α-L-fucosidase detection reagent or kit and preparation method thereof

Abstract

The present invention relates to the field of biological preparation technology, and more particularly to a kind of α-L-fucosidase detection reagent or kit and its preparation method. Which include: substrate, buffer solution with pH 4.5~5.5, metal ion chelator, inorganic compound containing metal ion, glycerol, Gemini surfactant, Tween and PEG. The present invention provides an α-L-fucosidase single reagent that improves substrate stability and resists the influence of multiple interfering substances. The single reagent can be stably stored for at least 20 months at 2-8 ° C, effectively removes the interference of multiple serum interfering substances such as bound bilirubin, free bilirubin, vitamin C, chyle and hemoglobin, and has high accuracy. Moreover, the single reagent of the present invention is convenient, simple and easy to operate, reduces the use of instrument reagent positions, and reduces detection costs.

Description

Technical Field

[0001] The present invention relates to the technical field of biological preparations, and in particular to an α-L-fucosidase detection reagent or kit and a preparation method thereof. Background Art

[0002] α-L-fucosidase (AFU) is a lysosomal acid hydrolase widely present in human tissues, cellular lysosomes, and body fluids. It is also found in the placenta, fetal tissue, brain, lung, liver, pancreas, kidney, as well as serum, urine, saliva, and tears. AFU hydrolyzes α-1,2, α-1,3, α-1,4, and α-1,6 glycosidic bonds. Its physiological function is to participate in the catabolism of various fucosidic glycolipids, glycoproteins, mucopolysaccharides, and other macromolecules. Its primary clinical significance is that serum AFU levels are significantly elevated in patients with liver cancer, leading it to be considered a new marker for primary hepatocellular carcinoma (PHC). Serum AFU activity in patients with primary HCC is significantly higher than that in normal controls and is also significantly higher than in patients with metastatic liver cancer, cirrhosis, cholangiocarcinoma, malignant mesothelioma, malignant hemangioendothelioma, congenital hepatic cysts, and other benign liver space-occupying lesions. AFU is generally considered to have a higher sensitivity than alpha-fetoprotein (AFP) but a lower specificity. While there is no significant correlation between AFU and AFP, combined monitoring of the two can improve the detection rate of liver cancer, particularly for AFP-negative patients and small cell liver cancer. AFU is also an ideal indicator for postoperative monitoring and follow-up of PHC. Its changes parallel the severity of the disease and precede clinical manifestations by 1-2 months, making it a useful indicator for assessing PHC efficacy and prognosis.

[0003] There are three methods for determining AFU in serum: fluorescence, colorimetry, and rate. The first two are difficult to automate and are rarely used. The rate method is the most commonly used method in combination with a fully automatic biochemical analyzer. The detection principle is: AFU in serum catalyzes the hydrolysis of 2-chloro-p-nitrophenol-α-L-fucopyranoside (CNPF) substrate to generate 2-chloro-p-nitrophenol (CNP), causing an increase in absorbance at a wavelength of 405 nm. The rate of absorbance increase is proportional to the activity of AFU in the sample.

[0004] The pH range for AFU-catalyzed substrate hydrolysis is 4.1–6.2, with an optimal pH of 5.0, at which enzyme activity is highest. However, the AFU-catalyzed substrate, CNPF, is relatively unstable in acidic aqueous solutions and easily hydrolyzed by acid. Consequently, commonly used commercially available reagents for AFU determination are dual-reagent reagents, which are cumbersome to use and occupy excessive instrument reagent spaces. Furthermore, existing reagents have poor anti-interference properties, and interfering substances such as bound bilirubin, free bilirubin, vitamin C, hemoglobin, and chyle can significantly impact sample test values.

[0005] Therefore, how to provide a single reagent of α-L-fucosidase that improves substrate stability and resists the influence of multiple interfering substances has become a technical problem that urgently needs to be solved in this field. Summary of the Invention

[0006] In order to solve the above technical difficulties, first, the present invention provides an α-L-fucosidase detection reagent (single reagent) or kit, comprising: an α-L-fucosidase activity assay substrate, a buffer solution with a pH of 4.5 to 5.5, a metal ion chelator, an inorganic compound containing metal ions, a protective agent, a surfactant and PEG; the protective agent is glycerol, and the surfactant is a Gemini surfactant and Tween.

[0007] The present invention discovered that a surfactant compounded from a Gemini surfactant and Tween, combined with the protective agent glycerol, can effectively slow substrate acidolysis, thereby improving substrate stability. Furthermore, the present invention utilizes a buffer solution with a pH of 4.5 to 5.5, effectively increasing the enzyme reaction rate. This ensures both the optimal pH for the enzyme reaction in serum and substrate stability. Furthermore, the reagent or kit of the present invention incorporates PEG to provide resistance to various interfering substances, including chyle; the metal ion-containing inorganic compound in the reagent or kit acts as an enzyme activator.

[0008] Preferably, the buffer is any one of glycine-sodium hydroxide buffer, disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer, disodium hydrogen phosphate-citric acid buffer, citric acid-sodium citrate buffer, citric acid-sodium hydroxide buffer, MES-sodium hydroxide buffer, acetic acid-sodium acetate buffer, and potassium hydrogen phthalate-sodium hydroxide buffer.

[0009] Preferably, the metal ion chelating agent is at least one of EDTA, EDTA-2Na, EDTA-4Na, and NTA.

[0010] Preferably, the inorganic compound containing metal ions is at least one of potassium chloride, sodium chloride, calcium chloride, magnesium sulfate, and sodium sulfate.

[0011] Preferably, the Gemini-type surfactant is at least one of octyl polyoxyethylene hexadecyl ammonium chloride, octyl polyoxyethylene octadecyl ammonium chloride, dodecanol polyoxyethylene ether dimethyl methyl ammonium chloride, hexadecanol polyoxyethylene ether dimethyl octyl ammonium chloride, ethylene didodecyl polyoxyethylene polyoxypropylene blocked ammonium bromide, octadecylamine polyoxyethylene ether diquaternary ammonium salt, and hexadecanol polyoxyethylene ether dimethyl octyl ammonium chloride; and the Tween is Tween-20 or Tween-80.

[0012] Preferably, the molecular weight of the PEG is 1000-20000 (e.g., PEG-1000, PEG-1500, PEG-2000, PEG-4000, PEG-6000, PEG-8000, PEG20000).

[0013] Preferably, the reagent or kit further comprises a preservative.

[0014] Preferably, the preservative is at least one of sodium azide, antibiotics (such as kanamycin, gentamicin), Proclin 300, and Krovin 100; and / or the concentration of the preservative is 0.5-5 g / L.

[0015] The addition of preservatives can further improve the stability of reagents or kits and prevent the growth of microorganisms.

[0016] Preferably, the concentration of the substrate for the α-L-fucosidase activity assay is 0.2-2 g / L, the concentration of the buffer is 20-200 mmol / L, the concentration of the metal ion chelator is 0.5-5 mmol / L, the concentration of the inorganic compound containing metal ions is 10-100 mmol / L, the concentration of the protective agent is 2-20 g / L, the concentration of the surfactant is 1-10 g / L, and the concentration of PEG is 0.5-10 g / L.

[0017] Furthermore, the present invention provides a method for preparing any of the above-mentioned reagents or kits, comprising: mixing a buffer, a metal ion chelator, and an inorganic compound containing metal ions, adjusting the pH to 4.5-5.5, and then mixing with an α-L-fucosidase activity assay substrate, a protective agent, a surfactant, and PEG to prepare the reagent or kit.

[0018] In the present invention, the substrate for determining the activity of α-L-fucosidase is 2-chloro-5-nitrobenzene-α-L-fucopyranoside (CNPF) or 2-chloro-4-nitrobenzene-α-L-fucoside (MG-CNPF).

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention provides a single-agent α-L-fucosidase assay that improves substrate stability and resistance to multiple interfering substances. This single-agent can be stably stored at 2-8°C for at least 20 months, effectively removing interference from multiple serum interfering substances, such as bound bilirubin, free bilirubin, vitamin C, chyle, and hemoglobin, with high accuracy. Furthermore, the single-agent assay is convenient, simple, and easy to operate, reducing the use of instrument reagent stations and lowering testing costs. DETAILED DESCRIPTION

[0021] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be described clearly and completely below. Obviously, the embodiments described are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention. The preparation methods of all embodiments and comparative examples are the same as those of Example 1.

[0022] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0023] Example 1

[0024] This example provides a single reagent for detecting α-L-fucosidase, comprising the following components: 0.5 g / L CNPF (Shanghai Xibao Biotechnology Co., Ltd.), 100 mmol / L glycine-sodium hydroxide buffer (pH 5.0), 2 mmol / L EDTA, 50 mmol / L magnesium sulfate, 10 g / L glycerol, 0.5 g / L Gemini surfactant octadecylamine polyoxyethylene ether diquaternary ammonium salt, 2.5 g / L TWEEN-80, 1.5 g / L PEG-6000, and 2.5 g / L sodium azide, with water as the solvent.

[0025] The preparation method is as follows: a buffer solid component, a metal ion chelating agent, and an inorganic compound containing metal ions are weighed according to the above concentrations and dissolved in water, the pH is adjusted to 5.0 after mixing, and then water is added to make the volume to the above concentration, and then an α-L-fucosidase activity assay substrate, a protective agent, a surfactant PEG, and a preservative are added to prepare the reagent or kit.

[0026] Example 2

[0027] This example provides a single reagent for detecting α-L-fucosidase, the components of which differ from those of Example 1 only in that the Gemini surfactant is replaced with hexadecanol polyoxyethylene ether dimethyl octyl ammonium chloride at the same concentration.

[0028] Example 3

[0029] This example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that the Gemini surfactant is replaced with octyl polyoxyethylene hexadecyl ammonium chloride of the same concentration.

[0030] Example 4

[0031] This example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that TWEEN-80 is replaced by TWEEN-20 of the same concentration.

[0032] Example 5

[0033] This example provides a single reagent for detecting α-L-fucosidase, the components of which differ from those of Example 1 only in that the buffer is replaced with a sodium hydrogen phosphate-sodium dihydrogen phosphate buffer of the same concentration and pH value.

[0034] Example 6

[0035] This example provides a single reagent for detecting α-L-fucosidase, the components of which differ from those of Example 1 only in that the buffer is replaced with a citric acid-sodium hydroxide buffer of the same concentration and pH value.

[0036] Example 7

[0037] This example provides a single reagent for detecting α-L-fucosidase, the components of which differ from those of Example 1 only in that the buffer is replaced with an acetic acid-sodium acetate buffer of the same concentration and pH value.

[0038] Example 8

[0039] This example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that magnesium sulfate is replaced by sodium chloride of the same concentration.

[0040] Example 9

[0041] This example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that PEG is PEG-2000.

[0042] Example 10

[0043] This example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that sodium azide is replaced with kanamycin of the same concentration.

[0044] Comparative Example 1

[0045] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that glycerol is replaced by mannitol of the same concentration.

[0046] Comparative Example 2

[0047] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that glycerol is replaced by sorbitol of the same concentration.

[0048] Comparative Example 3

[0049] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that glycerol is replaced by trehalose of the same concentration.

[0050] Comparative Example 4

[0051] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that the Gemini surfactant is replaced with an alkylphenol polyoxyethylene ether quaternary ammonium salt of the same concentration.

[0052] Comparative Example 5

[0053] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those of Example 1 only in that the Gemini surfactant is replaced with TRITON X-100 of the same concentration.

[0054] Comparative Example 6

[0055] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those of Example 1 only in that TWEEN-80 is replaced by Span-80 of the same concentration.

[0056] Comparative Example 7

[0057] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those of Example 1 only in that TWEEN-80 is replaced with TRITON X-100 of the same concentration.

[0058] Comparative Example 8

[0059] This comparative example provides a single reagent for detecting α-L-fucosidase, the components of which are different from those in Example 1 only in that no PEG component is added.

[0060] Test Example 1

[0061] This test example tested the stability of the α-L-fucosidase detection reagents prepared in the above examples and comparative examples 1-7 using an accelerated stability test. The specific steps were as follows: Each of the above α-L-fucosidase detection reagents was placed in a 37°C environment. The same test sample was measured on days 0, 1, 3, 5, 7, and 14 using a fully automatic biochemical analyzer (Hitachi 7180). The test parameters were as follows: dominant wavelength 405 nm, temperature 37°C, cuvette light diameter 1 cm, ascending reaction direction, reaction time 10 min, and analysis type Rate A. 20 μL of physiological saline was added to a blank tube, 20 μL of calibrator was added to a calibration tube, and 20 μL of test sample was added to a sample tube. These were then mixed with 180 μL of each reagent and incubated at 37°C for 10 minutes. The absorbance was then read. The blank tube was zeroed, and the AFU concentration in the sample was calculated. The test was repeated three times, and the data were averaged. The relative deviation (%) of the test sample from the average value of the day 0 test was calculated. The experimental results are shown in Table 1.

[0062] Table 1

[0063]

[0064] The results showed that a combination of a Gemini surfactant and Tween surfactant, combined with the protective agent glycerol, effectively slowed substrate acidolysis, thereby improving substrate stability. The single reagent prepared in this embodiment of the present invention maintained minimal relative deviation after 14 days in an accelerated stability test at 37°C, demonstrating that the single reagent of the present invention can be stably stored at 2-8°C for 20 months.

[0065] Test Example 2

[0066] This test example evaluated the anti-interference performance of the single reagent for α-L-fucosidase detection prepared in the above examples and comparative example 8. The specific steps are as follows: different concentrations of interfering substances were added to the serum, and the above single reagent was used to measure samples without added interfering substances and samples with added interfering substances at different concentrations on a fully automatic biochemical analyzer (brand and model are the same as those in Test Example 1). The test was repeated three times and the data were averaged. The deviation (%) between the test values ​​of the samples with added interfering substances at different concentrations and the test values ​​of the samples without added interfering substances was calculated. The experimental results are shown in Table 2.

[0067] Table 2

[0068]

[0069] The results demonstrate that the single reagent of the present invention is resistant to the effects of multiple interfering substances, including chyle, and effectively removes interference from concentrations of bound bilirubin ≤40 mg / dl, free bilirubin ≤40 mg / dl, vitamin C ≤20 mg / dl, chyle ≤250 mg / dl, and hemoglobin ≤400 mg / dl, demonstrating high sensitivity. In addition to the aforementioned tests, the present invention also employed multiple other samples from different sources, achieving consistent technical results.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. 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 various embodiments of the present invention.

Claims

1. An α-L-fucosidase detection reagent or kit, characterized in that: The components are as follows: an α-L-fucosidase activity assay substrate, a buffer solution with a pH of 4.5 to 5.5, a metal ion chelating agent, an inorganic compound containing metal ions, a protective agent, a surfactant, and PEG; the protective agent is glycerol, the surfactant is a Gemini surfactant and Tween; the detection reagent or the detection reagent in the kit is a single reagent; the buffer solution is any one of a glycine-sodium hydroxide buffer solution, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, a citric acid-sodium hydroxide buffer solution, and an acetic acid-sodium acetate buffer solution; and the inorganic compound containing metal ions is magnesium sulfate or sodium chloride. The concentration of the α-L-fucosidase activity assay substrate was 0.5 g / L, the concentration of the buffer was 100 mmol / L, the concentration of the metal ion chelator was 2 mmol / L, the concentration of the metal ion-containing inorganic compound was 50 mmol / L, the concentration of the protective agent was 10 g / L, the concentration of the Gemini surfactant was 0.5 g / L, the concentration of Tween was 2.5 g / L, and the concentration of PEG was 1.5 g / L.

2. The reagent or kit according to claim 1, characterized in that The metal ion chelating agent is at least one of EDTA, EDTA-2Na, EDTA-4Na, and NTA.

3. The reagent or kit according to claim 1, characterized in that The Gemini-type surfactant is at least one of octyl polyoxyethylene hexadecyl ammonium chloride, octyl polyoxyethylene octadecyl ammonium chloride, dodecanol polyoxyethylene ether dimethyl methyl ammonium chloride, hexadecanol polyoxyethylene ether dimethyl octyl ammonium chloride, ethylene didodecyl polyoxyethylene polyoxypropylene blocked ammonium bromide, octadecylamine polyoxyethylene ether diquaternary ammonium salt, and hexadecanol polyoxyethylene ether dimethyl octyl ammonium chloride; the Tween is Tween-20 or Tween-80.

4. The reagent or kit according to claim 1, characterized in that The molecular weight of the PEG is 1000-20000.

5. The reagent or kit according to claim 1, characterized in that The reagents or kits are added with preservatives.

6. The reagent or kit according to claim 5, characterized in that The preservative is at least one of sodium azide, antibiotics, Proclin 300, and Krovin 100; and / or the concentration of the preservative is 0.5-5 g / L.

7. The method for preparing the reagent or kit according to any one of claims 1 to 6, characterized in that: include: A buffer solution, a metal ion chelating agent, and an inorganic compound containing metal ions are mixed, the pH value is adjusted to 4.5-5.5, and then the mixture is mixed with an α-L-fucosidase activity assay substrate, a protective agent, a surfactant, and PEG to prepare the reagent or kit.

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