Kit for detecting folic acid and application thereof

Abstract

The invention discloses a kit for detecting folic acid and application of the kit. The kit comprises a folic acid binding protein signal marker capable of being specifically combined with folic acid to be detected, a solid-phase medium capable of being specifically combined with a compound formed by the folic acid to be detected and folic acid binding protein, and a sample treatment solution. When the method is used for detecting folic acid, higher-level sensitivity, precision and specificity are achieved, operation is easy and convenient, the reaction time is short, and the reaction time can be 10-15 min in the whole process.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to a kit for detecting folic acid and an application thereof. Background Art

[0002] Folic acid is one of the essential vitamins for the growth and reproduction of body cells. Its deficiency will affect the normal physiological activities of the human body. Folic acid deficiency is directly related to neural tube defects, megaloblastic anemia, cleft lip and palate, depression, tumors and other diseases. Folic acid and vitamin B12 are two important members of the water-soluble B vitamins. They play a synergistic role in the body. The effects of their combined use mainly include DNA synthesis and cell division, red blood cell formation, nervous system health, lowering homocysteine ​​levels, and immune system support. Vitamin B12 deficiency will greatly reduce the red blood cells' ability to absorb folic acid. Even if a large amount of folic acid is artificially supplemented, the folic acid content in red blood cells is still low. Therefore, it is necessary to measure vitamin B12 and folic acid and determine the cause of the disease before the right medicine can be prescribed.

[0003] Currently, the detection methods of folic acid include microbiological method, colorimetry, high performance liquid chromatography, mass spectrometry, electrochemical analysis and chemiluminescence.

[0004] Among them, chemiluminescence has the advantages of high automation, high sensitivity, wide detection range, simple and fast operation, good stability of markers, and no pollution, and has gradually become a development trend. Furthermore, magnetic particle chemiluminescence analysis combines chemiluminescence analysis technology with immunomagnetic particle technology, greatly improving detection sensitivity, accuracy and automation.

[0005] Folic acid exists in the blood in the form of a combination of folic acid and protein. When testing, it is necessary to first convert folic acid from a bound state to a free state through a dissociation step. The reagent dissociation solution usually exists separately in two reagent bottles, which leads to an increase in the components of the test kit, a complex reaction process, and a long detection time. In addition, the reagent is usually a liquid reagent and requires cold chain storage. The reagent is valid for one year if it is unopened, and the reagent is generally valid for one month after opening the bottle. A set of reagent bottles generally contains tests for more than 50 people.

[0006] Therefore, there is an urgent need for a novel folic acid detection kit in the art. Summary of the invention

[0007] Based on this, it is necessary to provide a folic acid detection kit with high specificity and sensitivity, short reaction time and easy operation.

[0008] In a first aspect, the present invention provides a kit for detecting folic acid, comprising a folate binding protein signal marker capable of specifically binding to the folic acid to be detected, a solid phase medium capable of specifically binding to a complex formed by the folic acid to be detected and the folate binding protein, and a sample processing solution.

[0009] In some embodiments, the signal marker is linked to a first antibody / aptamer that specifically binds to the folate to be detected.

[0010] In some embodiments, the signal marker is a luminescent signal marker.

[0011] In some embodiments, the luminescent signal marker is an acridinium ester.

[0012] In some embodiments, the solid phase medium comprises a solid phase support and a second antibody / aptamer connected to the solid phase support and capable of specifically binding to the complex.

[0013] In some embodiments, the solid support is selected from the group consisting of magnetic beads, plates, and membranes.

[0014] In some embodiments, the second antibody / aptamer is connected to the solid phase carrier by covalent binding of surface functional groups or non-covalent passive adsorption.

[0015] In some embodiments, the surface functional groups are selected from the group consisting of amino groups and carboxyl groups.

[0016] In some embodiments, the second antibody / aptamer is linked to the solid support via a tosyl group.

[0017] In some embodiments, the folate binding protein signal marker capable of specifically binding to the folate to be detected is a freeze-dried frozen microsphere 1.

[0018] In some embodiments, the method for preparing the cryomicrospheres 1 comprises: using a cryoprotectant to dilute a folate binding protein signal marker that can specifically bind to the folate to be detected and then freeze-drying the diluted folate binding protein signal marker.

[0019] In some embodiments, the concentration of the folate binding protein signal marker capable of specifically binding to the folate to be detected after dilution is 0.0005-0.002 mg / mL, for example 0.001 mg / mL.

[0020] In some embodiments, the solid phase medium capable of specifically binding to the complex is a freeze-dried frozen microsphere 2 .

[0021] In some embodiments, the method for preparing the frozen microspheres 2 comprises: using a cryoprotectant to dilute the solid phase medium that can specifically bind to the complex and then freeze-drying it.

[0022] In some embodiments, the concentration of the solid phase medium capable of specifically binding to the complex after dilution is 0.1-1 mg / mL, such as 0.5 mg / mL.

[0023] In some embodiments, based on the total mass of the cryoprotectant, the cryoprotectant comprises: 0.2wt%-1wt% mannitol or casein, 4wt%-20wt% trehalose and / or sucrose, 1wt%-5wt% bovine serum albumin or glucose, 0.03wt%-0.5wt% surfactant, 0.05wt%-1wt% gelatin or dextran, 0.1wt%-1wt% PEG8000 or PEG 20000, 0.5wt%-2wt% sodium chloride, 0.03wt%-0.5wt% preservative, 0.2wt%-1wt% buffer, and 68wt%-93.89wt% water.

[0024] In certain embodiments, in the cryoprotectant, the buffer is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, tris(hydroxymethylaminomethane), 2-(N-morpholino)ethanesulfonic acid, citrate, acetate and borate.

[0025] In certain embodiments, in the cryoprotectant, the surfactant is selected from the group consisting of Tween 20, Tween 80, Triton 100 and S9.

[0026] In certain embodiments, in the cryoprotectant, the preservative is selected from the group consisting of PC950, BND-10, PC300, gentamicin sulfate and proclin300.

[0027] In some embodiments, the components in the cryoprotectant and their mass proportions are as follows: mannitol 0.2wt%-1wt%, trehalose 4wt%-10wt%, bovine serum albumin 1wt%-5wt%, surfactant 0.03wt%-0.3wt%, gelatin 0.05wt%-1wt%, PEG 8000 0.1wt%-0.8wt%, sodium chloride 0.5wt%-1.5wt%, preservative 0.04wt%-0.5wt%, buffer 0.2wt%-1wt%, and the balance is water.

[0028] As some examples, the cryoprotectant is an aqueous solution and contains: mannitol 5g / L, trehalose 50g / L, sucrose 50g / L, bovine serum albumin 30g / L, Tween 20 0.5g / L, gelatin 1g / L, PEG 80002g / L, sodium chloride 9g / L, and preservative proclin300 0.5g / L, and 4.986g / L 4-hydroxyethylpiperazineethanesulfonic acid.

[0029] In some embodiments, based on the total mass of the sample processing liquid, the sample processing liquid includes 2wt%-4wt% of a buffer, 0.5wt%-2wt% of sodium chloride, 0.05wt%-0.2wt% of a surfactant, 0.5wt%-2wt% of a reducing agent, 0.05wt%-0.2wt% of a preservative PC950, 0.05wt%-0.2wt% of a preservative BND-10, and 91.4wt%-96.85wt% of water.

[0030] In certain embodiments, in the sample processing solution, the buffer is selected from the group consisting of 2-(N-morpholine)ethanesulfonic acid, tris(hydroxymethylaminomethane), citrate and acetate.

[0031] In certain embodiments, in the sample processing solution, the reducing agent is selected from the group consisting of sodium 2-mercaptoethane sulfonate, dithiothreitol, tris(2-chloroethyl) phosphate and thioglycerol.

[0032] In certain embodiments, in the sample processing solution, the surfactant is selected from the group consisting of Triton 100, Tween 20, Tween 80 and S9.

[0033] In some embodiments, the components in the sample processing solution and their mass proportions are as follows: 2-(N-morpholine)ethanesulfonic acid 2wt%-4wt%, sodium chloride 0.5wt%-2wt%, Triton 100 0.05wt%-0.2wt%, sodium 2-mercaptoethane sulfonate 0.5wt%-2wt%, preservative PC950 0.05wt%-0.2wt%, preservative BND-10 0.05wt%-0.2wt%, and the balance is water.

[0034] As some examples, the sample processing solution is an aqueous solution and contains: 21.4 g / L 2-(N-morpholine)ethanesulfonic acid, 9 g / L sodium chloride, 1 g / L Triton 100, 10 g / L sodium 2-mercaptoethanesulfonate, 0.5 g / L preservative PC950, and 0.5 g / L preservative BND-10.

[0035] In some embodiments, the water is purified water.

[0036] The second aspect of the present invention provides a method for detecting folic acid using the kit described in the first aspect, comprising the following steps:

[0037] (1) mixing and incubating a sample containing the folic acid to be tested, a folate binding protein signal marker capable of specifically binding to the folic acid to be tested, a solid phase medium capable of specifically binding to a complex formed by the folic acid to be tested and the folate binding protein, and a sample processing solution;

[0038] (2) Make the signal marker display a signal and read the signal value.

[0039] In some embodiments, the method comprises the steps of:

[0040] (1) mixing and incubating a sample containing folic acid to be tested with acridinium ester-labeled folate binding protein, a magnetic particle-folate-folate binding protein complex antibody conjugate, and a sample processing solution;

[0041] (2) Add acridinium ester excitation solution and read the luminescence value.

[0042] In step (2), the acridinium ester is stimulated to emit chemiluminescence by using an excitation solution, and the light signal is read. The luminescence value of the signal marker is read to determine the folic acid content.

[0043] In some embodiments, the folate binding protein is from Beijing Baixinyi Biotechnology Co., Ltd., with the product number A09M02-N.

[0044] In some embodiments, the magnetic microparticle-folate-folate binding protein complex antibody conjugate is sourced from Nanjing Okey Biotechnology Co., Ltd., with a catalog number of Q301q1.

[0045] In some embodiments, the method for preparing acridinium ester-labeled folate binding protein comprises the following steps:

[0046] S1: Mix folate binding protein and acridine sulfonamide at a molar ratio of 1:(5-20), and add 50 mM carbonate (CB) buffer at pH 9.6 for coupling;

[0047] S2: Add lysine to block for 1 to 2 hours and dialyze.

[0048] In some embodiments, the molar ratio in step S1 is 1:15.

[0049] In some embodiments, in step S1, after the mixing, the concentration of the folate binding protein is 0.5 to 2 mg / mL, for example, 1 mg / mL.

[0050] In some embodiments, in step S1, the coupling is performed at 2-8°C.

[0051] In some embodiments, in step S1, the coupling duration is 3 to 6 hours.

[0052] In some embodiments, in step S2, the dialysis is performed 4 to 6 times, each time for 1 to 2 hours.

[0053] The third aspect of the present invention provides a method for diagnosing a disease, comprising: determining the content of folic acid in a sample based on the method provided in the first aspect of the present invention, and then determining whether the subject from which the sample is derived suffers from a certain disease. Specifically:

[0054] (1) mixing and incubating a sample containing the folic acid to be tested, a folate binding protein signal marker capable of specifically binding to the folic acid to be tested, a solid phase medium capable of specifically binding to a complex formed by the folic acid to be tested and the folate binding protein, and a sample processing solution;

[0055] (2) reading the signal marker to determine the folic acid content;

[0056] (3) Determine whether the subject from which the sample was obtained suffers from a certain disease based on the folic acid content.

[0057] In the fourth aspect of the present invention, a method for treating a disease is provided, comprising: after determining that the subject from which the sample is derived suffers from a certain disease based on the diagnostic method provided in the third aspect of the present invention, treating the subject.

[0058] The method of the present invention is used to detect folic acid, which has a higher level of sensitivity, precision and specificity, and is easy to operate and has a short reaction time, which can be 10 to 15 minutes.

[0059] In some embodiments, the folate binding protein signal marker that can specifically bind to the folate to be tested and the solid phase medium that can specifically bind to the complex are both in a lyophilized form, which is easy to operate during use; and the product is stable in form and has mild storage conditions (e.g., 2 to 30° C.), which greatly extends the shelf life of the reagent (up to one year under normal circumstances). In addition, the lyophilized reagent can be used for a single person. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 The figure is a result of comparing the reagent of the present invention with the Roche electrochemical folic acid detection reagent in one embodiment of the present invention. DETAILED DESCRIPTION

[0061] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0062] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be an intermediate element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be an intermediate element at the same time.

[0063] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0064] definition

[0065] The term "sample" in the present invention may be a conventional biological sample, including but not limited to various liquids such as body fluids, cell culture supernatants, and tissue cell disruption solutions. The body fluids include but are not limited to whole blood, serum, plasma, various blood cells, blood clots, platelets, etc., as well as urine, semen, breast milk, sweat, interstitial fluid, interstitial lymph, bone marrow fluid, tissue fluid, saliva, gastric juice, joint fluid, pleural effusion, bile, ascites, amniotic fluid, etc. In some embodiments, the sample is serum. In addition, the source of the biological sample may be conventional mammals, including but not limited to humans, mice, rats, rabbits, dogs, pigs, sheep, cattle, horses, camels, monkeys, etc. In some embodiments, the sample is derived from humans. In some embodiments, the sample is serum derived from humans.

[0066] The term "solid phase carrier" in the present invention may be a conventional solid phase carrier in the art, and its material includes, but is not limited to, polymer compounds, glass, metal, magnetic body, resin composition containing magnetic body and combination thereof; wherein the polymer compound is, for example, polystyrene, polyethylene, polypropylene, polyester, poly (meth) acrylonitrile, styrene-butadiene copolymer, poly (meth) acrylate, fluororesin, cross-linked dextran, polysaccharide, etc. The shape of the solid phase medium in the present invention is not particularly limited, and examples thereof include tray-shaped, spherical (such as large-diameter silica spheres), particle-shaped, fibrous, rod-shaped, disk-shaped, container-shaped, microchannel, and plate. In the present invention, for the sake of solid-liquid separation, ease of washing, etc., it is preferred to use magnetic particles as the solid phase medium. The above-mentioned magnetic particles may be composed of ferrosoferric oxide (Fe 3 O 4 ), ferric oxide (γ-Fe 2 O 3 ), various ferrites, magnetic particles formed of metals such as iron, manganese, nickel, cobalt, and chromium, magnetic particles formed of alloys of these metals, and magnetic particles containing these magnetic particles in resins. As resins, for example, hydrophobic polymers, hydrophilic polymers, etc. can be cited.

[0067] The "adaptor" in the present invention specifically refers to a protein, the structure of which contains at least one domain that can specifically interact with other specific proteins.

[0068] The term "antibody" in the present invention includes any immunoglobulin, monoclonal antibody, polyclonal antibody, multivalent antibody, bivalent antibody, monovalent antibody, multispecific antibody or bispecific antibody that binds to a specific antigen. A natural complete antibody comprises two heavy chains and two light chains. Mammalian heavy chains are divided into α, δ, ε, γ and μ, each heavy chain comprising a variable region (VH) and a first, second, third and optional fourth constant region (CH1, CH2, CH3, CH4, respectively); mammalian light chains are divided into λ and κ, and each light chain comprises a variable region (VL) and a constant region. The antibody has a "Y" shape, and the stem of the Y consists of the second and third constant regions of two heavy chains bound together by disulfide bonds. Each arm of the Y comprises the variable region and the first constant region of a single heavy chain bound to the variable region and constant region of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in the two chains usually contain three highly variable loops, called complementarity determining regions (CDRs) (light chain CDRs include LCDR1, LCDR2 and LCDR3, and heavy chain CDRs include HCDR1, HCDR2, and HCDR3). The three CDRs are located between flanking segments called framework regions (FRs) (light chain FRs include LFR1, LFR2, LFR3, and LFR4, and heavy chain FRs include HFR1, HFR2, HFR3, and HFR4), which are highly conserved compared to CDRs. The constant regions of the heavy and light chains do not participate in antigen binding, but exhibit a variety of effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

[0069] If not otherwise specified, the term "cryoprotectant" in the present invention is a preparation used to protect biologically active ingredients with minimal or no loss of their active ingredients during the freezing process, and is used interchangeably with the "reagent storage agent" in the context.

[0070] In addition, it should be understood that the terms "first" and "second" in the present invention have no practical meaning and are only used to distinguish the same terms. For example, the first antibody / aptamer and the second antibody / aptamer can be the same antibody / aptamer or different antibodies / aptamers. The first antibody / aptamer and the second antibody / aptamer in the present invention can be classified according to the antibody specificity and can be one or more of polyclonal antibodies and monoclonal antibodies; according to the source classification, they can be one or more of mouse antibodies, rabbit antibodies, sheep antibodies and alpaca antibodies.

[0071] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0072] In the following examples, the magnetic microparticle-folic acid-folic acid binding protein complex antibody conjugate was purchased from Nanjing Oukai Biotechnology Co., Ltd., item number: Q301q1.

[0073] Folate binding protein was purchased from Beijing Baixinyi Biotechnology Co., Ltd., catalog number: A09M02-N.

[0074] Acridinium sulfonamide was purchased from Shenzhen Meikaite Technology Co., Ltd. as acridinium ester Intlus-B03, CAS No.: 199293-83-9.

[0075] Example 1

[0076] 1. Solution preparation

[0077] (1) Preparation of reagent storage agent: 5 g of mannitol, 50 g of trehalose, 50 g of sucrose, 30 g of bovine serum albumin, 200.5 g of Tween, 1 g of gelatin, 2 g of PEG 8000, 9 g of sodium chloride, and preservatives 3000.5 g of proclin and 4.986 g of 4-hydroxyethylpiperazineethanesulfonic acid. Add water to make up to 1 L and mix the above ingredients.

[0078] (2) Sample treatment solution: 21.4 g of 2-(N-morpholine)ethanesulfonic acid, 9 g of sodium chloride, 1 g of Triton 100, 10 g of sodium 2-mercaptoethanesulfonate, 0.5 g of preservative PC950, 0.5 g of preservative BND-10, add water to 1 L.

[0079] (3) Preparation of the excitation solution

[0080] Pre-excitation solution A: 0.25% (m / m) hydrogen peroxide plus 0.5% (m / m) nitric acid solution;

[0081] Exciting solution B: 1% (m / m) sodium hydroxide plus 0.1% (m / m) Triton 100.

[0082] 2. Reagent Preparation

[0083] (1) Preparation of reagent 1

[0084] Take the magnetic microparticle-folate-folate binding protein complex antibody conjugate, add the reagent storage agent to dilute the magnetic microparticle-folate-folate binding protein complex antibody conjugate to a magnetic microparticle working concentration of 0.5 mg / mL, and prepare reagent 1, i.e., folic acid magnetic microparticle coating raw material reagent.

[0085] (2) Preparation of Reagent 2

[0086] The folate binding protein and acridinium sulfonamide were mixed at a molar ratio of 1:15, and 50 mM CB buffer at pH 9.6 was added to make the final concentration of the folate binding protein 1 mg / mL, and coupled at 2-8°C for 4 hours; after coupling, lysine was added for blocking for 1 hour; after blocking, dialyzed 6 times, each time for 1 hour, and the folate binding protein labeled with acridinium ester was obtained after dialysis. The acridinium ester marker was diluted with a reagent storage agent to a folate binding protein concentration of 0.001 mg / mL to prepare reagent 2, i.e., folate acridinium ester labeled raw material reagent.

[0087] (3) Reagent spot freeze-drying

[0088] Reagent 1 and reagent 2 were added to the liquid nitrogen spotter respectively, and the spotting volume was set to 20 μL per drop, and the samples were spotted to form frozen microspheres 1 and frozen microspheres 2 respectively; frozen microspheres 1 and frozen microspheres 2 were then transferred to the freeze dryer respectively, and freeze-dried microsphere preparations 1 and microsphere freeze-dried preparations 2 were obtained after freeze drying. One of each of the two microsphere freeze-dried preparations was taken and placed in a reaction cup and sealed for storage.

[0089] 3. Detection time analysis

[0090] (1) Reaction process

[0091] Take 5 μL of folic acid clinical sample and add 125 μL of sample treatment solution prepared in "1. Solution preparation" to the reaction cup with microsphere lyophilized preparation 1 and microsphere lyophilized preparation 2; after incubation at 37°C, wash 4 times with washing solution; finally, add 100 μL of pre-excitation solution A and 100 μL of excitation solution B, and detect the relative luminescence intensity.

[0092] (2) Test results

[0093] Six clinical folic acid samples with gradient concentration changes (hereinafter referred to as "test samples") were taken. The research reagent was reacted according to the above reaction procedure for 5 minutes, 10 minutes and 15 minutes respectively. The sample signal value ratio and the signal value gradient (high value / low value) ratio were compared. The results are shown in Table 1. It can be seen from the sample signal value ratio and the signal value gradient (high value / low value) ratio that the sample signal value tends to be stable at 10 minutes to 15 minutes and is higher than the sample signal value at 5 minutes. The signal value gradient (high value / low value) ratio tends to be stable at 10 minutes to 15 minutes and is higher than the sample signal value gradient (high value / low value) ratio at 5 minutes. It can be seen that the reagent in this study has reached the optimal sensitivity of the reagent at 10 minutes to 15 minutes.

[0094] Table 1

[0095]

[0096] 4. Reagent performance test analysis

[0097] (1) Reaction process

[0098] Research reagents: Take 5μL of folic acid clinical sample, add 125μL of sample treatment solution prepared in "1. Solution preparation" above to the reaction cup with two microsphere freeze-dried preparations; incubate at 37℃ for 10min, wash 4 times with washing solution; finally add 100μL of pre-excitation solution A and 100μL of excitation solution B, detect relative luminescence intensity, and the detected luminescence value is proportional to the sample concentration value. The parameters of the fully automatic chemiluminescence instrument are set according to the above steps.

[0099] In the immune response, the company's linear reference is measured in three parallel wells, the average value is taken, the relative luminescence intensity value (RLU) and the corresponding concentration are linearly fitted, the main calibration curve is established, and scanned into the instrument. Through the saved calibration data, the system software automatically determines the patient's test results when the corresponding sample is tested. The result unit gives the test results of clinical samples in the form of ng / mL.

[0100] Control reagent: Roche electrochemical folic acid reagent is automatically tested according to the built-in reaction program of the fully automatic chemiluminescence instrument, and the test results of clinical samples are given in ng / mL.

[0101] (2) Test results

[0102] a. Comparison of sample detection gradient and detection signal value

[0103] Six clinical folic acid samples with gradient concentration values ​​(hereinafter referred to as "test samples") were collected and tested simultaneously with the research reagent and Roche electrochemical folic acid test reagent. The results are shown in Table 2. From the ratio of sample signal value and signal value gradient (high value / low value), it can be seen that the sample signal value of the research reagent is higher than that of the Roche reagent and the signal value gradient of the same concentration is better than that of the Roche reagent. It can be seen that the detection sensitivity of the research reagent is higher than that of the competitive reagent.

[0104] Table 2

[0105]

[0106] b. Clinical evaluation

[0107] 66 clinical serum samples were collected and tested with the kit in this study. The results were compared with those of Roche electrochemical folic acid test reagent. Figure 1 , regression equation y = 0.9851x-0.0468, correlation coefficient R 2 =0.9765, k of the two groups of reagents = 0.9851, and the k value is in the range of 0.9 to 1.1, indicating that the test results of the two groups of reagents are equivalent, R 2 =0.9765>0.95, indicating that the two groups of reagents have good correlation. The clinical evaluation results show that the two groups of reagents are equivalent in determining the content of folic acid in serum ( Figure 1 ).

[0108] c. Detection sensitivity

[0109] Use the zero concentration calibrator as the sample for testing, repeat the measurement 20 times, obtain the RLU value of the 20 test results, and calculate the average value and standard deviation (SD), The corresponding RLU value is substituted into the main calibration curve to find the corresponding concentration value, which is no higher than 0.1 ng / mL. 0.1 ng / mL is set as the minimum detection limit. The detection sensitivity of competitive reagents on the market is generally above 0.3 ng / mL.

[0110] d. Stability

[0111] The chemiluminescent immune microsphere freeze-dried preparation prepared in this example was placed at 45°C for 30 days, and the linear reference of the enterprise was tested. The relative luminescence intensity was compared with that before placement, and it was determined that the change in thermal stability after 30 days of storage at 45°C was ≤10%. Compared with conventional liquid chemiluminescent reagents, the stability was significantly improved. The change in thermal stability of conventional chemiluminescent reagents (liquid phase) after 3 days of storage at 45°C exceeded 30%.

[0112] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0113] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention shall be based on the attached claims, and the specification and the drawings are used to interpret the claims.

Claims

1. A kit for detecting folic acid, characterized in that: The method comprises a folate binding protein signal marker capable of specifically binding to the folate to be detected, a solid phase medium capable of specifically binding to a complex formed by the folate to be detected and the folate binding protein, and a sample processing solution.

2. The kit according to claim 1, characterized in that The signal marker is connected to the first antibody / aptamer that specifically binds to the folic acid to be detected.

3. The kit according to claim 1 or 2, characterized in that The signal marker is a luminescent signal marker; Optionally, the luminescent signal marker is an acridinium ester.

4. The kit according to any one of claims 1 to 3, characterized in that The solid phase medium comprises a solid phase carrier and a second antibody / aptamer connected to the solid phase carrier and capable of specifically binding to the complex; Optionally, the solid support is selected from the group consisting of magnetic beads, plates and membranes; Optionally, the second antibody / aptamer is connected to the solid phase carrier by covalent binding of surface functional groups or non-covalent passive adsorption; More optionally, the surface functional group is selected from the group consisting of amino and carboxyl groups; More optionally, the second antibody / aptamer is linked to the solid phase carrier via a tosyl group.

5. The kit according to any one of claims 1 to 4, characterized in that The folate binding protein signal marker capable of specifically binding to the folic acid to be detected is a freeze-dried frozen microsphere 1; Optionally, the preparation method of the cryomicrospheres 1 comprises: using a cryoprotectant to dilute a folate binding protein signal marker that can specifically bind to the folate to be detected and then freeze-drying the diluted folate binding protein signal marker; More optionally, after dilution, the concentration of the folate binding protein signal marker capable of specifically binding to the folic acid to be detected is 0.0005-0.002 mg / mL; and / or, The solid phase medium capable of specifically binding to the complex formed by the folic acid to be detected and the folic acid binding protein is a freeze-dried frozen microsphere 2; Optionally, the preparation method of the cryo-microspheres 2 comprises: using a cryoprotectant to dilute the solid phase medium capable of specifically binding to the complex and then freeze-drying it; More optionally, the concentration of the solid phase medium capable of specifically binding to the complex after dilution is 0.1-1 mg / mL.

6. The kit according to claim 5, characterized in that Based on the total mass of the cryoprotectant, the cryoprotectant comprises: Mannitol or casein 0.2wt%-1wt%, trehalose and / or sucrose 4wt%-20wt%, bovine serum albumin or glucose 1wt%-5wt%, surfactant 0.03wt%-0.5wt%, gelatin or dextran 0.05wt%-1wt%, PEG 8000 or PEG 20000 0.1wt%-1wt%, sodium chloride 0.5wt%-2wt%, preservative 0.03wt%-0.5wt%, buffer 0.2wt%-1wt%, and water 68wt%-93.89wt%; Optionally, the cryoprotectant satisfies one or more of the following conditions: The buffer is selected from the group consisting of 4-hydroxyethylpiperazineethanesulfonic acid, trishydroxymethylaminomethane, 2-(N-morpholine)ethanesulfonic acid, citrate, acetate and borate; The surfactant is selected from the group consisting of Tween 20, Tween 80, Triton 100 and S9; The preservative is selected from the group consisting of PC950, BND-10, PC300, gentamicin sulfate and proclin300.

7. The kit according to any one of claims 1 to 6, characterized in that Based on the total mass of the sample processing liquid, the sample processing liquid includes: 2wt%-4wt% of a buffer, 0.5wt%-2wt% of sodium chloride, 0.05wt%-0.2wt% of a surfactant, 0.5wt%-2wt% of a reducing agent, 0.05wt%-0.2wt% of a preservative PC950, 0.05wt%-0.2wt% of a preservative BND-10, and 91.4wt%-96.85wt% of water.

8. The kit according to claim 7, characterized in that The sample processing solution meets one or more of the following conditions: The buffer is selected from the group consisting of 2-(N-morpholine)ethanesulfonic acid, tris(hydroxymethylaminomethane), citrate and acetate; The reducing agent is selected from the group consisting of sodium 2-mercaptoethane sulfonate, dithiothreitol, tris(2-chloroethyl) phosphate and thioglycerol; The surfactant is selected from the group consisting of Triton 100, Tween 20, Tween 80 and S9.

9. A method for detecting folic acid using the kit according to any one of claims 1 to 8, characterized in that: It includes the following steps: (1) mixing and incubating a sample containing the folic acid to be tested, a folate binding protein signal marker capable of specifically binding to the folic acid to be tested, a solid phase medium capable of specifically binding to a complex formed by the folic acid to be tested and the folate binding protein, and a sample processing solution; (2) Read the signal marker to determine the folic acid content.

10. The method according to claim 9, characterized in that It includes the following steps: (1) mixing and incubating a sample containing folic acid to be tested with acridinium ester-labeled folate binding protein, tosyl-modified magnetic microparticle-folate-folate binding protein complex antibody conjugate, and a sample processing solution; (2) Add acridinium ester excitation solution and read the luminescence value.

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