Method for detecting enzyme activity of lysosomes and application of method

Abstract

The invention particularly discloses a method for detecting enzyme activity of lysosomes and application of the method, and relates to the technical field of biological detection. The invention provides a method for detecting enzyme activity of lysosomes. The method specifically comprises the following steps: S1, mixing a sample with a buffer solution, an internal standard and a substrate, and reacting; s2, adding a stop solution into the reaction, and centrifuging to obtain supernate; and S3, detecting the supernate through a liquid chromatography-tandem mass spectrometry technology to obtain an enzyme activity result. A protein precipitation method is used, and a complex treatment process of liquid-liquid extraction is not needed. A chromatographic column separation mode is used, a mobile phase and a gradient are optimized, detection can be completed in a short time, meanwhile, the influence of interference and substrate cracking is reduced, the detection accuracy is improved, linear correlation coefficients r of detection results are all larger than 0.99, and variable coefficients CV are all smaller than 18%. The invention provides a theoretical basis and an experimental basis for developing a new treatment strategy, and has good clinical transformation and clinical application prospects.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a method for detecting lysosomal enzyme activity and application thereof. Background Art

[0002] Lysosomal storage disorders (LSDs) are a group of rare genetic diseases caused by defects in glycosidases, proteases, membrane proteins, transporters, enzyme modifiers or activators in lysosomes, which prevent the enzyme substrates from being degraded and are stored in lysosomes. Commonly used examination methods include enzyme activity detection, biomarker detection, gene detection, etc.

[0003] Currently, the Advisory Committee on Hereditary Disorders in Newborns and Children (ACHDNC) has included Pompe disease and mucopolysaccharidosis type I in the "Recommended Uniform Screening Panel (RUSP)", and the inclusion of Fabry disease, Niemann-Pick disease type A / B, and Krabbe disease is also under discussion. New York, Illinois, Missouri and other states in the United States have already carried out newborn screening for six LSDs (Fabry disease, Pompe disease, Gaucher disease, mucopolysaccharidosis type I, Niemann-Pick disease type A / B, and Krabbe disease), and other states are also actively preparing and implementing corresponding plans.

[0004] Lysosomal enzyme activity measurement is the gold standard for diagnosing LSD, but the traditional leukocyte method for detecting enzyme activity is not suitable for large-scale neonatal disease screening due to its complex operation. Currently, the commonly used neonatal LSD screening methods are fluorescence method and tandem mass spectrometry.

[0005] Fluorescence method: Currently, LSD fluorescence screening is mainly achieved through digital microfluidics (DMF), which has the advantages of simple instrumentation, low maintenance cost, and short incubation time. However, the fluorescence method has some inherent defects and limitations: most of the widely used substrates have the same fluorescent group (non-specific), which will interfere with each other and easily lead to a high false positive rate. Although the introduction of DMF can achieve the so-called "multiple analysis", it is essentially still a separate detection of each reaction product, that is, the enzyme activity reaction systems of different LSDs need to be independent of each other. In addition, the substrate used in this method must be fluorescent (containing fluorescent groups), and the detection sensitivity of this method is relatively low. These factors also make it difficult to expand the types of LSD screening in the future.

[0006] Tandem mass spectrometry: Based on the highly sensitive, highly specific and highly selective liquid chromatography-tandem mass spectrometry technology (LC-MS / MS), it can realize the simultaneous detection of multiple LSD enzyme activities without mutual interference; at the same time, it is conducive to the detection of low enzyme activity samples (positive samples). In addition, artificial synthetic substrates that are closer to natural substrates can be used, and the introduction of isotope internal standards also further improves the accuracy of quantitative results. These factors also make the use of tandem mass spectrometry technology have the potential and prospects to further expand the screening of diseases. At present, most countries and regions that have carried out neonatal LSD screening use tandem mass spectrometry technology, which is also the future development trend.

[0007] The six existing mass spectrometry detection methods for enzyme activity basically use the liquid-liquid extraction method after incubation, and its pretreatment is relatively time-consuming. In addition, the existing methods use flow injection without connecting to a chromatographic column, which is relatively demanding on instrument conditions. It mainly sets source parameters to reduce the in-source cleavage of the substrate, and has very high requirements on the instrument. Interference in the matrix and in-source cleavage of the substrate will affect the accuracy of the detection results. Summary of the invention

[0008] 1. Technical issues to be solved

[0009] In view of this, one of the main purposes of the present invention is to provide a method for detecting lysosomal enzyme activity, the method specifically comprising:

[0010] S1: Mix the sample with buffer, internal standard and substrate and react;

[0011] S2: adding a stop solution to the reaction and centrifuging to obtain a supernatant;

[0012] S3: detecting the supernatant by liquid chromatography-tandem mass spectrometry to obtain enzyme activity results;

[0013] Wherein, the conditions of liquid chromatography in S3 are:

[0014] (1) Mobile phase A: 0.1% (volume ratio) formic acid in water;

[0015] (2) Mobile phase B: 0.1% (volume ratio) formic acid acetonitrile;

[0016] (3) Gradient setting: 0-0.8 min, 10%-40% (volume ratio) mobile phase B; 1 min, 40%-60% (volume ratio) mobile phase B; 2 min, 70%-90% (volume ratio) mobile phase B; 2.2-3 min, 90%-100% (volume ratio) mobile phase B; 3.01-3.5 min, 10%-40% (volume ratio) mobile phase B.

[0017] The method provided by the present invention adopts a method of direct extraction and instrumentation after incubation, which improves the accessibility of detection. A chromatographic column is used for separation, and through gradient elution, interference and source cleavage products can be effectively separated in a short time, which greatly improves the accuracy of detection.

[0018] (II) Technical solution

[0019] In order to solve the above problems, the present invention provides a method for detecting lysosomal enzyme activity, which specifically comprises:

[0020] S1: The subject's sample is mixed with buffer, internal standard and substrate for reaction;

[0021] S2: adding a stop solution to the reaction and centrifuging to obtain a supernatant;

[0022] S3: detecting the supernatant by liquid chromatography-tandem mass spectrometry to obtain enzyme activity results;

[0023] Wherein, the conditions of liquid chromatography in S3 are:

[0024] (1) Mobile phase A: 0.1% formic acid in water;

[0025] (2) Mobile phase B: 0.1% formic acid acetonitrile;

[0026] (3) Gradient setting: 0-0.8 min, 10%-40% mobile phase B; 1 min, 40%-60% mobile phase B; 2 min, 70%-90% mobile phase B; 2.2-3 min, 90%-100% mobile phase B; 3.01-3.5 min, 10%-40% mobile phase B.

[0027] In one embodiment, the method involves only detection for non-diagnostic purposes.

[0028] In one embodiment, the solvent of the 0.1% formic acid and 0.1% formic acid acetonitrile is water.

[0029] In one embodiment, the gradient is set as follows: 0-0.8 min, 28% mobile phase B; 1 min, 50% mobile phase B; 2 min, 75% mobile phase B; 2.2-3 min, 100% mobile phase B; 3.01-3.5 min, 28% mobile phase B.

[0030] In one embodiment, the gradient is set as follows: 0-0.8 min, 72% mobile phase A; 1 min, 50% mobile phase A; 2 min, 25% mobile phase A; 2.2-3 min, 0% mobile phase A; 3.01-3.5 min, 72% mobile phase A.

[0031] In one embodiment, the gradient is set as: 0-0.8 min, 72% mobile phase A, 28% mobile phase B; 1 min, 50% mobile phase A, 50% mobile phase B; 2 min, 25% mobile phase A, 75% mobile phase B; 2.2-3 min, 0% mobile phase A, 100% mobile phase B; 3.01-3.5 min, 72% mobile phase A, 28% mobile phase B.

[0032] In one embodiment, the lysosomal enzyme in S1 includes one or a combination of α-L-iduronidase, acid α-glucosidase, α-galactosidase, β-galactocerebrosidase, acid sphingomyelinase and / or acid β-glucocerebrosidase.

[0033] In one embodiment, the S1 lysosome comprises α-L-iduronidase, acid α-glucosidase, α-galactosidase, β-galactocerebroside, acid sphingomyelinase and acid β-glucocerebrosidase.

[0034] The corresponding relationship between the lysosomes and LSDs is shown in Table 1:

[0035] Table 1

[0036]

[0037] In one embodiment, the subject is a mammal. Preferably, the mammal is a human.

[0038] In one embodiment, the sample in S1 is one or a combination of tissue, cell or fluid.

[0039] In one embodiment, the sample comprises one or a combination of tissue, blood, serum, plasma, blood-derived cells, lymph, synovial fluid, cerebrospinal fluid, pleural fluid, peritoneal fluid, bladder washings, secretions (e.g., breast secretions), oral washes, swabs (e.g., buccal swabs), touch preparations, fine needle aspirates, and cell extracts.

[0040] In one embodiment, the sample is blood.

[0041] In one embodiment, the sample is a dried blood spot.

[0042] In one embodiment, S1 also includes a quality control sample, and the preparation method of the quality control sample includes: using whole blood for centrifugation, high-concentration quality control is prepared by extracting the middle white blood cell layer and adjusting the cell volume to 0.475±0.025 to prepare dry blood spots; low-concentration and medium-concentration quality control are prepared by diluting the high-concentration quality control with red blood cells whose cell volume is adjusted to 0.475±0.025 to prepare dry blood spots.

[0043] In one embodiment, the concentrations of the substrate (S) and the internal standard (IS) in S1 are shown in Table 2:

[0044] Table 2

[0045] No. Comp.ID Concentration (μmol / L) 1 IDUA-S 3000 2 IDUA-IS 50 3 GAA-S 3000 4 GAA-IS 50 5 GLA-S 3000 6 GLA-IS 50 7 GALC-S 3000 8 GALC-IS 50 9 ASM-S 3000 10 ASM-IS 50 11 GBA-S 3000 12 GBA-IS 50

[0046] In one embodiment, the composition of the buffer in S1 is shown in Table 3:

[0047] Table 3

[0048]

[0049] In one embodiment, the volume of the buffer is 50 μL, and the sum of the volumes of the internal standard and the substrate is 10 μL.

[0050] In one embodiment, the volumes of the internal standard and substrate are 1 μL and 9 μL, respectively.

[0051] In one embodiment, the stop solution in S2 is one or a combination of 100% methanol, 100% acetonitrile, 0.1% formic acid in methanol, 0.1% formic acid in acetonitrile, 90% methanol or 90% acetonitrile; preferably, the stop solution is 0.1% formic acid in acetonitrile.

[0052] In one embodiment, the stop solution solvent is water.

[0053] In one embodiment, S3 further comprises adding a stop solution to the supernatant and mixing.

[0054] In one embodiment, the method includes:

[0055] S1: Mix the sample with buffer, internal standard and substrate and react;

[0056] S2: adding a stop solution to the reaction and centrifuging to obtain a supernatant;

[0057] S3: adding a stop solution to the supernatant and mixing, and detecting the enzyme activity result by liquid chromatography-tandem mass spectrometry;

[0058] Wherein, the conditions of liquid chromatography in S3 are:

[0059] (1) Mobile phase A: 0.1% formic acid in water;

[0060] (2) Mobile phase B: 0.1% formic acid acetonitrile;

[0061] (3) Gradient setting: 0-0.8 min, 10%-40% mobile phase B; 1 min, 40%-60% mobile phase B; 2 min, 70%-90% mobile phase B; 2.2-3 min, 90%-100% mobile phase B; 3.01-3.5 min, 10%-40% mobile phase B.

[0062] In one embodiment, the gradient is set as follows: 0-0.8 min, 28% mobile phase B; 1 min, 50% mobile phase B; 2 min, 75% mobile phase B; 2.2-3 min, 100% mobile phase B; 3.01-3.5 min, 28% mobile phase B.

[0063] In one embodiment, the gradient is set as follows: 0-0.8 min, 72% mobile phase A; 1 min, 50% mobile phase A; 2 min, 25% mobile phase A; 2.2-3 min, 0% mobile phase A; 3.01-3.5 min, 72% mobile phase A.

[0064] In one embodiment, the gradient is set as: 0-0.8 min, 72% mobile phase A, 28% mobile phase B; 1 min, 50% mobile phase A, 50% mobile phase B; 2 min, 25% mobile phase A, 75% mobile phase B; 2.2-3 min, 0% mobile phase A, 100% mobile phase B; 3.01-3.5 min, 72% mobile phase A, 28% mobile phase B.

[0065] In one embodiment, the reaction in S1 is a shaking extraction, and the conditions are: 37° C., 400 rpm, 18±2 h.

[0066] In one embodiment, the centrifugation condition in S2 is: 4000 rpm, 5 min.

[0067] In one embodiment, the S2 further includes shaking before centrifugation, and the conditions are: 600 rpm, 10 min.

[0068] In one embodiment, the mixing in S3 is oscillating mixing, and the conditions are: 600 rpm, 5 min.

[0069] In one embodiment, the LC-MS / MS detection parameters are shown in Table 4:

[0070] Table 4

[0071]

[0072]

[0073] In another aspect, the present invention provides a lysosomal enzyme activity detection kit, wherein the detection target of the kit is α-L-iduronidase, acid α-glucosidase, α-galactosidase, β-galactocerebroside, acid sphingomyelinase and / or acid β-glucocerebrosidase;

[0074] The kit includes an internal standard, a substrate, a buffer, a stop solution and a quality control sample;

[0075] The buffer comprises KH2PO4, K2HPO4 and Triton X-100;

[0076] The stop solution includes formic acid and methanol.

[0077] In one embodiment, the reagents in the kit are used to perform detection according to the above method.

[0078] (III) Beneficial effects

[0079] The present invention provides a method for detecting lysosomal enzyme activity. Compared with the prior art, the method has the following beneficial effects:

[0080] 1. The pretreatment uses protein precipitation method, which does not require the complicated treatment process of liquid-liquid extraction.

[0081] 2. Using chromatographic column separation, optimizing the mobile phase and gradient, the detection can be completed in a short time, while reducing the influence of interference and substrate cleavage, improving the detection accuracy. The linear correlation coefficient r of the detection results is greater than 0.99, and the coefficient of variation CV is less than 18%.

[0082] (IV) Terms and Definitions

[0083] As used herein, the term "buffer" may be any buffer known in the art that is suitable for use in protease reactions. The buffer disclosed in the present invention may contain one or more buffer reagents selected from the group consisting of Tris, TrisHCl, 3-{[tris(hydroxymethyl)methyl]amino}propanesulfonic acid (TAPS), N,N-bis(2-hydroxyethyl)glycine (Bicine), N-tris(hydroxymethyl)methylglycine (tricine), 3-[N-tris(hydroxymethyl)methylamino]-2-hydroxypropanesulfonic acid (TAPSO), HEPES, 2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid (TES), 3-(N-morpholino)propanesulfonic acid (MOPS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), dimethylarsonic acid (cacodate), sodium citrate (SSC), 2-(N-morpholino)ethanesulfonic acid (MES), 2(R)-2-(methylamino)succinic acid (succinic acid), borate, phosphate, acetate, glycine, magnesium carbonate or calcium carbonate and bicarbonate. Preferably, the buffer disclosed in the present invention comprises KH2PO4, K2HPO4 and Triton X-100.

[0084] The pH of the buffer can vary. For example, the buffer in the method of the present invention can be adjusted to about 1.0 to about 12.0. Preferably, it is adjusted to about 1.0 to about 6.0. Preferably, it is adjusted to 4.5.

[0085] As used herein, the term "100% methanol" refers to a methanol whose volume fraction is infinitely close to 100%, for example, a methanol aqueous solution greater than 99%, for example, a 99.9% methanol aqueous solution, a 99.99% methanol aqueous solution, and the like.

[0086] As used herein, the term "100% acetonitrile" refers to acetonitrile with a volume fraction infinitely close to 100%, for example, an acetonitrile aqueous solution greater than 99%, for example, a 99.9% acetonitrile aqueous solution, a 99.99% acetonitrile aqueous solution, and the like.

[0087] As used herein, the terms "substrate" and "internal standard" are well known and optional to those skilled in the art, for example, as disclosed in Gelb MH, Turecek F, Scott CR, Chamoles NA. Direct multiplex assay of enzymes indried blood spots by tandem mass spectrometry for the newborn screening of lysosomal storage disorders. J Inherit Metab Dis. 2006 Apr-Jun; 29(2-3): 397-404. doi: 10.1007

[0088] / s10545-006-0265-4. PMID: 16763908; PMCID: PMC2488386. The entire text is incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0089] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0090] Figure 1 is the LC parameter gradient setting diagram;

[0091] Figure 2 This is the IDUA test result diagram;

[0092] Figure 3 This is the GAA test result chart;

[0093] Figure 4 This is the GLA test result chart;

[0094] Figure 5 This is the GALC test result graph;

[0095] Figure 6 This is the ASM test result diagram;

[0096] Figure 7 This is the GBA test result chart. DETAILED DESCRIPTION

[0097] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. 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.

[0098] As used herein, “containing,” “having,” or “including” include “comprising,” “mainly consisting of,” “substantially consisting of,” and “consisting of;” “mainly consisting of,” “substantially consisting of,” and “consisting of” are subordinate concepts of “containing,” “having,” or “including.”

[0099] The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the reagents, methods and equipment used are all conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0100] Example 1

[0101] Detection of lysosomal enzyme activity:

[0102] 1. Preparation of quality control samples:

[0103] Whole blood was centrifuged, and high-concentration quality control was prepared by extracting the middle white blood cell layer and adjusting the cell volume to 0.475±0.025 to prepare dried blood spots. Low-concentration and medium-concentration quality control were prepared by diluting the high-concentration quality control with red blood cells adjusted to 0.475±0.025 to prepare dried blood spots.

[0104] 2. Detection of lysosomal enzyme activity

[0105] S1: Use a manual or automatic dry blood spot puncher to punch 1 spot on the sample dry blood spot and the quality control sample dry blood spot. (1 / 8 inch) samples were placed in a U-shaped 96-well plate;

[0106] S2: Add 50 μL of the buffer shown in Table 3 and 10 μL of the internal standard and substrate (ChemBind, Catalog No. CBPE001-012) shown in Table 2 to each plate well containing samples, cover the plate with aluminum foil to ensure good sealing and minimize volatiles; place the 96-well plate on an oscillator, shake at 37°C, 400 rpm for 18±2 hours;

[0107] S3: Add 120 μL of 0.1% formic acid methanol as stop solution, oscillate at 600 rpm for 10 min; centrifuge at 4000 rpm for 5 min; take 30 μL of supernatant, add 90 μL of stop solution, oscillate at 600 rpm for 5 min, and perform detection according to the parameters shown in Table 4.

[0108] Test results such as Figure 2-7 As shown, the detection method provided by the present invention can effectively separate cleavage and interference in the substrate source.

[0109] 3. Verification of analytical performance

[0110] The detection method provided by the present invention was verified from aspects such as blank limit, linear correlation coefficient, precision and accuracy. The results are shown in Tables 5 to 8:

[0111] Table 5

[0112]

[0113] Table 6

[0114]

[0115] Table 7

[0116]

[0117] Table 8

[0118]

[0119] Example 2

[0120] Effect of stop solution type on test results:

[0121] The difference between this embodiment and embodiment 1 is that the stop solution is 100% methanol, 100% acetonitrile, 0.1% formic acid in methanol, 0.1% formic acid in acetonitrile, 90% methanol or 90% acetonitrile solution, and the other parts are exactly the same as those in embodiment 1.

[0122] The test results are shown in Table 9:

[0123] Table 9

[0124]

[0125] As shown in Table 9, the test results obtained by selecting 0.1% formic acid methanol as the stop solution meet the expected requirements, and the repeatability, stop reaction effect and peak shape are good.

[0126] Example 3

[0127] Effect of buffer volume on test results:

[0128] The difference between this embodiment and embodiment 1 is that the volume of the buffer solution is 30 μL, 50 μL and 100 μL, and the other parts are exactly the same as those in embodiment 1.

[0129] The test results are shown in Table 10:

[0130] Table 10

[0131]

[0132]

[0133] It can be seen from Table 10 that the test results obtained by selecting 50 μL of buffer meet the expected requirements.

[0134] Example 4

[0135] The influence of mobile phase on the test results:

[0136] The difference between this embodiment and embodiment 1 is that the mobile phase is changed, and the other parts are exactly the same as embodiment 1.

[0137] The test results are shown in Table 11:

[0138] Table 11

[0139]

[0140] As shown in Table 11, the peak shape of the test results obtained by selecting 0.1% formic acid water as phase A and 0.1% formic acid acetonitrile as phase B is better and can be separated in a short time.

[0141] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "comprise a ..." do not exclude the existence of other identical elements in the process, method, article or device including the elements.

[0142] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. 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 method for detecting lysosomal enzyme activity, characterized in that: The method specifically comprises: S1: The subject's sample is mixed with buffer, internal standard and substrate for reaction; S2: adding a stop solution to the reaction and centrifuging to obtain a supernatant; S3: detecting the supernatant by liquid chromatography-tandem mass spectrometry to obtain enzyme activity results; Wherein, the conditions of liquid chromatography in S3 are: (1) Mobile phase A: 0.1% formic acid in water; (2) Mobile phase B: 0.1% formic acid acetonitrile; (3) Gradient setting: 0-0.8 min, 10%-40% mobile phase B; 1 min, 40%-60% mobile phase B; 2 min, 70%-90% mobile phase B; 2.2-3 min, 90%-100% mobile phase B; 3.01-3.5 min, 10%-40% mobile phase B.

2. The method according to claim 1, characterized in that The lysosomal enzyme is selected from one or a combination of α-L-iduronidase, acid α-glucosidase, α-galactosidase, β-galactocerebrosidase, acid sphingomyelinase and / or acid β-glucocerebrosidase.

3. The method according to claim 2, characterized in that The lysosomal enzymes include α-L-iduronidase, acid α-glucosidase, α-galactosidase, β-galactocerebroside, acid sphingomyelinase and acid β-glucocerebrosidase.

4. The method according to claim 1, characterized in that The sample in S1 is one or a combination of tissue, cell or fluid.

5. The method according to claim 4, characterized in that The sample was a dried blood spot.

6. The method according to claim 1, characterized in that The stop solution in S2 is one of 100% methanol, 100% acetonitrile, 0.1% formic acid in methanol, 0.1% formic acid in acetonitrile, 90% methanol or 90% acetonitrile, or a combination thereof.

7. The method according to claim 6, characterized in that The stop solution in S2 is 0.1% formic acid in methanol.

8. The method according to claim 1, characterized in that The step S3 further includes adding a stop solution to the supernatant and mixing the solution.

9. A lysosomal enzyme activity detection kit, characterized in that: The detection target of the kit is α-L-iduronidase, acid α-glucosidase, α-galactosidase, β-galactocerebroside, acid sphingomyelinase and / or acid β-glucocerebrosidase; The kit includes an internal standard, a substrate, a buffer, a stop solution and a quality control sample; The buffer comprises KH2PO4, K2HPO4 and Triton X-100; The stop solution includes formic acid and methanol.

10. The kit according to claim 9, characterized in that The reagents in the kit are used to perform detection according to the method described in any one of claims 1 to 8.

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