Reagent and kit for detecting alpha-L-fucosidase

Abstract

The embodiment of the invention belongs to the technical field of biological agents with enzyme content, and relates to a reagent and a kit for detecting alpha-L-fucosidase, and the reagent comprises a reagent R1 and a reagent R2 which are independent; wherein the reagent R1 comprises the following components: a first ion buffer solution, a metal ion complex, a first surfactant and a first preservative; and the reagent R2 comprises the following components: a second ion buffer solution, an alpha-L-fucosidase reaction substrate, a second surfactant and a second preservative. According to the application, the hemoglobin interference resistance of the reagent for detecting the alpha-L-fucosidase can be improved.

Description

Technical Field

[0001] The present application relates to the technical field of enzyme content biological preparations, and in particular to a reagent and a kit for detecting α-L-fucosidase. Background Art

[0002] AFU (α-L-fucosidase) is a bioactive macromolecular hydrolase that catalyzes the hydrolysis of fucosylated glycoproteins. It is widely distributed in human tissues, cells, body fluids, and blood, and is highly active in tissues such as the liver and kidneys. AFU is an enzyme encoded by the human FUCA1 gene that catalyzes the decomposition of L-fucose.

[0003] Studies have found that AFU is an autosomal recessive hereditary disease. Genetic defects lead to a neurovisceral storage disease called fucosylation. Various glycoproteins and glycolipids, including sugars, accumulate in the lysosomes of cells throughout the body, leading to progressive psychomotor retardation. The incidence of this disease is particularly low among lysosomal diseases. Patients are distributed all over the world, but only a few hundred cases have been reported. Clinically, it is usually divided into two types. Severe type I can show psychomotor retardation in infancy, which is characterized by increased sweat NaCl concentration. Mild type II does not show psychomotor retardation until 1-2 years old, but is characterized by angular hemangioma.

[0004] 81.2% of patients with primary liver cancer have increased serum AFU levels. Studies have shown that the combined diagnosis of AFU with AFP, TSGF, and IL-8 is of great significance for liver cancer patients and can make up for the deficiency of AFP alone. The combined detection of AFU, AFP, TSGF, and IL-8 can increase the positive diagnosis rate of primary liver cancer by 93.1%. Compared with normal people, the increase in serum AFU in patients with liver cancer is statistically significant. Dynamic observation is of great significance for judging the efficacy, prognosis, and recurrence of liver cancer. Serum AFU can also increase in metastatic liver cancer, lung cancer, breast cancer, ovarian cancer, and uterine cancer; it also increases slightly in cirrhosis, chronic hepatitis, gastrointestinal bleeding, etc.

[0005] At present, there are many methods for measuring AFU in clinical practice, such as fluorescence method, endpoint method and CNPF substrate method. The fluorescence method has complicated steps and is not conducive to widespread use; the endpoint colorimetric method is easy to operate and does not require high equipment, but the enzyme itself has poor anti-interference ability and cannot solve the influence of serum hemolysis, lipemia and other factors on the determination. Its main determination operation is cumbersome, the required reaction time is also long, and it cannot be automatically detected, so it is difficult to promote; the CNPF substrate method has the advantages of simplicity, rapidity, sensitivity, and anti-interference ability. It can be used in various semi- / fully automated analytical instruments and is easy to promote and apply.

[0006] However, in the CNPF substrate method, the AFU reagent has poor resistance to hemoglobin interference and is not stable enough, resulting in low accuracy in the detection of α-L-fucosidase in clinical samples. Summary of the invention

[0007] The purpose of the embodiments of the present application is to provide a reagent and a kit for detecting α-L-fucosidase to solve the technical problem of poor anti-hemoglobin interference in the detection of α-L-fucosidase in the prior art.

[0008] In order to solve the above technical problems, the present application embodiment provides a reagent for detecting α-L-fucosidase, which adopts the following technical solution:

[0009] Including independent R1 reagent and R2 reagent;

[0010] Wherein, the components of the R1 reagent include a first ion buffer, a metal ion complex, a first surfactant and a first preservative;

[0011] The components of the R2 reagent include a second ion buffer, an α-L-fucosidase reaction substrate, a second surfactant and a second preservative.

[0012] Furthermore, the first ion buffer is selected from at least one of a phosphate buffer solution, a Tris buffer solution and a citric acid buffer solution;

[0013] The metal ion complex is at least one of disodium ethylenediaminetetraacetate, ethylenediaminetetraacetic acid and disodium nitrilotriacetate;

[0014] At least one of the first surfactant, alkoxy polyethylene hydroxyethanol, and dodecyl polyethylene glycol ether, or a combination of alkoxy polyethylene hydroxyethanol and dodecyl polyethylene glycol ether;

[0015] The first preservative is at least one of sodium azide, ProClin300, kanamycin sulfate and ampicillin.

[0016] Furthermore, the concentration of the first ion buffer is 50 to 300 mmol / L, and the pH value is 7.2 to 7.4;

[0017] The concentration of the metal ion complex is 0.1 to 5 g / L;

[0018] The concentration of the first surfactant is 1 to 5 g / L;

[0019] The concentration of the first preservative is 0.5-1 g / L.

[0020] Furthermore, the second ion buffer is selected from at least one of a phosphate buffer solution, a Tris buffer solution and a citric acid buffer solution;

[0021] The α-L-fucosidase reaction substrate is at least one of MG-2-chloro-4-nitrobenzene-α-L-fucoside, novel 2-chloro-4-nitrophenyl-β-D-pyranofucoidosylglycoside and 4-nitrophenyl-α-L-fucoside;

[0022] The second surfactant is polyoxyethylene lauryl ether;

[0023] The second preservative is at least one of sodium azide and ProClin300.

[0024] Further, the second ion buffer has a concentration of 50 to 300 mmol / L and a pH value of 7.2 to 7.4;

[0025] The concentration of the α-L-fucosidase reaction substrate is 2 to 5 g / L;

[0026] The concentration of the second surfactant is 1 to 5 g / L;

[0027] The concentration of the second preservative is 0.5-1 g / L.

[0028] Furthermore, the volume ratio of the R1 reagent to the R2 reagent is 3:1.

[0029] In order to solve the above technical problems, the present application also provides a kit for detecting α-L-fucosidase, which adopts the following technical solution:

[0030] comprising reagents, calibrators and quality controls for detecting α-L-fucosidase as described above;

[0031] The calibrator and the quality control product are used to construct a detection procedure for α-L-fucosidase, and the components of the calibrator and the quality control product are independently α-L-fucosidase.

[0032] Compared with the prior art, the embodiments of the present application have the following beneficial effects:

[0033] The present application provides a reagent for detecting α-L-fucosidase, comprising independent R1 reagent and R2 reagent; wherein the components of the R1 reagent include a first ion buffer, a metal ion complex, a first surfactant and a first preservative; the components of the R2 reagent include a second ion buffer, an α-L-fucosidase reaction substrate, a second surfactant and a second preservative; the reagent of the present application has the advantages of good stability, long shelf life, strong anti-interference ability and high sensitivity, and can improve the anti-hemoglobin interference ability of the α-L-fucosidase detection reagent, has a good detection effect on low-value samples, and is helpful for the auxiliary diagnosis of autosomal recessive hereditary diseases of α-L-fucosidase. DETAILED DESCRIPTION

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs; the terms used in the specification of the application herein are only for the purpose of describing specific embodiments and are not intended to limit this application; the terms "including" and "having" and any variations thereof in the specification and claims of this application are intended to cover non-exclusive inclusions. The terms "first", "second", etc. in the specification and claims of this application are used to distinguish different objects, not to describe a specific order.

[0035] Reference to "embodiments" herein means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present application. The appearance of the phrase in various locations in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those skilled in the art that the embodiments described herein may be combined with other embodiments.

[0036] In order to enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below.

[0037] The present application provides a reagent for detecting α-L-fucosidase, comprising independent R1 reagent and R2 reagent.

[0038] The components of the R1 reagent include a first ion buffer, a metal ion complex, a first surfactant and a first preservative; the components of the R2 reagent include a second ion buffer, an α-L-fucosidase reaction substrate, a second surfactant and a second preservative.

[0039] The first ion buffer and the second ion buffer can stabilize the pH value of the reagent within a certain range and improve the buffering capacity. The metal ion complex is an important chelating agent that can chelate metal ions in the solution to form a soluble chelate without producing any precipitation, thereby improving the antioxidant property of the solvent. The first surfactant and the second surfactant have wetting, emulsifying and solubilizing effects. The first preservative and the second preservative can inhibit the growth of microorganisms in the reagent and extend the shelf life of the reagent. The α-L-fucosidase reaction substrate has the property of allosterically activating the α-L-fucosidase reaction, which can improve the sensitivity of the reagent.

[0040] In some embodiments, the first ion buffer is selected from at least one of phosphate buffer solution, Tris buffer solution and citric acid buffer solution; the metal ion complex is at least one of disodium ethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid (EDTA) and disodium nitrilotriacetic acid; the first surfactant is at least one of Triton X-100, alkoxypolyethylene glycol hydroxyethanol (15-S-9), dodecyl polyethylene glycol ether (Brij35) or a combination of 15-S-9 and Brij35; the first preservative is at least one of sodium azide, ProClin300, kanamycin sulfate and ampicillin.

[0041] Preferably, the first ion buffer is a combination of a phosphate buffer solution and a citric acid buffer solution. The combination of the two buffers can stabilize the pH value of the solution within a certain range, and the buffering capacity is also stronger, which plays a key role in stabilizing the activity of the enzyme.

[0042] It should be noted that the pH value of the first ion buffer can be adjusted using various known pH adjusters.

[0043] The metal ion complex is disodium ethylenediaminetetraacetate, which can react with Mg 2+ , Ca 2+ , Mn 2+ , Fe 2+ Combines with divalent metal ions to form soluble chelates.

[0044] In this embodiment, Triton X-100, 15-S-9 and Brij35 are non-ionic surfactants with good solubilization, low irritation, washing, antistatic, calcium soap dispersion and other properties. They are low in irritation and have excellent wetting and washing functions. Within a certain concentration range, they can be fully dissolved in various surfactants and have a significant thickening effect.

[0045] In some embodiments, the concentration of the first ion buffer is 50-300 mmol / L, and the pH value is 7.2-7.4; the concentration of the metal ion complex is 0.1-5 g / L; the concentration of the first surfactant is 1-5 g / L; and the concentration of the first preservative is 0.5-5 g / L.

[0046] In some embodiments, the second ion buffer is selected from at least one of phosphate buffer solution, Tris buffer solution and citrate buffer solution; the α-L-fucosidase reaction substrate is at least one of MG-2-chloro-4-nitrobenzene-α-L-fucoside, novel 2-chloro-4-nitrophenyl-β-D-pyranofucoidosylglycoside and 4-nitrophenyl-α-L-fucopyranoside; the second surfactant is polyoxyethylene lauryl ether; and the second preservative is at least one of sodium azide and ProClin300.

[0047] In some embodiments, the second ion buffer is 50-300 mmol / L, and the pH value is 7.2-7.4; the concentration of the α-L-fucosidase reaction substrate is 2-5 g / L; the concentration of the second surfactant is 0.5-1 g / L; and the concentration of the second preservative is 0.5-1 g / L.

[0048] In some embodiments, the volume ratio of reagent R1 to reagent R2 is 3:1, which can further improve the sensitivity of α-L-fucosidase detection.

[0049] The above reagent for detecting α-L-fucosidase has the advantages of good stability, long shelf life, strong anti-interference ability and high sensitivity. It has a good detection effect on low-value samples and is helpful for the auxiliary diagnosis of autosomal recessive genetic diseases of α-L-fucosidase.

[0050] The present application also provides a kit for detecting α-L-fucosidase, comprising the reagent, calibrator and quality control product for detecting α-L-fucosidase as described above.

[0051] The calibrator and the quality control product are used to construct a detection procedure for α-L-fucosidase, and the components of the calibrator and the quality control product are independently α-L-fucosidase.

[0052] The following is a more specific description of the content of the present application in conjunction with specific embodiments, and further elaboration of the present application, but these embodiments are by no means limiting the present application.

[0053] Example 1

[0054] This example is a kit for detecting α-L-fucosidase provided by the present application.

[0055] The components of reagent R1 are shown in Table 1.

[0056] Table 1

[0057] Components concentration Disodium hydrogen phosphate dodecahydrate 200mmol / L Triton X-100 3g / L Ethylenediaminetetraacetic acid disodium salt 0.5g / L Sodium Azide 0.5g / L

[0058] The components of R2 reagent are shown in Table 2.

[0059] Table 2

[0060]

[0061]

[0062] Example 2

[0063] This example is another kit for detecting α-L-fucosidase provided by the present application. The components of the R1 reagent are shown in Table 3.

[0064] Table 3

[0065] Components concentration Tris 200mmol / L 15-S-9 3g / L Ethylenediaminetetraacetic acid disodium salt 0.5g / L Sodium Azide 0.5g / L

[0066] The components of R2 reagent are shown in Table 4.

[0067] Table 4

[0068]

[0069] Example 3

[0070] This example is another kit for detecting α-L-fucosidase provided by the present application. The components of the R1 reagent are shown in Table 5.

[0071] Table 5

[0072] Components concentration Disodium hydrogen phosphate dodecahydrate 200mmol / L Brij35 3g / L Ethylenediaminetetraacetic acid disodium salt 0.5g / L Sodium Azide 0.5g / L

[0073] The components of R2 reagent are shown in Table 6.

[0074] Table 6

[0075]

[0076] Example 4

[0077] This example is a kit for detecting α-L-fucosidase provided by the present application. The components of the R1 reagent are shown in Table 7.

[0078] Table 7

[0079]

[0080]

[0081] The components of R2 reagent are shown in Table 8.

[0082] Table 8

[0083]

[0084] The test principle is that the α-L-fucosidase in the sample acts on the α-L-fucosidase reaction substrate to generate 4-nitrophenol compounds. The 4-nitrophenol compounds have strong absorption at a wavelength of 405nm. By detecting the 4-nitrophenol generation rate (absorbance increase rate), the AFU activity in the sample can be determined, that is, the AFU content can be obtained.

[0085] The calculation method is as follows:

[0086]

[0087] Establish a test procedure, the test volume ratio of the above reagent R1 and reagent R2 is 3:1, enter the AFU parameters in the fully automatic biochemical analyzer, taking Hitachi 7180 as an example:

[0088] Table 9 Test procedure parameters

[0089]

[0090]

[0091] During the test, the amounts of R1 reagent, R2 reagent, quality control product, standard product and sample used are shown in Table 10.

[0092] Table 10

[0093]

[0094]

[0095] Subsequently, after the inspection procedure is initially set, the quality control solution in the quality control tube is tested, and the experimental results are measured through the above steps. The experimental results are compared with the quality control target values. If they are within the range, the inspection procedure is correct.

[0096] The performance tests of the kits of Examples 1-4 were performed, including blank absorbance, analytical sensitivity, blank limit, linearity, repeatability, and anti-interference experiments.

[0097] 1. The test performance indicators are as follows:

[0098] 1.1 Reagent Blank Absorbance

[0099] The reagent blank absorbance should be ≤0.3000.

[0100] 1.2 Analytical sensitivity

[0101] When testing samples with a concentration of 30.0U / L, the absorbance change rate (ΔA / min) should be ≥0.0050.

[0102] 1.3 Blank limit

[0103] The limit of blank (LoB) should be ≤1.0U / L.

[0104] 1.4 Linearity

[0105] The linear range of the kit is [5.0, 300.0] U / L and meets the following requirements:

[0106] a) The linear correlation coefficient (r) of the kit should be ≥ 0.990;

[0107] b) The absolute linear deviation in the range of [5.0, 50.0]U / L should not exceed ±5.0U / L;

[0108] c) The relative linear deviation in the range of (50.0, 300.0]U / L should not exceed ±10.0%.

[0109] 1.5 Repeatability

[0110] The intra-assay coefficient of variation (CV) should be ≤5.0%.

[0111] 1.6 Interference

[0112] When hemoglobin ≤2g / L, the relative deviation shall not exceed ±10.0%.

[0113] 2. Test results

[0114] (1) Blank absorbance

[0115] Use the reagent to test purified water, record the absorbance when the test is started (A1) and the absorbance after 5 minutes (t) (A2), measure twice, and take the latter A2 test result as the reagent blank absorbance measurement value. The result should meet the requirements of 1.1.

[0116] Table 11 Blank absorbance test results

[0117]

[0118] The results showed that the blank absorbance of the reagent in Example 1 was 0.0467, and the blank absorbance of the reagent in Example 2 was 0.0465. The blank absorbances were both < 0.3000, so the blank absorbances met the design requirements.

[0119] (2) Analytical sensitivity

[0120] Use the reagent to test the sample with a concentration of 30.0U / L±10.0U / L, measure once, and record the absorbance change produced by the reagent under the specified parameters. Convert it to the absorbance change rate (ΔA / min) of 30.0U / L α-L-fucosidase according to the formula, and the result should meet the requirements of 2.5.

[0121] The calculation formula is as follows:

[0122]

[0123] Where:

[0124] ΔA / min—absorbance change rate;

[0125] ΔA0 / min—absorbance change rate of the sample being tested;

[0126] X—concentration of the sample being tested.

[0127] Table 12 Analytical sensitivity test results

[0128] Test concentration <![CDATA[A1]]> <![CDATA[A2]]> <![CDATA[△A0]]> Conversion concentration △A △A / min Example 1 30.5 0.0665 0.0853 0.0188 30.0 0.0185 0.0090 Example 2 30.5 0.0654 0.0808 0.0154 30.0 0.0151 0.0074 Example 3 30.5 0.0665 0.0822 0.0157 30.0 0.0154 0.0075 Example 4 30.5 0.0677 0.0847 0.0170 30.0 0.0167 0.0081

[0129] The results show that the analytical sensitivity of the reagent in Example 1 is 0.0090, the analytical sensitivity of the reagent in Example 2 is 0.0074, the analytical sensitivity of the reagent in Example 3 is 0.0075, and the analytical sensitivity of the reagent in Example 2 is 0.0081. The analytical sensitivities are all greater than 0.0050, so the analytical sensitivities meet the design requirements.

[0130] (3) Blank limit

[0131] The test was repeated 20 times using purified water as the sample.

[0132] Table 13 Blank limit test results

[0133]

[0134]

[0135] The results show that the blank limit of the reagent in Example 1 is 0.6U / L, the blank limit of the reagent in Example 2 is 0.6U / L, the blank limit of the reagent in Example 3 is 0.4U / L, and the blank limit of the reagent in Example 4 is 0.6U / L. The blank limit (LoB) is all <1.0U / L, so the blank limit (LoB) meets the design requirements.

[0136] (4) Linear range

[0137] A low-concentration sample of 5.0 U / L, which is close to the lower limit of the linear interval, and a high-concentration sample of 300.0 U / L, which is close to the upper limit of the linear interval, were mixed in a certain proportion to form 5 concentration samples.

[0138] The mixing ratio is as follows:

[0139] Table 14 Sample Mixing Ratio

[0140] Sample No. 1 2 3 4 5 Low concentration sample (mL) 1.0 0.9 0.6 0.2 0.0 High concentration sample (mL) 0.0 0.1 0.4 0.8 1.0

[0141] Table 15 Linear range experimental results

[0142]

[0143]

[0144] The results showed that the linear correlation coefficient of the kit of Example 1 was 0.9989, the linear correlation coefficient of the kit of Example 2 was 0.9985, the linear correlation coefficient of the kit of Example 3 was 0.9985, and the linear correlation coefficient of the kit of Example 4 was 0.9991. The correlation coefficients were all > 0.990, so the linear range met the design requirements.

[0145] (5) Repeatability

[0146] The sample with a concentration of (45.0±15.0) U / L was tested and repeated 10 times to calculate the coefficient of variation (CV).

[0147] Table 16 Repeatability test results

[0148]

[0149] The results showed that the CV of the kit of Example 1 was 2.8%, the linear CV of the kit of Example 2 was 3.4%, the CV of the kit of Example 3 was 3.1%, and the linear CV of the kit of Example 4 was 1.6%. The CVs were all less than 5.0%, so the repeatability met the design requirements.

[0150] (6) Anti-interference experiment

[0151] The α-L-fucosidase detection kit was used to test hemoglobin, a common interfering substance in clinical practice, and the interfering substance was configured into five concentration gradients for interference experiments.

[0152] Table 17 Experimental results

[0153]

[0154]

[0155] The results show that the maximum relative deviation of the reagent in Example 1 is -35.1%, the maximum relative deviation of the reagent in Example 2 is -17.9%, the maximum relative deviation of the reagent in Example 3 is -21.4%, and the maximum relative deviation of the reagent in Example 4 is -8.9%. The relative deviation of Example 4 does not exceed ±10.0%, so the interference of the reagent in Example 4 meets the design requirements.

[0156] Obviously, the embodiments described above are only a part of the embodiments of the present application, rather than all the embodiments, but do not limit the patent scope of the present application. The present application can be implemented in many different forms. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive. Although the present application is described in detail with reference to the aforementioned embodiments, for those skilled in the art, it is still possible to modify the technical solutions recorded in the aforementioned specific implementation methods, or to perform equivalent replacement of some of the technical features therein. Any equivalent structure made using the specification of this application, directly or indirectly used in other related technical fields, is similarly within the scope of patent protection of this application.

Claims

1. A reagent for detecting α-L-fucosidase, characterized in that: include: Independent R1 reagent and R2 reagent; Wherein, the components of the R1 reagent include a first ion buffer, a metal ion complex, a first surfactant and a first preservative; The components of the R2 reagent include a second ion buffer, an α-L-fucosidase reaction substrate, a second surfactant and a second preservative.

2. The reagent for detecting α-L-fucosidase according to claim 1, characterized in that: The first ion buffer is selected from at least one of a phosphate buffer solution, a Tris buffer solution and a citric acid buffer solution; The metal ion complex is at least one of disodium ethylenediaminetetraacetate, ethylenediaminetetraacetic acid and disodium nitrilotriacetate; The first surfactant is at least one of Triton X-100, alkoxy polyethylene hydroxyethanol, and dodecyl polyethylene glycol ether, or a combination of alkoxy polyethylene hydroxyethanol and dodecyl polyethylene glycol ether; The first preservative is at least one of sodium azide, ProClin300, kanamycin sulfate and ampicillin.

3. The reagent for detecting α-L-fucosidase according to claim 1, characterized in that: The concentration of the first ion buffer is 50 to 300 mmol / L, and the pH value is 7.2 to 7.4; The concentration of the metal ion complex is 0.1 to 5 g / L; The concentration of the first surfactant is 1 to 5 g / L; The concentration of the first preservative is 0.5-1 g / L.

4. The reagent for detecting α-L-fucosidase according to claim 1, characterized in that: The second ion buffer is selected from at least one of a phosphate buffer solution, a Tris buffer solution and a citric acid buffer solution; The α-L-fucosidase reaction substrate is at least one of MG-2-chloro-4-nitrobenzene-α-L-fucoside, novel 2-chloro-4-nitrophenyl-β-D-pyranofucoidosylglycoside and 4-nitrophenyl-α-L-fucoside; The second surfactant is polyoxyethylene lauryl ether; The second preservative is at least one of sodium azide and ProClin300.

5. The reagent for detecting α-L-fucosidase according to claim 1, characterized in that: The second ion buffer has a pH value of 50 to 300 mmol / L and a pH value of 7.2 to 7.4; The concentration of the α-L-fucosidase reaction substrate is 2 to 5 g / L; The concentration of the second surfactant is 1 to 5 g / L; The concentration of the second preservative is 0.5-1 g / L.

6. The reagent for detecting α-L-fucosidase according to any one of claims 1 to 5, characterized in that: The volume ratio of the R1 reagent to the R2 reagent is 3:

1.

7. A kit for detecting α-L-fucosidase, characterized in that: Comprising the reagent, calibrator and quality control product for detecting α-L-fucosidase according to any one of claims 1 to 6; The calibrator and the quality control product are used to construct a detection procedure for α-L-fucosidase, and the components of the calibrator and the quality control product are independently α-L-fucosidase.

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