A folic acid detection kit and a detection method

Abstract

The application relates to the technical field of medical analysis, in particular to a folic acid detection kit and a detection method. R1 reagent and R2 reagent are reacted with a sample, and a treatment agent contained in the R1 reagent and the R2 reagent can dissociate folic acid in the sample from folic acid binding protein. The sample does not need to be treated by strong alkali, the reaction condition is mild, and since the strong alkali treatment is not used, the interference of the strong alkali reagent on the immune reaction and detection is avoided, the detection precision and accuracy are high, meanwhile, the step of neutralizing the strong alkali by using a neutralizing reagent is omitted, the method is simplified, and the detection time is short.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medical analysis, in particular to a folic acid detection kit and a detection method. BACKGROUND

[0002] Folic acid is a water-soluble vitamin, and its molecular formula is C 19 H 19 N7O6. Folic acid exists in spinach, broccoli, green leafy vegetables such as rape, and fruits such as grapes, pears, and grapefruits, and the meat and offal of many animals also contain folic acid. Folic acid is composed of a residue of pteridine, p-aminobenzoic acid and glutamic acid, and is an essential coenzyme in DNA replication. Lack of folic acid can cause megaloblastic anemia and some stomatitis, dermatitis, etc. Pregnant women lack of folic acid will not only cause megaloblastic anemia, but also cause fetal development retardation, low intelligence and other deformities, so the detection of folic acid has important clinical significance.

[0003] At present, the method for detecting serum folic acid is earliest microbial method, which has high accuracy but poor repeatability. There is also a radioisotope labeling method, but the operation of this method is complicated, the stability is poor, and there is greater pollution. There are also ion capture method, gas chromatography-mass spectrometry method, enzyme-linked immunoassay method, chemiluminescence method, etc. At present, the chemiluminescence method is a method widely used in clinic, which has high detection precision and accuracy. Since the folic acid in the sample is combined with protein, the folic acid needs to be dissociated from the combined protein before the next step. At present, the dissociation of folic acid from the combined protein mostly adopts a dissociation method of strong alkali combined with a reducing agent, however, the strong alkali will affect the pH and cause the protein in the reaction system to precipitate, affecting the detection precision and accuracy. In addition, in order to reduce the influence of strong alkali, a neutralizing agent is added for neutralization, resulting in complicated steps and long operation time. SUMMARY

[0004] Therefore, the technical problem to be solved by the present application is to overcome the defects of low detection precision and accuracy, complicated steps and long time of the folic acid detection method in the prior art, so as to provide a folic acid detection kit and a detection method, which have high detection precision and accuracy, simplified steps and short detection time.

[0005] A folic acid detection kit, comprising: R1 reagent and R2 reagent;

[0006] The R1 reagent comprises magnetic microparticles coated with folic acid and a treatment agent a;

[0007] The R2 reagent comprises folic acid binding protein labeled with chemiluminescent substance and a treatment agent b;

[0008] The treatment agent a and treatment agent b each comprise at least one of tris(2-carbonylethyl)phosphine, mercaptoethanol, guanidine hydrochloride, sodium 2-mercaptoethanesulfonate, messo, α-thioglycerol, phosphate, ascorbate, and dithiothreitol.

[0009] Optionally, treatment agent a and treatment agent b each further include an alkali, and the pH value of each of reagents R1 and R2 is ≤10;

[0010] Optionally, the alkali includes sodium hydroxide or potassium hydroxide.

[0011] Optionally, in the R1 reagent, the concentration of treatment agent a is 0.01% to 5% w / v; and / or

[0012] In the R1 reagent, the concentration of folic acid-coated magnetic microparticles is 100–1000 μg / mL; and / or

[0013] The R1 reagent further includes a buffer solution with a concentration of 10 mM to 200 mM and a pH of 5.0 to 10.0; and / or

[0014] The R1 reagent also includes a sensitizer at a concentration of 0.1% to 5% w / v; and / or

[0015] The R1 reagent also includes a protective agent at a concentration of 0.05% to 10% w / v; and / or

[0016] The R1 reagent also includes a surfactant at a concentration of 0.01% to 0.5% w / v; and / or

[0017] The R1 reagent also includes a preservative with a concentration of 0.1% to 5% w / v.

[0018] Optionally, in the R1 reagent, the magnetic microparticles used to coat folic acid are hydrophilic magnetic microparticles with a particle size of 0.5–5.0 μm; and / or

[0019] In the R1 reagent, the folic acid-coated magnetic microparticles are obtained by coating folic acid and IgG conjugates onto magnetic microparticles; the IgG is at least one of human IgG, rabbit IgG, bovine IgG, and sheep IgG; and / or

[0020] In the R1 reagent, the buffer solution is at least one selected from acetic acid, phosphoric acid, phosphate, boric acid, boric acid-borax, citric acid, citrate, Tris, Tris-HCl, 4-morpholine ethanesulfonic acid, 4-hydroxyethylpiperazine ethanesulfonic acid, and piperazine-1,4-diethanesulfonic acid buffer solution; and / or

[0021] In the R1 reagent, the sensitizer is at least one selected from PEG 2000, PEG 4000, PEG 6000, PEG 8000, PEG 10000, and PEG 20000; and / or

[0022] In the R1 reagent, the protective agent is at least one selected from bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent (which may be from JSR) and CE210 blocking agent (which may be from JSR); and / or

[0023] In the R1 reagent, the surfactant is at least one selected from nonylphenol polyoxyethylene ether, secondary alcohol ethoxylate series, secondary alkanol polyether-9, polysorbate-20, polysorbate-80, Triton X-100, octylphenol polyoxyethylene ether, and hexadecyl ammonium chloride; and / or

[0024] In the R1 reagent, the preservative is at least one of gentamicin, KroVin series preservatives, NaN3, Proclin 300, Proclin 950, and BND series antibacterial agents.

[0025] Optionally, in the R2 reagent, the concentration of treatment agent b is 0.01% to 5% w / v; and / or

[0026] In the R2 reagent, the concentration of chemiluminescent folic acid-binding protein labeled with the substance is 1–10 μg / mL; and / or

[0027] The R2 reagent further includes a buffer solution with a concentration of 10 mM to 200 mM and a pH of 5.0 to 10.0; and / or

[0028] The R2 reagent also includes a sensitizer at a concentration of 0.1% to 5% w / v; and / or

[0029] The R2 reagent also includes a protective agent at a concentration of 0.05%–10% w / v; and / or

[0030] The R2 reagent also includes a surfactant at a concentration of 0.01% to 0.5% w / v; and / or

[0031] The R2 reagent also includes a preservative at a concentration of 0.1% to 5% w / v.

[0032] Optionally, in the R2 reagent, the chemiluminescent substance in the chemiluminescent folic acid-binding protein labeled with the chemiluminescent substance is an acridine ester compound; optionally, it is an acridine sulfonamide-labeled folic acid-binding protein; and / or

[0033] In the R2 reagent, the buffer solution is at least one selected from acetic acid, phosphoric acid, phosphate, boric acid, boric acid-borax, citric acid, citrate, Tris, 4-morpholine ethanesulfonic acid, 4-hydroxyethylpiperazine ethanesulfonic acid, and piperazine-1,4-diethanesulfonic acid buffer solutions; and / or

[0034] In the R2 reagent, the sensitizer is at least one selected from PEG 2000, PEG 4000, PEG 6000, PEG 8000, PEG 10000, and PEG 20000; and / or

[0035] In the R2 reagent, the protective agent is at least one selected from bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent (which may be from JSR) and CE210 blocking agent (which may be from JSR); and / or

[0036] In the R2 reagent, the surfactant is at least one selected from nonylphenol polyoxyethylene ether, secondary alcohol ethoxylate series, secondary alkanol polyether-9, polysorbate-20, polysorbate-80, Triton X-100, octylphenol polyoxyethylene ether, and hexadecyl ammonium chloride; and / or

[0037] In the R2 reagent, the preservative is at least one of gentamicin, KroVin series preservatives, NaN3, Proclin 300, Proclin 950, and BND series antibacterial agents.

[0038] Optionally, calibrators, which are folic acid solutions of known concentrations, may also be included;

[0039] Optionally, the calibrator contains a folic acid solution of 0–20 ng / ml;

[0040] Optionally, the calibrator may further include a buffer solution with a concentration of 10 mM to 200 mM and a pH of 5.0 to 10.0;

[0041] Optionally, the calibrator may also include a protective agent at a concentration of 0.05% to 10% w / v;

[0042] Optionally, the calibrator may further include a surfactant at a concentration of 0.01% to 0.5% w / v;

[0043] Optionally, the calibrator also includes a preservative at a concentration of 0.1% to 5% w / v;

[0044] Optionally, in the calibrator, the buffer solution is at least one of acetic acid, phosphoric acid, phosphate, boric acid, boric acid-borax, citric acid, Tris, Tris-HCl, 4-morpholine ethanesulfonic acid, 4-hydroxyethylpiperazine ethanesulfonic acid, and piperazine-1,4-diethanesulfonic acid buffer solution;

[0045] Optionally, the protective agent in the calibrator is at least one selected from bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent (which may be from JSR) and CE210 blocking agent (which may be from JSR); and / or

[0046] Optionally, in the calibrator, the surfactant is at least one selected from nonylphenol polyoxyethylene ether, secondary alcohol ethoxylate series, secondary alkanol polyether-9, polysorbate-20, polysorbate-80, Triton X-100, octylphenol polyoxyethylene ether, and hexadecyl ammonium chloride; and / or

[0047] Optionally, in the calibrator, the preservative is at least one of gentamicin, KroVin series preservatives, NaN3, Proclin 300, Proclin 950, and BND series antibacterial agents.

[0048] A method for detecting folic acid using the aforementioned folic acid detection kit includes the following steps:

[0049] S1. Mix the sample and reagent R2, incubate, and obtain the first reaction solution;

[0050] S2. Add reagent R1 to the first reaction solution, mix, and incubate to obtain the second reaction solution;

[0051] S3. Perform magnetic separation on the second reaction liquid, wash the magnetically separated magnetic particles, then add a pre-exciter, incubate, then add an exciter and detect the luminescence signal value;

[0052] S4. Perform the calibrator operation according to steps S1-S3, plot a standard curve based on the obtained luminescence signal value and the concentration of the calibrator, and then calculate the folic acid content in the sample to be tested based on the standard curve and the luminescence signal value of the sample.

[0053] Optionally, the volume ratio of sample, R2 reagent, R1 reagent, pre-activator, and activator is 60:60:60:100:100.

[0054] Optionally, in step S1, the incubation conditions are 37°C for 25 minutes; and / or

[0055] In step S2, the incubation conditions are 37°C for 20 minutes; and / or

[0056] In step S3, the incubation conditions are 37°C for 2 minutes; and / or

[0057] In step S1, the sample is a serum sample.

[0058] The technical solution of this invention has the following advantages:

[0059] 1. The folic acid detection kit provided by this invention utilizes reagents R1 and R2 to react with the sample. The treatment agents contained in reagents R1 and R2 can dissociate folic acid from folic acid-binding proteins in the sample, eliminating the need to treat the sample with a strong alkali at a high pH value (such as pH 13-14). The reaction conditions are mild, and because no strong alkali treatment at a high pH value is used, the interference of strong alkali reagents at high pH values ​​on the immune reaction and detection is avoided, resulting in high detection accuracy and precision. At the same time, the step of neutralizing strong alkali with a neutralizing reagent is omitted, simplifying the method and shortening the detection time. Attached Figure Description

[0060] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0061] Figure 1 This is the result of the accuracy detection method in the experimental example of this invention. Detailed Implementation

[0062] The following embodiments are provided to better understand the present invention and are not limited to the preferred embodiments described. They do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the protection scope of the present invention.

[0063] For experiments not specifically described in the examples, the procedures or conditions should be followed according to the conventional experimental procedures described in the literature in this field. Reagents or instruments whose manufacturers are not specified are all commercially available conventional reagent products.

[0064] The magnetic bead stock solution was purchased from JSR Corporation; the folic acid binding protein was purchased from Jiangsu Baiming Biotechnology Co., Ltd.; and acridine sulfonamide was a commercially available product.

[0065] In the examples below, the pre-activator was purchased from substrate solution I of Yantai Demai Biotechnology Co., Ltd.; the activator was purchased from substrate solution II of Yantai Demai Biotechnology Co., Ltd.

[0066] When preparing the calibrators, the folic acid used was purchased pure folic acid substance, weighed, and mixed thoroughly with 5M sodium hydroxide solution to obtain a concentrated solution, which was then prepared into calibrators of the required concentration.

[0067] The folic acid-IgG conjugate was purchased from Hangzhou Boyue Biotechnology Co., Ltd.

[0068] The preparation method of the folic acid-coated magnetic microparticles in the following examples is as follows:

[0069] Treatment of magnetic microparticles: Use 4-morpholinoethanesulfonic acid (MES) solution with pH 4.5-6.0 to wash the magnetic beads with 10 times the original volume of the solution. Remove the supernatant after magnetic adsorption. Repeat 3 times.

[0070] Activation: Add 10 mg / mL 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) solution, 20 mg / mL N-hydroxysuccinimide (NHS) solution, and 7 times the volume of the original magnetic bead solution. Shake and mix for 30 min. Wash once with MES solution, and then bring the volume up to 10 times the original volume of the magnetic bead solution with MES solution.

[0071] Coating: Add a conjugate of folic acid and IgG at a ratio of 1 mg magnetic beads / 20 mg, and shake to coat for 4 hours;

[0072] Blocking: Add 10% w / v bovine serum albumin (BSA) and shake to block for 1 hour;

[0073] Collection: Discard the supernatant magnetically, wash once with MES solution, and bring to volume with buffer from reagent R1 for storage.

[0074] The preparation method of folic acid-binding protein labeled with acridine esters is as follows:

[0075] Treatment of folic acid-binding protein: Add folic acid-binding protein to an ultrafiltration tube, add 0.1M PBS solution, mix well, centrifuge at 10000 rpm for 5 min, remove the supernatant, add 0.1M PBS solution again, repeat this process 3 times, and take the remaining concentrate into an EP tube.

[0076] Labeling: Add 0.1 mg folic acid binding protein / 4 μL acridine ester (specifically acridine sulfonamide), and react with shaking in the dark for 1 h;

[0077] Purification: Take the labeling solution and add it to an ultrafiltration tube. Add 0.1M PBS solution, mix well, centrifuge at 10000 rpm for 15 min, remove the supernatant, and add 0.1M PBS solution again. Repeat this process 5 times. Take the remaining concentrate into an EP tube, add buffer from reagent R2 to make up the volume and store.

[0078] Example 1: A folic acid detection kit and detection method

[0079] This embodiment provides a folic acid detection kit, comprising:

[0080] R1 reagent comprises the following components:

[0081] Folic acid-coated magnetic microparticles: concentration of 500 μg / mL, particle size of magnetic microparticles of 3.0 μm, consisting of a conjugate of rabbit IgG and folic acid coated on magnetic microparticles;

[0082] Buffer solution: 100mM PBS, pH 5.0–10.0;

[0083] Sensitizer: 0.3% w / v PEG 8000;

[0084] Protectants: 0.5% w / v ovalbumin, 0.5% w / v lactose, 0.5% w / v mannitol;

[0085] Treatment agents: 0.02% w / v dithiothreitol, 0.01% w / v mercaptoethanol, 0.03% w / v α-thioglycerol;

[0086] Surfactant: 0.05% w / v polysorbate-80;

[0087] Preservatives: 0.1% w / v gentamicin, 0.1% w / v Proclin 300;

[0088] The remainder is purified water;

[0089] The pH value of the above reagent R1 is 7.4.

[0090] R2 reagent comprises the following components:

[0091] Acridinium ester-labeled folic acid-binding protein at a concentration of 1 μg / mL, with acridinium sulfonamide as the luminescent substance;

[0092] Buffer solution: 50 mM 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES), pH 5.0–10.0;

[0093] Sensitizer: 0.2% w / v PEG 8000;

[0094] Protectants: 0.2% w / v ovalbumin, 0.2% w / v sucrose, 0.5% w / v sorbitol;

[0095] Treatment agents: 0.02% w / v sodium ascorbate, 0.01% w / v sodium 2-mercaptoethanesulfonate;

[0096] Surfactant: 0.05% w / v polysorbate-80;

[0097] Preservatives: 0.1% w / v gentamicin, 0.1% w / v Proclin 300;

[0098] The remaining components are purified water;

[0099] The pH value of the above-mentioned reagent R2 is 6.1.

[0100] Calibrators, including:

[0101] Folic acid: concentrations of 0, 1, 2, 5, 10, or 20 ng / mL;

[0102] Buffer solution: 100mM Tris-HCl, pH 5.0–10.0;

[0103] Protectant: 1% w / v ovalbumin;

[0104] Surfactant: 0.1% w / v polysorbate-80;

[0105] Preservatives: 0.1% w / v gentamicin, 0.1% w / v Proclin 300;

[0106] The remaining components are purified water. The pH of the calibrator is 8.0.

[0107] The calibrator series consists of reagents containing folic acid at concentrations of 0, 1, 2, 5, 10, and 20 ng / mL.

[0108] This embodiment provides a method for detecting folic acid using the above-mentioned folic acid detection kit, including the following steps:

[0109] S1. Add 60 μL of sample and 60 μL of reagent R2 to the reaction vessel, shake to mix, and incubate at 37°C for 25 min to obtain the first reaction solution;

[0110] S2. Add 60 μL of reagent R1 to the first reaction solution, shake to mix, and incubate at 37°C for 20 min to obtain the second reaction solution.

[0111] S3. Perform magnetic separation on the second reaction solution, wash the magnetically separated magnetic particles three times, then add 100 μL of pre-exciter, incubate at 37°C for 2 min, then add 100 μL of exciter and detect the luminescence signal value.

[0112] S4. Perform the calibrator operation according to steps S1-S3, plot a standard curve based on the obtained luminescence signal value and the concentration of the calibrator, and then calculate the folic acid content in the sample to be tested based on the standard curve and the luminescence signal value of the sample.

[0113] Example 2: A folic acid detection kit and detection method

[0114] This embodiment provides a folic acid detection kit, comprising:

[0115] R1 reagent includes:

[0116] The magnetic microparticles coated with folic acid, with a concentration of 300 μg / mL and a particle size of 2.0 μm, are a conjugate of folic acid and human IgG coated on magnetic microparticles.

[0117] Buffer solution: 50 mM Tris-HCl, pH 5.0–10.0;

[0118] Sensitizer: 0.3% w / v PEG 2000;

[0119] Protective agents: 0.5% w / v human serum albumin, 0.5% w / v trehalose, 0.2% w / v gelatin;

[0120] Treatment agents: 0.02% w / v dithiothreitol, 0.02% w / v potassium hydroxide, 0.03% w / v messodium (sodium 2-mercaptoethanesulfonate);

[0121] Surfactant: 0.05% w / v Triton X-100;

[0122] Preservatives: 0.1% w / v BND (5-bromo-5-nitro-1,3-dioxane) antibacterial agent, 0.1% w / v Proclin 950;

[0123] The remaining components are purified water;

[0124] The pH value of the above reagent R1 is 8.0.

[0125] R2 reagent includes:

[0126] Acridinium ester-labeled folic acid-binding protein at a concentration of 1.5 μg / mL, with acridinium sulfonamide as the luminescent substance;

[0127] Buffer solution: 50 mM sodium citrate, pH 5.0–10.0;

[0128] Sensitizer: 0.3% w / v PEG 8000;

[0129] Protective agents: 0.2% w / v human serum albumin, 0.3% w / v lactose, 0.2% w / v mannitol;

[0130] Treatment agents: 0.02% w / v sodium ascorbate, 0.02% w / v tris(2-carbonylethyl)phosphine (TCEP);

[0131] Surfactant: 0.05% w / v Triton X-100;

[0132] Preservatives: 0.1% w / v BND antibacterial agent, 0.1% w / v Proclin 950;

[0133] The remaining components are purified water;

[0134] The pH value of the above-mentioned reagent R2 is 5.8.

[0135] Calibrators, including:

[0136] Folic acid: concentrations of 0, 1, 2, 5, 10, or 20 ng / mL;

[0137] Buffer solution: 50 mM PBS, pH 5.0–10.0;

[0138] Protectant: 1% w / v human serum albumin;

[0139] Surfactant: 0.1% w / v Triton X-100;

[0140] Preservatives: 0.1% w / v BND antibacterial agent, 0.1% w / v Proclin 950;

[0141] The remaining components are purified water. The pH of the calibrator is 7.4.

[0142] The calibrator series consists of reagents with concentrations of 0, 1, 2, 5, 10, and 20 ng / mL.

[0143] This embodiment provides a method for detecting folic acid using the above-mentioned folic acid detection kit, including the following steps:

[0144] S1. Add 60 μL of sample and 60 μL of reagent R2 to the reaction vessel, shake to mix, and incubate at 37°C for 25 min to obtain the first reaction solution;

[0145] S2. Add 60 μL of reagent R1 to the first reaction solution, shake to mix, and incubate at 37°C for 20 min to obtain the second reaction solution.

[0146] S3. Perform magnetic separation on the second reaction solution, wash the magnetically separated magnetic particles three times, then add 100 μL of pre-exciter, incubate at 37°C for 2 min, then add 100 μL of exciter and detect the luminescence signal value.

[0147] S4. Perform the calibrator operation according to steps S1-S3, plot a standard curve based on the obtained luminescence signal value and the concentration of the calibrator, and then calculate the folic acid content in the sample to be tested based on the standard curve and the luminescence signal value of the sample.

[0148] Example 3

[0149] This embodiment provides a folic acid detection kit, comprising:

[0150] R1 reagent comprises the following components:

[0151] Folic acid-coated magnetic microparticles: concentration of 100 μg / mL, particle size of magnetic microparticles of 0.5 μm, which are bovine IgG and folic acid conjugates coated on magnetic microparticles;

[0152] Buffer solution: 10 mM piperazine-1,4-diethanesulfonic acid (PIPES), pH 5.0–10.0;

[0153] Sensitizer: 0.1% w / v PEG 20000;

[0154] Protective agent: 0.05% w / v CE510 sealant (JSR Corporation);

[0155] Treatment agent: 0.01% w / v tris(2-carbonylethyl)phosphine (TCEP);

[0156] Surfactant: 0.01% w / v nonylphenol polyoxyethylene ether;

[0157] Preservative: 0.1% w / v NaN3;

[0158] The rest is purified water.

[0159] R2 reagent comprises the following components:

[0160] Acridinium ester-labeled folic acid-binding protein at a concentration of 2 μg / mL, with acridinium sulfonamide as the luminescent substance;

[0161] Buffer solution: 10 mM piperazine-1,4-diethanesulfonic acid (PIPES), pH 5.0–10.0;

[0162] Sensitizer: 0.1% w / v PEG 20000;

[0163] Protective agents: 0.01% w / v bovine serum albumin (BSA), 0.01% w / v trehalose, 0.03% w / v gelatin;

[0164] Treatment agents: 0.005% w / v α-thioglycerol, 0.005% w / v mercaptoethanol;

[0165] Surfactant: 0.01% w / v cetyl ammonium chloride;

[0166] Preservative: 0.1% w / v BND-10 antibacterial agent;

[0167] The remaining components are purified water.

[0168] Calibrators, including:

[0169] Folic acid: concentrations of 0, 1, 2, 5, 10, or 20 ng / mL;

[0170] Buffer solution: 10 mM PBS, pH 5.0–10.0;

[0171] Protectant: 0.05% w / v gelatin;

[0172] Surfactant: 0.01% w / v Triton X-100;

[0173] Preservative: 0.1% w / v gentamicin;

[0174] The remaining components are purified water.

[0175] The calibrator series consists of reagents containing folic acid at concentrations of 0, 1, 2, 5, 10, and 20 ng / mL.

[0176] This embodiment provides a method for detecting folic acid using the above-mentioned folic acid detection kit, including the following steps:

[0177] S1. Add 60 μL of sample and 60 μL of reagent R2 to the reaction vessel, shake to mix, and incubate at 37°C for 25 min to obtain the first reaction solution;

[0178] S2. Add 60 μL of reagent R1 to the first reaction solution, shake to mix, and incubate at 37°C for 20 min to obtain the second reaction solution.

[0179] S3. Perform magnetic separation on the second reaction solution, wash the magnetically separated magnetic particles three times, then add 100 μL of pre-exciter, incubate at 37°C for 2 min, then add 100 μL of exciter and detect the luminescence signal value.

[0180] S4. Perform the calibrator operation according to steps S1-S3, plot a standard curve based on the obtained luminescence signal value and the concentration of the calibrator, and then calculate the folic acid content in the sample to be tested based on the standard curve and the luminescence signal value of the sample.

[0181] Example 4

[0182] This embodiment provides a folic acid detection kit, comprising:

[0183] R1 reagent comprises the following components:

[0184] Folic acid-coated magnetic microparticles: concentration of 1000 μg / mL, particle size of magnetic microparticles of 5.0 μm, consisting of a conjugate of sheep IgG and folic acid coated on magnetic microparticles;

[0185] Buffer solution: 200mM acetate buffer, pH 5.0–10.0;

[0186] Sensitizers: 1% w / v PEG 4000, 1% w / v PEG 6000, 1% w / v PEG 8000, 2% w / v PEG 10000;

[0187] Protective agents: 0.5% w / v bovine serum albumin (BSA), 5% w / v galactose, 4.5% w / v CE210 blocking agent;

[0188] Treatment agents: 1% w / v sodium ascorbate, 4% w / v sodium 2-mercaptoethanesulfonate;

[0189] Surfactants: 0.1% w / v secondary alkanol polyether-9, 0.1% w / v hexadecyl ammonium chloride, 0.1% w / v polysorbate-20, 0.2% w / v Triton X-100;

[0190] Preservative: 5% w / v gentamicin;

[0191] The rest is purified water.

[0192] R2 reagent comprises the following components:

[0193] Acridinium ester-labeled folic acid-binding protein at a concentration of 10 μg / mL, with acridinium sulfonamide as the luminescent substance;

[0194] Buffer solution: 200 mM 4-morpholinoethanesulfonic acid (MES), pH 5.0–10.0;

[0195] Sensitizer: 5% w / v PEG10000;

[0196] Protective agents: 3% w / v ovalbumin, 3% w / v lactose, 4% w / v CE210 blocking agent;

[0197] Treatment agents: 2.5% w / v dithiothreitol, 2.5% w / v mercaptoethanol;

[0198] Surfactant: 0.5% w / v octylphenol polyoxyethylene ether;

[0199] Preservative: 5% w / v Proclin 950;

[0200] The remaining components are purified water.

[0201] Calibrators, including:

[0202] Folic acid: concentrations of 0, 1, 2, 5, 10, or 20 ng / mL;

[0203] Buffer solution: 200mM citrate buffer, pH 5.0–10.0;

[0204] Protectant: 10% w / v mannitol;

[0205] Surfactant: 0.5% w / v secondary alkanol polyether-9;

[0206] Preservative: 5% w / v Proclin 300;

[0207] The remaining components are purified water.

[0208] The calibrator series consists of reagents containing folic acid at concentrations of 0, 1, 2, 5, 10, and 20 ng / mL.

[0209] This embodiment provides a method for detecting folic acid using the above-mentioned folic acid detection kit, including the following steps:

[0210] S1. Add 60 μL of sample and 60 μL of reagent R2 to the reaction vessel, shake to mix, and incubate at 37°C for 25 min to obtain the first reaction solution;

[0211] S2. Add 60 μL of reagent R1 to the first reaction solution, shake to mix, and incubate at 37°C for 20 min to obtain the second reaction solution.

[0212] S3. Perform magnetic separation on the second reaction solution, wash the magnetically separated magnetic particles three times, then add 100 μL of pre-exciter, incubate at 37°C for 2 min, then add 100 μL of exciter and detect the luminescence signal value.

[0213] S4. Perform the calibrator operation according to steps S1-S3, plot a standard curve based on the obtained luminescence signal value and the concentration of the calibrator, and then calculate the folic acid content in the sample to be tested based on the standard curve and the luminescence signal value of the sample.

[0214] Experimental Example

[0215] (1) Precision testing

[0216] The kits of Examples 1 and 2 were subjected to precision testing. Four blood samples with folic acid content within the linear range were selected for precision testing (using Abbott's folic acid assay kit (chemiluminescence method). The folic acid content of the four blood samples was 2.6 ng / ml, 5.6 ng / ml, 9.3 ng / ml, and 14.5 ng / ml, respectively). The intra-day precision testing method was as follows: on days 1, 2, and 3, the blood samples were tested 10 times using the kit of Example 1 or Example 2. The test results were statistically analyzed, and the mean AV and coefficient of variation CV were calculated. CV = standard deviation / mean × 100%. The inter-day precision testing method was as follows: the AV values ​​of the blood samples tested using Example 1 or Example 2 on days 1, 2, and 3 were statistically analyzed, and the mean AV and coefficient of variation CV of the inter-day precision were calculated. CV = standard deviation / mean × 100%. The results are shown in Table 1 below, indicating that the kits of Examples 1 and 2 of this invention have high precision.

[0217]

[0218]

[0219]

[0220] (2) Accuracy testing

[0221] Eighty-eight blood samples with folic acid levels within the linear range were analyzed using Abbott's folic acid assay kit (chemiluminescence method). The detected folic acid concentration was plotted on the ordinate. The kits from Example 1 or Example 2 were used to analyze the folic acid concentration in the 88 blood samples, and the plotted on the abscissa. A linear relationship graph was constructed, and the results are shown below. Figure 1 ,from Figure 1 As can be seen, the correlation between the kits of Examples 1-2 of this invention and existing detection reagents is R, respectively. 2 =0.9877 and R 2 =0.9657, indicating that the kits of Examples 1-2 of the present invention have high consistency and high accuracy with existing detection reagents.

[0222] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A folic acid detection kit, characterized by, The application relates to a reagent for detecting folate receptor, comprising: R1 reagent and R2 reagent; the R1 reagent comprises folate-coated magnetic microparticles and treatment agent a; the R2 reagent comprises chemiluminescent substance-labeled folate binding protein and treatment agent b; the treatment agent a and the treatment agent b each comprise at least one of tris(2-carboxyethyl) phosphine, mercaptoethanol, guanidine hydrochloride, 2-mercaptoethanesulfonic acid sodium, alpha-thioglycerol, ascorbate and dithiothreitol; the pH of the R1 reagent and the R2 reagent is less than or equal to 10.

2. The folic acid detection kit according to claim 1, characterized by the treatment agent a and the treatment agent b each further comprise an alkali, and the alkali comprises sodium hydroxide or potassium hydroxide.

3. The folic acid detection kit according to claim 1 or 2, characterized by in the R1 reagent, the concentration of the treatment agent a is 0.01% to 5% w / v; and / or in the R1 reagent, the concentration of the folate-coated magnetic microparticles is 100 to 1000 micrograms per milliliter; and / or in the R1 reagent, a buffer is further included, and the concentration of the buffer is 10 mM to 200 mM, and the pH is 5.0 to 10.0; and / or in the R1 reagent, a sensitizer is further included, and the concentration of the sensitizer is 0.1% to 5% w / v; and / or in the R1 reagent, a protective agent is further included, and the concentration of the protective agent is 0.05% to 10% w / v; and / or in the R1 reagent, a surfactant is further included, and the concentration of the surfactant is 0.01% to 0.5% w / v; and / or in the R1 reagent, a preservative is further included, and the concentration of the preservative is 0.1% to 5% w / v.

4. The folic acid detection kit according to claim 3, characterized by in the R1 reagent, the folate-coated magnetic microparticles are hydrophilic magnetic microparticles, and the particle size is 0.5 to 5.0 micrometers; and / or in the R1 reagent, the folate-coated magnetic microparticles are obtained by coating folate and IgG conjugates on magnetic microparticles, and the IgG is at least one of human IgG, rabbit IgG, bovine IgG and goat IgG; and / or in the R1 reagent, the buffer is at least one of acetic acid, phosphoric acid, phosphate, boric acid, boric acid-borax, citric acid, citrate, Tris, Tris-HCl, 4-morpholine ethanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, piperazine-1,4-diethanesulfonic acid; and / or in the R1 reagent, the sensitizer is at least one of PEG 2000, PEG 4000, PEG 6000, PEG 8000, PEG 10000 and PEG 20000; and / or in the R1 reagent, the protective agent is at least one of bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent and CE210 blocking agent; and / or in the R1 reagent, the surfactant is at least one of nonylphenol polyoxyethylene ether, secondary alcohol ethoxylate series, secondary alkyl alcohol polyether-9, polysorbate-20, polysorbate-80, Triton X-100, octylphenol polyoxyethylene ether and cetyl ammonium chloride; and / or in the R1 reagent, the preservative is at least one of gentamicin, KroVin series preservative, NaN3, Proclin 300, Proclin 950 and BND series bacteriostatic agent.

5. The folic acid detection kit according to claim 1 or 2, characterized by, The concentration of the processing agent b in the R2 reagent is 0.01% to 5% w / v; and / or The concentration of the chemiluminescent substance-labeled folate binding protein in the R2 reagent is 1 to 10 μg / mL; and / or The R2 reagent further comprises a buffer, and the concentration of the buffer is 10 mM to 200 mM, and the pH is 5.0 to 10.0; and / or The R2 reagent further comprises a sensitizing agent, and the concentration of the sensitizing agent is 0.1% to 5% w / v; and / or The R2 reagent further comprises a protective agent, and the concentration of the protective agent is 0.05% to 10% w / v; and / or The R2 reagent further comprises a surfactant, and the concentration of the surfactant is 0.01% to 0.5% w / v; and / or The R2 reagent further comprises a preservative, and the concentration of the preservative is 0.1% to 5% w / v.

6. The folic acid detection kit according to claim 5, characterized by The chemiluminescent substance in the chemiluminescent substance-labeled folate binding protein in the R2 reagent is an acrid ester compound; and / or The buffer in the R2 reagent is at least one of an acetic acid buffer, a phosphoric acid buffer, a phosphate buffer, a boric acid buffer, a boric acid-borax buffer, a citric acid buffer, a citrate buffer, a Tris buffer, a 4-morpholine ethanesulfonic acid buffer, a 4-hydroxyethyl piperazine ethanesulfonic acid buffer, and a piperazine-1, 4-diethanesulfonic acid buffer; and / or The sensitizing agent in the R2 reagent is at least one of PEG 2000, PEG 4000, PEG 6000, PEG 8000, PEG 10000, and PEG 20000; and / or The protective agent in the R2 reagent is at least one of bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent, and CE210 blocking agent; and / or The surfactant in the R2 reagent is at least one of nonylphenol polyoxyethylene ether, secondary alcohol ethoxylate series, secondary alkyl alcohol polyether-9, polysorbate-20, polysorbate-80, Triton X-100, octylphenol polyoxyethylene ether, and cetyl ammonium chloride; and / or The preservative in the R2 reagent is at least one of gentamicin, KroVin series preservative, NaN3, Proclin 300, Proclin 950, and BND series bacteriostatic agent.

7. The folic acid detection kit according to claim 5, characterized by The chemiluminescent substance-labeled folate binding protein in the R2 reagent is an acridinesulfonamide-labeled folate binding protein; and / or The buffer in the R2 reagent is at least one of an acetic acid buffer, a phosphoric acid buffer, a phosphate buffer, a boric acid buffer, a boric acid-borax buffer, a citric acid buffer, a citrate buffer, a Tris buffer, a 4-morpholine ethanesulfonic acid buffer, a 4-hydroxyethyl piperazine ethanesulfonic acid buffer, and a piperazine-1, 4-diethanesulfonic acid buffer; and / or The sensitizing agent in the R2 reagent is at least one of PEG 2000, PEG 4000, PEG 6000, PEG 8000, PEG 10000, and PEG 20000; and / or The protective agent in the R2 reagent is at least one of bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent, and CE210 blocking agent. The protective agent in the R2 reagent is at least one of bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent and CE210 blocking agent; and / or The surfactant in the R2 reagent is at least one of nonylphenol polyoxyethylene ether, secondary alcohol ethoxylate series, secondary alkyl polyether-9, polysorbate-20, polysorbate-80, Triton X-100, octylphenol polyoxyethylene ether and cetyl ammonium chloride; and / or The preservative in the R2 reagent is at least one of gentamicin, KroVin series preservative, NaN3, Proclin 300, Proclin 950, BND series bacteriostatic agent.

8. The folic acid detection kit according to claim 1 or 2, characterized by, Also included is a calibrator, which is a solution of folic acid with a known concentration.

9. The folic acid detection kit according to claim 8, wherein, The calibrator contains 0-20 ng / ml of folic acid solution; And / or, the calibrator further contains a buffer with a concentration of 10 mM-200 mM and a pH of 5.0-10.0; And / or, the calibrator further contains a protective agent with a concentration of 0.05%-10% w / v; And / or, the calibrator further contains a surfactant with a concentration of 0.01%-0.5% w / v; And / or, the calibrator further contains a preservative with a concentration of 0.1%-5% w / v.

10. The folic acid detection kit according to claim 9, wherein, The buffer in the calibrator is at least one of acetic acid, phosphoric acid, phosphate, boric acid, boric acid-borax, citric acid, Tris, Tris-HCl, 4-morpholine ethanesulfonic acid, 4-hydroxyethyl piperazine ethanesulfonic acid, piperazine-1, 4-diethanesulfonic acid; And / or, the protective agent in the calibrator is at least one of bovine serum albumin, ovalbumin, human serum albumin, sucrose, lactose, galactose, trehalose, sorbitol, mannitol, gelatin, CE510 blocking agent and CE210 blocking agent; And / or, the surfactant in the calibrator is at least one of nonylphenol polyoxyethylene ether, secondary alcohol ethoxylate series, secondary alkyl polyether-9, polysorbate-20, polysorbate-80, Triton X-100, octylphenol polyoxyethylene ether and cetyl ammonium chloride; And / or, the preservative in the calibrator is at least one of gentamicin, KroVin series preservative, NaN3, Proclin 300, Proclin 950, BND series bacteriostatic agent.

11. A method for detecting folate using the folate detection kit according to any one of claims 1 to 10, characterized in that, The steps include: S1, mixing the sample and the R2 reagent, incubating to obtain a first reaction solution; S2, adding the R1 reagent to the first reaction solution, mixing, incubating to obtain a second reaction solution; S3, performing magnetic separation on the second reaction solution, washing the magnetic particles separated by magnetism, then adding a pre-activator, incubating, subsequently adding an activator and detecting the luminescence signal value; S4, operating the calibrators according to steps S1-S3, drawing a standard curve according to the obtained luminescence signal value and the concentration of the calibrators, and then calculating the folate content in the sample to be measured according to the standard curve and the luminescence signal value of the sample.

12. The method of detecting folate according to claim 11, wherein, The volume ratio of the sample, the R2 reagent, the R1 reagent, the pre-excitation reagent and the excitation reagent is 60:60:60:100:

100.

13. The method of detecting folate according to claim 12, wherein, In the S1 step, the incubation condition is 37℃, 25min; and / or In the S2 step, the incubation condition is 37℃, 20min; and / or In the S3 step, the incubation condition is 37℃, 2min; and / or In the S1 step, the sample is a serum sample.

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