Reagent kit for determination of alpha-fetoprotein content by nephelometry

Abstract

The application discloses a kit for determining alpha-fetoprotein content by a scattering turbidimetric method, which comprises reagent 1 and reagent 2; the reagent 1 comprises 0.02wt%-0.1wt% lipid substance blocking agent, 5mmol / L-50mmol / L buffer, 0.5g / L-10g / L surfactant and 0.5-5g / L preservative; and the reagent 2 comprises 0.1w / v%-1w / v% anti-alpha-fetoprotein antibody latex microspheres. The kit for determining alpha-fetoprotein content by the scattering turbidimetric method comprises 0.02wt%-0.1wt% lipid substance blocking agent, 5mmol / L-50mmol / L buffer, 0.5g / L-10g / L surfactant and 0.5-5g / L preservative, the lipid substance blocking agent can combine with lipid substance to form a molecular complex, thereby reducing the concentration of lipid substance in the sample, and effectively eliminating the interference of the lipid substance, so that the test result is more accurate. In combination with specific embodiments, the kit for determining alpha-fetoprotein content by the scattering turbidimetric method is more accurate in detection.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostic reagent technology, and in particular to a kit for determining alpha-fetoprotein levels using a turbidimetric method. Background Technology

[0002] Alpha-fetoprotein (AFP) is currently the most widely used serum biomarker for the auxiliary diagnosis of liver cancer in clinical practice. AFP is mainly divided into three types: AFP-L1 is mainly derived from benign hepatocytes; AFP-L2 is mostly produced by yolk sac tumors and originates from pregnant women; and AFP-L3 is mainly derived from cancerous hepatocytes. AFP-L3 is a highly specific indicator for liver cancer, with a specificity exceeding 95%, making it the most specific tumor marker for diagnosing liver cancer. The ratio of serum AFP-L3 to total AFP is referred to as the AFP-L3% ratio.

[0003] The turbidimetric method determines the detection endpoint based on the change in scattered light during the coagulation process of a sample. In this method, the monochromatic light source in the detection channel is at a 90° angle to the photodetector. When a coagulation activator is added to the sample, the intensity of scattered light gradually increases as fibrin clots form. Once the sample is completely coagulated, the intensity of scattered light no longer changes. Typically, the coagulation start point is taken as 0%, the coagulation end point as 100%, and 50% is taken as the coagulation time. The photodetector receives this optical change, converts it into an electrical signal, amplifies it, and transmits it to the monitor for processing, plotting the coagulation curve.

[0004] When turbidimetric assay is used to determine alpha-fetoprotein (AFP) levels, lipids in the sample can interfere with the detection process, leading to a decrease in the accuracy of the test results. Summary of the Invention

[0005] Therefore, it is necessary to provide a kit for determining alpha-fetoprotein content by turbidimetric scattering that can solve the above problems.

[0006] A kit for determining alpha-fetoprotein levels using a turbidimetric method, comprising reagent 1 and reagent 2;

[0007] The reagent 1 comprises 0.02wt% to 0.1wt% of a lipid blocking agent, 5mmol / L to 50mmol / L of a buffer, 0.5g / L to 10g / L of a surfactant, and 0.5g / L to 5g / L of a preservative.

[0008] The reagent 2 comprises 0.1 w / v% to 1 w / v% of anti-alpha-fetoprotein antibody latex microspheres.

[0009] In one embodiment, the lipid blocker is a phosphodiesterase.

[0010] In one embodiment, the lipid blocker is selected from at least one of soybean phosphodiesterase, Gynostemma pentaphyllum phosphodiesterase, and ginseng phosphodiesterase.

[0011] In one embodiment, reagent 2 further includes a stabilizer of 0.1 g / L to 5 g / L.

[0012] In one embodiment, the stabilizer is selected from at least one of Tritonx-100, D-mannitol, and isopropanol.

[0013] In one embodiment, reagent 2 further includes 5 mmol / L to 50 mmol / L of the buffer, 0.5 g / L to 10 g / L of the surfactant, and 0.5 to 5 g / L of the preservative.

[0014] In one embodiment, the buffer is selected from at least one of PBS buffer, Tris buffer, glycine buffer, BS buffer, HEPES buffer, MOPSO buffer, and MES buffer;

[0015] The surfactant is selected from at least one of polyvinylpyrrolidone, glycerol, Triton 100, PEG6000, PEG8000, PEG10000, PEG20000, Tween-20, Tween-40 and Tween-80;

[0016] The preservative is selected from at least one of sodium azide, Proclin-950, Proclin-300, krovin100, krovin300, krovin500, krovin750 and thimerosal.

[0017] In one embodiment, the anti-alpha-fetoprotein antibody latex microspheres are anti-alpha-fetoprotein isoform 1 antibody latex microspheres, anti-alpha-fetoprotein isoform 2 antibody latex microspheres, or anti-alpha-fetoprotein isoform 3 antibody latex microspheres.

[0018] In one embodiment, the pH of both reagent 1 and reagent 2 is 6.0 to 9.0.

[0019] In one embodiment, the volume ratio of reagent 1 to reagent 2 is 1:1 to 5:1.

[0020] The reagent 1 of the kit for determining alpha-fetoprotein content by turbidimetric scattering of the present invention comprises 0.02wt% to 0.1wt% of a lipid blocking agent, 5mmol / L to 50mmol / L of a buffer, 0.5g / L to 10g / L of a surfactant, and 0.5g / L to 5g / L of a preservative. The lipid blocking agent can bind with lipids to form molecular complexes, thereby reducing the concentration of lipids in the sample and effectively eliminating the interference of lipids, thus improving the accuracy of the test results.

[0021] In conjunction with specific embodiments, the kit for determining alpha-fetoprotein content using the turbidimetric method of the present invention has better detection accuracy.

[0022] The kit for determining alpha-fetoprotein (AFP) content by turbidimetric scattering of the present invention has a simple composition. It adds a lipid blocking agent to bind with lipids to form a molecular complex, thereby reducing the concentration of lipids in the sample and effectively eliminating the interference of lipids. It has the advantages of high accuracy, good stability and wide linear range.

[0023] Preferably, the lipid blocking agent is a phosphodiesterase. Phosphodiesterase can bind to lipids to form water-insoluble molecular complexes, thereby inhibiting the activity of lipids and eliminating interference from lipids in the sample in the test results of the kit, thus improving the accuracy of the test results.

[0024] Preferably, reagent 2 further includes a stabilizer at a concentration of 0.1 g / L to 5 g / L. The stabilizer must be a hydrophilic substance that does not dissociate when dissolved in water, while increasing the viscosity of the aqueous solution to protect the latex reagent.

[0025] Preferably, the stabilizer is selected from at least one of Tritonx-100, D-mannitol, and isopropanol.

[0026] Tritonx-100, D-mannitol, and isopropanol are nonionic surfactants. Their hydrophilic groups are mainly formed by hydrogen bonds between a certain number of oxygen-containing groups (ether groups or hydroxyl groups) and water, which increases the viscosity of the aqueous solution and protects the latex reagent. Therefore, adding Tritonx-100, D-mannitol, and isopropanol as stabilizers can make the test results of reagent 2 more stable. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] It should be noted that all directional indications (such as up, down, left, right, front, back, etc.) in the embodiments of the present invention are only used to explain the relative positional relationship and movement of each component in a specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0029] Furthermore, the use of terms such as "first" and "second" in this invention is for descriptive purposes only and should not be construed as indicating or implying their relative importance or implicitly specifying the number of technical features indicated. Therefore, features defined with "first" and "second" may explicitly or implicitly include at least one of the stated features. Additionally, the technical solutions of the various embodiments can be combined with each other, but only on the basis of being achievable by those skilled in the art. When the combination of technical solutions is contradictory or impossible to implement, such a combination of technical solutions should be considered non-existent and not within the scope of protection claimed by this invention.

[0030] This invention discloses a kit for determining alpha-fetoprotein content by turbidimetric scattering according to one embodiment, comprising reagent 1 and reagent 2.

[0031] Reagent 1 includes 0.02wt% to 0.1wt% of a lipid blocking agent, 5mmol / L to 50mmol / L of a buffer, 0.5g / L to 10g / L of a surfactant, and 0.5g / L to 5g / L of a preservative.

[0032] Reagent 2 consists of 0.1 w / v% to 1 w / v% anti-alpha-fetoprotein antibody latex microspheres.

[0033] The reagent 1 of the kit for determining alpha-fetoprotein content by turbidimetric scattering of the present invention comprises 0.02wt% to 0.1wt% of a lipid blocking agent, 5mmol / L to 50mmol / L of a buffer, 0.5g / L to 10g / L of a surfactant, and 0.5g / L to 5g / L of a preservative. The lipid blocking agent can bind with lipids to form molecular complexes, thereby reducing the concentration of lipids in the sample and effectively eliminating the interference of lipids, thus improving the accuracy of the test results.

[0034] In conjunction with specific embodiments, the kit for determining alpha-fetoprotein content using the turbidimetric method of the present invention has better detection accuracy.

[0035] The kit for determining alpha-fetoprotein (AFP) content by turbidimetric scattering of the present invention has a simple composition. It adds a lipid blocking agent to bind with lipids to form a molecular complex, thereby reducing the concentration of lipids in the sample and effectively eliminating the interference of lipids. It has the advantages of high accuracy, good stability and wide linear range.

[0036] Preferably, the lipid blocking agent is phosphodiesterase. Phosphodiesterase can bind to lipids to form water-insoluble molecular complexes, thereby inhibiting the activity of lipids. This eliminates interference from lipids in the sample on the test results of the kit, resulting in better accuracy of the test results.

[0037] Specifically, the lipid blocker is selected from at least one of soybean phosphodiesterase, Gynostemma pentaphyllum phosphodiesterase, and ginseng phosphodiesterase.

[0038] Preferably, in this embodiment, reagent 2 further includes a stabilizer at a concentration of 0.1 g / L to 5 g / L. The stabilizer must be a hydrophilic substance that does not dissociate when dissolved in water, while simultaneously increasing the viscosity of the aqueous solution to protect the latex reagent.

[0039] More preferably, in this embodiment, the stabilizer is selected from at least one of Tritonx-100, D-mannitol, and isopropanol.

[0040] Tritonx-100, D-mannitol, and isopropanol are nonionic surfactants. Their hydrophilic groups are mainly formed by hydrogen bonds between a certain number of oxygen-containing groups (ether groups or hydroxyl groups) and water, which increases the viscosity of the aqueous solution and protects the latex reagent. Therefore, adding Tritonx-100, D-mannitol, and isopropanol as stabilizers can make the test results of reagent 2 more stable.

[0041] Preferably, in this embodiment, reagent 2 further includes 5 mmol / L to 50 mmol / L of the buffer, 0.5 g / L to 10 g / L of the surfactant, and 0.5 to 5 g / L of the preservative.

[0042] In this embodiment, the buffer is selected from at least one of PBS buffer, Tris buffer, glycine buffer, BS buffer, HEPES buffer, MOPSO buffer, and MES buffer.

[0043] In this embodiment, the surfactant is selected from at least one of polyvinylpyrrolidone, glycerol, Triton 100, PEG6000, PEG8000, PEG10000, PEG20000, Tween-20, Tween-40 and Tween-80.

[0044] In this embodiment, the preservative is selected from at least one of sodium azide, Proclin-950, Proclin-300, krovin100, krovin300, krovin500, krovin750 and thimerosal.

[0045] Specifically, the anti-alpha-fetoprotein antibody latex microspheres are anti-alpha-fetoprotein isoform 1 antibody latex microspheres, anti-alpha-fetoprotein isoform 2 antibody latex microspheres, or anti-alpha-fetoprotein isoform 3 antibody latex microspheres.

[0046] Preferably, in this embodiment, the pH of both reagent 1 and reagent 2 is 6.0 to 9.0.

[0047] Preferably, in this embodiment, the volume ratio of reagent 1 to reagent 2 is 1:1 to 5:1.

[0048] The following are specific examples.

[0049] In the specific embodiment, PBS buffer, Tween-20, ProClin300, phosphodiesterase, isopropanol, Tritonx-100, and D-mannitol were all purchased from Sinopharm Group; anti-human alpha-fetoprotein isoform 3 antibody latex microspheres were purchased from Agilent Technologies, with a particle size of 60 nm to 220 nm.

[0050] Example 1

[0051] Prepare six sets of test reagents (1-1, 1-2, 1-3, 1-4, 1-5, and 1-6) according to Table 1 below.

[0052] Table 1

[0053]

[0054]

[0055] Different amounts of triglycerides (lipids) were added to serum samples containing 2.1 ng / mL alpha-fetoprotein isoform 3 (from a hospital in Shenzhen) to prepare solutions containing 0, 2 g / L, 4 g / L, 6 g / L, and 8 g / L of lipids, which were named Sample 1-1, Sample 1-2, Sample 1-3, Sample 1-4, and Sample 1-5, respectively.

[0056] Using the above six sets of test reagents, samples 1-1, 1-2, 1-3, 1-4, and 1-5 were tested using the Shine i2910 instrument to determine the effect of lipid blocker concentration on the results. The test results are shown in Table 2 below.

[0057] Table 2: Effect of different phosphodiesterase concentrations

[0058]

[0059] Referring to Table 2, it can be seen that the test results of Reagent 1 without added lipid blockers increase with the increase of lipid addition, changing from 2.1 ng / mL in the sample without added lipids to 5.65 ng / mL. For Reagent 1 with a lipid blocker concentration greater than or equal to 0.2 g / L, the lipid addition in the samples tested with Reagent 2 does not change with the increase of lipid addition, indicating that the blocker is effective.

[0060] Example 2

[0061] According to Table 3 below, prepare seven sets of test reagents: 2-1, 2-2, 2-3, 2-4, 2-5, 2-6, and 2-7.

[0062] Table 3

[0063]

[0064]

[0065]

[0066] Different amounts of triglycerides (lipids) were added to serum samples containing 2.1 ng / mL alpha-fetoprotein isoform 3 (from a hospital in Shenzhen) to prepare solutions containing 0, 2 g / L, 4 g / L, 6 g / L, and 8 g / L of lipids, which were named Sample 2-1, Sample 2-2, Sample 2-3, Sample 2-4, and Sample 2-5, respectively.

[0067] Using the seven sets of test reagents mentioned above, samples 2-1, 2-2, 2-3, 2-4, and 2-5 were tested using the Shine i2910 instrument to determine the effect of lipid blocker concentration on the results. The test results are shown in Table 4 below.

[0068] Table 4: Effect of different stabilizer concentrations

[0069]

[0070] Based on Table 4, it can be seen that test reagent 2-1 is reagent 2 without stabilizer, and the repeatability of the test results is poor. The repeatability of test samples of reagent 2 with stabilizer added (reagents 2-2 to 2-6) is improved, indicating that the stabilizer is effective.

[0071] Furthermore, the test results from reagents 2-6 and 2-7 show that the stabilizing effect begins to decrease when the stabilizer concentration exceeds 5 g / L.

[0072] Example 3

[0073] According to Table 5 below, prepare seven sets of test reagents: 3-1, 3-2, 3-3, 3-4, 3-5, 3-6, and 3-7.

[0074] Table 5

[0075]

[0076]

[0077] Different amounts of triglycerides (lipids) were added to serum samples containing 2.1 ng / mL alpha-fetoprotein isoform 3 (from a hospital in Shenzhen) to prepare solutions containing 0, 2 g / L, 4 g / L, 6 g / L, and 8 g / L of lipids, which were named Sample 3-1, Sample 3-2, Sample 3-3, Sample 3-4, and Sample 3-5, respectively.

[0078] Using the seven sets of test reagents mentioned above, samples 3-1, 3-2, 3-3, 3-4, and 3-5 were tested using the Shine i2910 instrument to determine the effect of lipid blocker concentration on the results. The test results are shown in Table 6 below.

[0079] Table 6: Effect of different stabilizer concentrations

[0080]

[0081]

[0082] Based on Table 6, it can be seen that reagent 2 of test reagent 3-1 lacks a stabilizer, resulting in poor repeatability of the test results. The repeatability of test samples of reagent 2 of test reagents 3-2 to 3-7, which contain stabilizers, is improved. This indicates that different types of stabilizers are effective when used alone or in combination.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A kit for determining alpha-fetoprotein (AFP) content using a turbidimetric scattering method, characterized in that, It includes reagent 1 and reagent 2; the volume ratio of reagent 1 to reagent 2 used is 1:1 to 5:1; The reagent 1 comprises 0.02wt%~0.1wt% of a lipid blocker, 5mmol / L~50mmol / L of a buffer, 0.5g / L~10g / L of a surfactant, and 0.5~5g / L of a preservative; the lipid blocker is phosphodiesterase; The reagent 2 comprises 0.1 w / v% to 1 w / v% of anti-alpha-fetoprotein antibody latex microspheres, 5 mmol / L to 50 mmol / L of the buffer, 0.5 g / L to 10 g / L of the surfactant, 0.5 to 5 g / L of the preservative, and 0.1 g / L to 5 g / L of the stabilizer; the stabilizer is selected from at least one of Tritonx-100, D-mannitol, and isopropanol.

2. The kit for determining alpha-fetoprotein content by turbidimetric scattering according to claim 1, characterized in that, The phosphodiesterase is selected from at least one of soybean phosphodiesterase, Gynostemma pentaphyllum phosphodiesterase, and ginseng phosphodiesterase.

3. The kit for determining alpha-fetoprotein content by turbidimetric scattering according to claim 1, characterized in that, The buffer is selected from at least one of PBS buffer, Tris buffer, glycine buffer, BS buffer, HEPES buffer, MOPSO buffer, and MES buffer; The surfactant is selected from at least one of polyvinylpyrrolidone, glycerol, Triton 100, PEG6000, PEG8000, PEG10000, PEG20000, Tween-20, Tween-40 and Tween-80; The preservative is selected from at least one of sodium azide, Proclin-950, Proclin-300, krovin100, krovin300, krovin500, krovin750 and thimerosal.

4. The kit for determining alpha-fetoprotein content by turbidimetric scattering according to claim 1, characterized in that, The anti-alpha-fetoprotein antibody latex microspheres are anti-alpha-fetoprotein isoform 1 antibody latex microspheres, anti-alpha-fetoprotein isoform 2 antibody latex microspheres, or anti-alpha-fetoprotein isoform 3 antibody latex microspheres.

5. The kit for determining alpha-fetoprotein content by turbidimetric scattering according to claim 1, characterized in that, The pH of both reagent 1 and reagent 2 is 6.0~9.0.

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