs(An-Am) / [(nm)*S*10000]*60 (S is the reading cycle of the instrument)

[0101] Calculate the absolute values ​​of the absorbance change rate △A1 / min, △A2 / min and △A3 / min three times as above, and calculate the average of the three results as the absorbance change rate △A / min.

[0102] Example 6 Basic performance test of Tertigol-based reagent combination

[0103] According to the method described in Example 5, the accuracy and analytical sensitivity of the reagent combination in the experimental group and the control group prepared in Example 2 were measured, and the results are shown in Table 5 below.

[0104] Table 5

[0105]

[0106] As shown in Table 5, the experimental groups with the addition of Tertigol and erythromycin or Tertigol and penicillin can maintain the accuracy at a good level and keep the analytical sensitivity within an acceptable range. Among them, the analytical sensitivity of the experimental group A1 with the addition of 1g / L Tertigol and 1g / L erythromycin and the experimental group A3 with the addition of 1g / L Tertigol and 0.5g / L erythromycin is better.

[0107] Example 7 Preparation of a reagent combination based on Tween 20 and erythromycin

[0108] The reagent combination of the experimental group and the reagent combination of the control group for detecting TBA were prepared according to Table 1 and Table 2, wherein the type and content of the surfactant and the type and content of the antibiotic preservative are shown in Table 6 below.

[0109] Table 6

[0110]

[0111] Example 8 Results of microbial challenge experiments based on a combination of Tween 20 and erythromycin

[0112] According to the method described in Example 3, the antiseptic ability of the reagent 2 in the experimental group and the control group prepared in Example 7 was measured. The results are shown in Table 7 below.

[0113] Table 7

[0114]

[0115] As shown in Table 7, after inoculation of Acremonium truncatum, the colony count of the control group B1 without adding surfactant and antibiotic preservatives once reached 2400. For the control group B2 with 1g / L erythromycin but no surfactant, the colony count once reached 614. When Tween20 and erythromycin were added at the same time, the colony count was maintained at a low level, among which the experimental group B3 with 7g / L Tween20 added had a better antiseptic effect.

[0116] Example 9 Basic performance test of the reagent combination based on Tween 20 and erythromycin

[0117] According to the method described in Example 5, the accuracy and analytical sensitivity of the reagent combination in the experimental group and the control group prepared in Example 7 were measured, and the results are shown in Table 8 below.

[0118] Table 8

[0119]

[0120] As shown in Table 8, the experimental groups with added Tween20 and erythromycin can maintain the accuracy at a better level and keep the analytical sensitivity within an acceptable range. In addition, the analytical sensitivity of the experimental group B1 with added 2g / L Tween20 and the experimental group B2 with added 4g / L Tween20 is better.

[0121] Example 10 Preparation of a reagent combination based on Tween 20 and cefotaxime sodium salt

[0122] The reagent combination of the experimental group and the reagent combination of the control group for detecting TBA were prepared according to Table 1 and Table 2, wherein the type and content of the surfactant and the type and content of the antibiotic preservative are shown in Table 9 below.

[0123] Table 9

[0124]

[0125] Example 11 Results of microbial challenge experiments based on a combination of Tween 20 and cefotaxime sodium salt

[0126] According to the method described in Example 3, the antiseptic ability of the reagent 2 in the experimental group and the control group prepared in Example 10 was measured. The results are shown in Table 10 below.

[0127] Table 10

[0128]

[0129] As shown in Table 10, after inoculation of Acremonium truncatum, the colony count of the control group C1 without adding surfactant and antibiotic preservatives once reached 1800. For the control group C2 with 1g / L cefotaxime sodium added but no surfactant, the colony count reached 101 at 7 days. When Tween20 and cefotaxime sodium were added at the same time, the colony count was maintained at a low level, among which the experimental group C1 with 7g / L Tween20 and 1g / L cefotaxime sodium added had a better preservative effect.

[0130] Example 12 Basic performance test of the reagent combination based on Tween 20 and cefotaxime sodium salt

[0131] According to the method described in Example 5, the accuracy and analytical sensitivity of the reagent combination in the experimental group and the control group prepared in Example 10 were measured, and the results are shown in Table 11 below.

[0132] Table 11

[0133]

[0134] As shown in Table 11, the experimental groups to which Tween 20 and cefotaxime sodium salt were added were able to maintain the accuracy and analytical sensitivity at a better level.

[0135] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent transformations made using the contents of the present invention specification under the inventive concept of the present invention, or direct / indirect applications in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A kit for detecting TBA, comprising: A first reagent comprising Thio-NAD, a pH adjuster, and a preservative; a second reagent comprising NADH, 3α-HSD, a buffer, a nonionic surfactant and an antibiotic preservative; Wherein, the antibiotic preservative is selected from one or more of erythromycin, penicillin and cefotaxime sodium salt; Wherein, the nonionic surfactant is Tertigol or Tween20.

2. The kit according to claim 1, characterized in that The pH adjuster in the first reagent adjusts the pH of the first reagent to 3-4.

5.

3. The kit according to claim 1, characterized in that The pH of the buffer solution in the second reagent is 8-10.

4. The kit according to claim 1, wherein The concentration of Tween 20 is 1-8 g / L.

5. The kit according to claim 4, wherein The concentration of Tween 20 is 3-8 g / L.

6. The kit according to claim 1, wherein The concentration of the Tertigol is 0.5-2.5 g / L.

7. The kit according to claim 6, wherein The concentration of the Tertigol is 0.5-1.5 g / L.

8. The kit according to claim 1, wherein The concentration of the antibiotic preservative is 0.1-1.5 g / L.

9. The kit according to claim 8, wherein The concentration of the antibiotic preservative is 0.2-1 g / L.

10. A method for detecting TBA using the kit according to any one of claims 1 to 9 for non-diagnostic purposes, comprising the following steps: 1) Mixing the sample to be tested with the first reagent and incubating; 2) adding the second reagent, mixing and incubating; 3) measuring a first absorbance; 4) After a certain period of time, measuring a second absorbance; and 5) Calculating TBA concentration according to the first absorbance and the second absorbance.

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