Vitamin D homogeneous detection method and reagent

Abstract

The invention discloses a homogeneous immunodetection method and reagent for vitamin D, and relates to the field of immunodiagnosis. The alkaline dissociation agent is adopted to treat a vitamin D sample, so that sensitive and accurate detection in a homogeneous immunoreaction system is realized.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] The present invention claims priority to Chinese patent application No. 202311331182.6 filed with the Chinese Patent Office on October 12, 2023, entitled “Homogeneous Detection Method and Reagent for Vitamin D”, the entire contents of which are incorporated by reference into the present disclosure. Technical Field

[0003] The present invention relates to the technical field of immunodiagnosis, and in particular to a vitamin D homogeneous detection method and reagent. Background Art

[0004] Vitamin D (Vitamin D, VD) is a lipophilic steroid derivative. Among the five compounds in the vitamin D family, the most important ones for humans are vitamin D2 (ergocalciferol) and vitamin D3 (also known as cholecalciferol). In terms of chemical structure, vitamin D2 has one more methyl group and carbon-carbon double bond than vitamin D3.

[0005] The main natural source of vitamin D is the chemical reaction in the lower layer of the skin epidermis after exposure to sunlight to produce cholecalciferol, which is further hydroxylated by the kidneys to form the biologically active form of vitamin D, calcitriol (also known as 1,25-dihydroxycholecalciferol). There is increasing evidence that vitamin D deficiency may be associated with the incidence and progression of many diseases, including osteoporosis, chronic diseases, cancer progression, autoimmunity, and type 2 diabetes. The content of vitamin D in the body is closely related to age and the degree of light received. The vitamin D deficiency rate is relatively low in people under the age of 14, and the deficiency rate continues to increase with age or lack of light. Vitamin D testing is an important task to prevent vitamin D deficiency.

[0006] The content of vitamin D in the human body is between 30ng / ml and 100ng / ml. If it exceeds 150ng / ml, it indicates vitamin D poisoning. If the vitamin D concentration is between 12ng / ml and 20ng / ml, it is insufficient. If it is less than 12ng / ml, it indicates deficiency. Vitamin D in the human body mainly exists in a state of binding to vitamin D carrier protein (Vitamin D Binding Protein, VDBP, 88%) or albumin (12%). Only 0.04% of vitamin D circulates in the body in a free form. Therefore, how to dissociate vitamin D bound to vitamin D carrier protein or albumin is the key to vitamin D detection.

[0007] Immunoassay is a fast and effective method for detecting vitamin D. Immunoassay can be divided into homogeneous and heterogeneous detection according to whether there is a phase separation step in the detection process. That is, heterogeneous detection involves a solid-liquid phase separation process. Through this step, the substances used for sample processing and affecting the subsequent vitamin D can be separated, so that the immune response is not interfered by the sample processing reagents, and the detection can be carried out accurately. Common heterogeneous immunoassay methods include chemiluminescence, fluorescence, enzyme-linked immunosorbent assay, etc. These methodologies can remove sample processing reagents and other components from the detection system through the phase separation process to avoid adverse effects on the immune response during the detection, thereby achieving higher detection accuracy.

[0008] Homogeneous immunoassay methods such as immunoturbidimetry can significantly reduce the complexity of detection and shorten the detection time because there is no phase separation step. However, at the same time, the homogeneous immunoassay reaction will have a dissociation agent that has a significant impact on the volume and reaction conditions of the immune system during the immunoassay, resulting in insufficient detection accuracy. In addition, in addition to the phase separation step, the existing vitamin D sample processing scheme also has problems such as low dissociation efficiency, long dissociation time, and large amount of dissociation agent, which does not meet the requirements of fast and efficient homogeneous reaction systems. Summary of the invention

[0009] The purpose of the present invention is to provide a method and reagent for homogeneous immunoassay of vitamin D, which solves the defects existing in the prior art and is used to detect the content of vitamin D in a sample.

[0010] In order to achieve the above object, the present invention provides a method for homogeneous immunoassay of vitamin D, comprising the following steps:

[0011] a) contacting the sample to be tested with an alkaline dissociation agent, wherein the pH of the dissociation agent is in the range of 7.0-12.0;

[0012] b) Detecting the presence and / or amount of vitamin D in the sample.

[0013] In an optional embodiment, the above method uses at least two antibodies; optionally, the above method is a non-competitive method; optionally, the above method is a sandwich method; optionally, the first antibody specifically binds to vitamin D, and the second antibody is a complex formed by specifically binding to the first antibody and vitamin D; optionally, the first antibody and the second antibody are added at the same time.

[0014] In an optional embodiment, the above method is double latex immunoturbidimetry; optionally, the above antibodies are coupled to latex respectively.

[0015] In an optional embodiment, the above method does not include an acid-base neutralization step.

[0016] In an optional embodiment, the pH range of the above-mentioned dissociation agent is 7.5-12; preferably, the pH range of the above-mentioned dissociation agent is 7.5-10.5; preferably, the pH range of the above-mentioned dissociation agent is 7.5-9.5; preferably, the pH range of the above-mentioned dissociation agent is 7.5-8.5; preferably, the pH range of the above-mentioned dissociation agent is 7.0-8.6; the pH range of the above-mentioned dissociation agent is 7.2-8.0.

[0017] In an optional embodiment, the above method comprises a sample, an alkaline dissociation agent, and an antibody, and the pH range of the entire detection system is between 7.0-13.0; preferably, the pH range of the aforementioned detection system is 7.5-12.0; preferably, the pH range of the aforementioned detection system is 7.5-10.5; preferably, the pH range of the aforementioned detection system is 7.5-9.5; preferably, the pH range of the aforementioned detection system is 7.5-8.5; preferably, the pH range of the aforementioned detection system is 7.0-9.0.

[0018] In an optional embodiment, the volume ratio of the dissociation agent to the sample to be tested is 100:1-10:1; preferably, the volume ratio of the dissociation agent to the sample to be tested is 80:1-20:1.

[0019] In an optional embodiment, the time for treating the sample with the dissociation agent is 1 min to 10 min; preferably, the time for treating the sample with the dissociation agent is 1 min to 5 min.

[0020] In an optional embodiment, the correlation coefficient R of the above method for vitamin D 2 Greater than 0.95.

[0021] In an optional embodiment, the vitamin D sample to be tested is whole blood, serum or plasma.

[0022] The present invention further provides a reagent for detecting vitamin D, wherein the reagent includes the dissociation agent used in the above vitamin D detection method.

[0023] In an optional embodiment, the above dissociating agent component includes one or more of a surfactant and guanidine;

[0024] Preferably, the above dissociating agent includes a perfluoroalkyl surfactant and guanidine hydrochloride;

[0025] Preferably, the above surfactant is S9;

[0026] Preferably, the above dissociation agent further comprises dithiothreitol;

[0027] Preferably, the above reagents further include one or more of C1-4 alcohol, EDTA, and dextran.

[0028] In an optional embodiment, the above reagent includes at least two antibodies; optionally, the first antibody specifically binds to vitamin D, and the second antibody specifically binds to a complex formed by the first antibody and vitamin D;

[0029] Preferably, the first antibody and / or the second antibody is coupled to nanoparticles; optionally, the average particle size of the nanoparticles is 150-400 nm; optionally, the nanoparticles are polystyrene latex microspheres;

[0030] Preferably, the reagent further comprises a protective agent, an antibacterial agent, and an electrolyte.

[0031] In an optional embodiment, the average particle size of the nanoparticles is in the range of 30-500nm; preferably, the nanoparticles are latex particles with an average particle size of 150-400nm, and the latex particles include polystyrene, polymethyl methacrylate, polymethylnaphthalene, polydivinylbenzene, polyethylenenaphthalene, styrene, divinylbenzene acrylate, naphthalene, silicon, and silicon dioxide.

[0032] The present invention unexpectedly discovered that in the homogeneous immunoassay process of vitamin D, the use of an alkaline dissociator with a pH range of 7.0-12.0 can quickly and efficiently dissociate vitamin D in the sample to be tested, and the dissociation effect is stable. The alkaline dissociator has little effect on the subsequent immune response, and is very suitable for application in a homogeneous reaction system without phase separation; and it can effectively reduce the influence of the alkaline reagent in the instrument's own liquid path cleaning system on the test results during the detection process, and has a very high correlation with the fixed sample reference value. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0034] Figure 1 is the test result of dissociation agent T1 (pH7.5);

[0035] Figure 2 is the test result of dissociation agent T2 (pH7.5);

[0036] Figure 3 It is the test result of dissociation agent T3 (pH7.0);

[0037] Figure 4 This is the test result of dissociation agent T4 (pH 8.6);

[0038] Figure 5 It is the test result of dissociation agent T5 (pH11.9);

[0039] Figure 6 This is the test result of dissociation agent T3 (pH 8.0);

[0040] Figure 7 This is the test result of dissociation agent T3 (pH 3.8). DETAILED DESCRIPTION

[0041] In a first aspect, the present invention provides a homogeneous immunoassay method for vitamin D, comprising the following steps:

[0042] a) contacting a sample with an alkaline dissociation agent, wherein the pH of the dissociation agent is in the range of 7.0-12.0; b) detecting the presence and / or content of vitamin D in the sample.

[0043] In an optional embodiment, the method for detecting the vitamin D content in a sample uses at least two antibodies; optionally, the method for detecting the vitamin D content in a sample is a non-competitive method; optionally, the method for detecting the vitamin D content in a sample is a sandwich method; optionally, the first antibody specifically binds to vitamin D, and the second antibody specifically binds to a complex formed by the first antibody and vitamin D; optionally, the first antibody and the second antibody are added at the same time.

[0044] In an optional embodiment, the method for detecting the vitamin D content in the sample is double latex immunoturbidimetry; optionally, the vitamin D content in the sample is detected using at least two antibodies coupled to latex respectively.

[0045] As used herein, "specific binding" refers to the specificity of a binding agent (e.g., an antibody) that allows it to preferentially bind to a determined analyte or target, and when other potential targets are present, the recognition of a specific analyte or target by a binding agent or antibody is a feature of this binding. Of course, it is understood that a certain degree of nonspecific interaction may occur between molecules and non-target molecules. In some embodiments, the binding agent that specifically binds to the analyte avoids binding to other interfering parts in the sample to be tested. The specific binding between the antibody and the antigen, the affinity between the two can be measured by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art, such as surface plasmon resonance (SPR) technology, analysis on a BIAcore instrument (Liljeblad et al., Glyco J 17, 323-329 (2000)) and traditional binding assays (Heeley, Endocr Res 28, 217-229 (2002)). In one embodiment, the extent of binding of the antibody to an unrelated antigen is less than about 10% of the extent of binding of the antibody to the antigen. In certain embodiments, the dissociation constant (KD) between the antigen and the antibody is ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or ≤0.001 nM (e.g., 10 -7 M or lower, e.g. 10 -8 M to 10 -13 M, for example 10 -9 M to 10 -13 M).

[0046] The concentration herein, unless otherwise specified, should be understood as mass volume percentage or mass volume percentage. For example, the concentration of 2% guanidine hydrochloride referred to in this application means that 100 ml contains 2 g guanidine hydrochloride.

[0047] In an optional embodiment, before detecting the vitamin D content in the sample, the step of separating the dissociation agent from vitamin D is not included; further, the step of separating the vitamin D binding protein from vitamin D is not included; further, any washing step is not included.

[0048] In an optional embodiment, the acid-base neutralization step is not included before detecting the vitamin D content in the sample.

[0049] In an optional embodiment, during the vitamin D detection process, the vitamin D sample is first contacted with an alkaline dissociator, and after incubation for a period of time, latex particles coated with two different antibodies are added; or the vitamin D sample is first contacted with an alkaline dissociator, and after incubation for a period of time, latex particles coated with the first antibody and latex particles coated with the second antibody are added in sequence.

[0050] In an alternative embodiment, the pH of the dissociation agent is 7.0, 7.2, 7.3, 7.5, 8.0, 8.5, 8.6, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0 or any value within the range of 7.0-12.0.

[0051] In an optional embodiment, the "detection system" refers to a reaction system comprising a sample, an alkaline pH dissociation agent, and a detection reagent during detection. The detection reagent includes a detection antibody, and the detection antibody can be a particle (e.g., a latex particle) coated with two specific antibodies. The pH can be any suitable value or range, for example, the pH range of the detection system is 7 to about 13, for example, it can be 7.0, 7.5, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11.5, 12.0, 13.0 or any value between 7.0 and 13.0.

[0052] In an optional embodiment, the dissociator can be boric acid-sodium tetraborate, disodium hydrogen phosphate-potassium dihydrogen phosphate, disodium hydrogen phosphate-citric acid, sodium carbonate-sodium bicarbonate or sodium tetraborate-sodium hydroxide, or other dissociators having a dissociation function in an alkaline pH range, and the concentration of the above dissociator is 10mM-120mM, for example, it can be 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM, 100mM, 110mM, 120mM or any value between 10mM and 120mM.

[0053] In an optional embodiment, the volume ratio of the dissociation agent to the sample to be tested is 100:1, 90:1, 80:1, 70:1, 60:1, 50:1, 40:1, 30:1, 20:1, 10:1 or any value between 100:1-10:1.

[0054] In an optional embodiment, the time for the dissociation agent to treat the sample to be tested is: 1 min, 3 min, 5 min, 7 min, 9 min, 10 min or any value between 1 min and 10 min.

[0055] The method can be carried out for any suitable reaction time. In certain embodiments, the total detection time of the method is about 60 min or shorter, for example, about 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 30, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 minute or any value between 59-1 min.

[0056] In an optional embodiment, detecting the amount of vitamin D in a sample comprises evaluating the binding between the antibody and the vitamin D to determine the presence, absence and / or amount of the vitamin D in the sample. Optionally, the antibody is different from the natural vitamin D binding protein of vitamin D.

[0057] In an optional embodiment, the dissociation agent further includes 0.05%-1% perfluoroalkyl surfactant, for example, it can be sodium perfluorooctanoate, for example, it can be 0.05%, 0.1%, 0.3%, 0.5%, 0.7%, 0.9%, 1% or any value between 0.05% and 1%. Optionally, the above dissociation agent also includes 0.1%-5% guanidine hydrochloride (GC), for example, it can be 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or any value between 0.1% and 5%; Optionally, the above dissociation agent also includes 0.01%-0.1% dithiothreitol (DTT); for example, it can be 0.01%, 0.05%, 0.1% or any value between 0.01% and 0.1%. Optionally, the reagent further comprises 0.1%-2% C1-4 alcohol, for example, 0.1%, 0.5%, 1%, 1.5%, 2% or any value between 0.1% and 2%. Optionally, the reagent further comprises 1-100mM EDTA (Ethylene Diamine Tetraacetic Acid), for example, 1mM, 10mM, 20mM, 30mM, 40mM, 50mM, 60mM, 70mM, 80mM, 90mM or any value between 1mM-100mM. Optionally, the reagent further comprises 0.1%-2% dextran, for example, 0.1%, 0.5%, 1%, 1.5%, 2% or any value between 0.1% and 2%. The molecular weight of dextran can be 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 50,000, 100,000, 200,000, 250,000, 300,000, 350,000, 400,000, 450,000, 500,000, 550,000, 600,00, 650,000 or 700,000. Optionally, the above dissociation agent also includes 0.05%-0.5% S9, for example, it can be 0.05%, 0.1%, 0.15%, 0.2%, 0.3%, 0.4%, 0.5% or any value between 0.05% and 0.5%. Optionally, the reagent further comprises a salt, such as sodium chloride, magnesium chloride, etc., and the concentration thereof is 200-600 mM, for example, 200, 300, 400, 500, 600 mM or any value between 200 mM and 600 mM.

[0058] Preferably, the two antibodies are coupled to nanoparticles, respectively, and the nanoparticles can have any suitable size or diameter. For example, the diameter of the nanoparticles is 30nm, 40nm, 50nm, 60nm, 70nm, 80nm, 90nm, 100nm, 150nm, 200nm, 250nm, 300nm, 350nm, 400nm, 450nm, 500nm or any value between 30 and 500nm.

[0059] In an optional embodiment, the above-mentioned nanoparticles are latex particles, and the aforementioned latex particles include polystyrene, polymethyl methacrylate, polymethylnaphthalene, polydivinylbenzene, polyethylenenaphthalene, copolymers of styrene, divinylbenzene acrylate, naphthalene, carbon 60, magnetic beads, gold, silver, silicon, silicon dioxide, chromium dioxide and / or titanium dioxide.

[0060] In an optional embodiment, the above-mentioned sample to be tested is a cell or cell line, a cell supernatant, a cell lysate, a platelet, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate and tissue culture fluid, tissue extracts such as homogenized tissue, tumor tissue, cell extracts, and combinations thereof.

[0061] Another aspect of the present invention provides a reagent for detecting vitamin D, wherein the reagent includes a dissociation agent used in the above-mentioned vitamin D homogeneous immunoassay method.

[0062] The detection methods and detection reagents of the present invention can achieve any suitable accuracy. In some embodiments, the correlation coefficient R between the detection results of the present invention and the reference value of the detection results of other methods is 2 Greater than 0.9, for example, can be 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or more, or any value between 0.9 and 1. For example, for vitamin D levels of 30 ng / ml or less, for example, about 30 ng / ml, 25 ng / ml, 20 ng / ml, 15 ng / ml, 10 ng / ml, 5 ng / ml, 4 ng / ml, 3 ng / ml, 2 ng / ml, 1 ng / ml or less, the method can achieve a correlation coefficient of about 0.9 or greater, for example, 0.9, 0.91, 0.92, 0.93, 0.94, 0.95, 0.96, 0.97, 0.98, 0.99 or any value between 0.9 and 1.

[0063] In some embodiments, the first antibody can be coated on the first nanoparticle, and another antibody or the second antibody can be labeled with the second nanoparticle. When vitamin D is present in the detection system, the second antibody binds to the vitamin D in the sample and the first antibody coated on the surface or particle to form a "sandwich" complex (first antibody-vitamin D-second antibody), and the amount of the "sandwich" complex formed on the particle is proportional to the amount of vitamin D in the sample, and can be quantitatively evaluated by detecting the change in the absorbance signal in the reaction system.

[0064] As used herein, "antibodies" include not only complete polyclonal or monoclonal antibodies, but also fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (ScFv), bispecific antibodies, multispecific antibodies formed by antibody fragments, mutants thereof, fusion proteins comprising antibody portions, and any other modified structures of immunoglobulin molecules comprising the desired specific antigen recognition site. Antibodies include antibodies of any class, such as IgG, IgA or IgM (or their subclasses), and antibodies do not necessarily belong to any particular class.

[0065] As used herein, the competition method includes direct competition method and indirect competition method. The direct competition method is a method in which an antibody against the target antigen to be determined is immobilized on a solid phase, and after a blocking treatment (treating the solid phase with a protein solution such as serum albumin) for preventing nonspecific adsorption, the antibody, the sample containing the target antigen and a certain amount of labeled antigen are reacted, and after washing, the label bound to the solid phase is quantified. The antigen and the labeled antigen in the sample are competitively bound to the antibody, so the more antigen in the sample, the less label is bound to the solid phase. Various known concentrations of antigen standard solutions are prepared, and the labeling amount is measured by immobilizing on the solid phase (determining the absorbance, luminescence intensity, fluorescence intensity, etc. according to the nature of the label), and a standard curve is prepared with the antigen concentration as the horizontal axis and the labeling amount as the vertical axis. For unknown samples, the labeling amount is measured, and the amount of antigen in the unknown sample can be measured by substituting the measured labeling amount into the standard curve. In the indirect competition method, the target antigen is immobilized on a solid phase. Next, after the blocking treatment of the solid phase, the test sample containing the target antigen and a certain amount of anti-target antigen antibody are mixed to react with the solid-phased antigen. After washing, the anti-target antigen antibody bound to the solid phase is quantified. This can be done by reacting a labeled secondary antibody against the anti-target antigen antibody, and measuring the labeled amount after washing. Prepare various antigen standard solutions of known concentrations, solid-phase them on the solid phase and measure the labeled amount to prepare a standard curve. For unknown test samples, the amount of antigen in the unknown test sample can be determined by measuring the labeled amount and substituting the measured labeled amount into the standard curve. It should be noted that it is also possible to use a labeled primary antibody instead of a labeled secondary antibody.

[0066] As used herein, the term "detection" is intended to include both quantitative and qualitative detection, i.e., obtaining an absolute value for the amount or concentration of an analyte in a sample, as well as obtaining an index, ratio, percentage, image or other value indicative of the level of the analyte in the sample. Detection may be direct or indirect, and the chemical substance actually detected need not be the analyte itself, but may be, for example, a derivative thereof or some other substance.

[0067] As used in this article, the term "fixed value sample" refers to a sample whose vitamin D concentration is measured by a recognized method. The specific measurement method includes measuring the concentration of the sample using mass spectrometry, commercially available vitamin D reagent detection, or other recognized vitamin D concentration detection methods.

[0068] In the present invention, "vitamin D" refers to all members or forms of the vitamin D family, which is a group of fat-soluble open-ring steroids responsible for intestinal absorption of calcium and phosphate. Exemplary vitamin D forms include vitamin D1, D2 (ergocalciferol), D3 (cholecalciferol), D4 and D5. Exemplary vitamin D also includes calcidiol (calcifediol, INN), also known as calcifediol (INN); 25-hydroxycholecalciferol, or 25-hydroxyvitamin D, referred to as 25 (OH) D, which is a specific vitamin D metabolite, and its content is detected in serum to determine a person's vitamin D status; and calcitriol, a biologically active form of vitamin D. In some embodiments, "vitamin D" refers to 25-hydroxyvitamin D, referred to as 25 (OH) D, including D2 and D3 forms.

[0069] It should be noted that the meaning of the reagent in the present invention and the meaning of the kit can be regarded as equivalent to each other.

[0070] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0071] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the present disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used for the practice or testing of the preparations or unit doses herein, some methods and materials are now described. Unless otherwise stated, the techniques adopted or considered herein are standard methods. Materials, methods and examples are illustrative and non-restrictive only.

[0072] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0073] Example 1

[0074] Dissociating agents of different pH are formed by varying the amounts of the components in the dissociating agent to adjust the pH to a specific value.

[0075] Table 1

[0076] Dissociation agent code Reagent components T1 20mM Boric acid-sodium tetraborate T2 20mM sodium hydrogen phosphate-potassium dihydrogen phosphate T3 100mM sodium hydrogen phosphate-citric acid T4 50mM Sodium carbonate-sodium bicarbonate T5 25mM Sodium Tetraborate-Sodium Hydroxide

[0077] Example 2

[0078] Vitamin D test reagent preparation method:

[0079] (1) Mix 10 ml of anti-vitamin D antibody (3.1 mg / ml) with 20 ml of 20 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate solution at room temperature for 10 minutes and set aside;

[0080] (2) Dissolve 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) in MES solution at pH 5.5 to a concentration of 10 mg / ml;

[0081] (3) 0.5 ml of latex particles with a particle size of 300-400 nm were added to 24.5 ml of MES solution at pH 5.5 to make the latex particles activated at a concentration of 0.2%. After adding 0.5 ml of 10 mg / ml EDC, the mixture was shaken at 37°C for 0.5 hours to form a sensitized latex particle buffer;

[0082] (4) adding anti-vitamin D antibody and anti-vitamin D antibody-vitamin D complex antibody to the sensitized latex particle buffer, and adding 40 ml of 20 mmol / L disodium hydrogen phosphate-sodium dihydrogen phosphate solution, and mixing in a shaker at 37° C. for 2 hours;

[0083] (5) Add 2 ml of blocking solution (glycine buffer containing 20% ​​Tween-20) and shake at room temperature overnight;

[0084] (6) Centrifugation at 15,000 rpm for 40 min at 4°C;

[0085] (7) Discard the supernatant, dissolve the latex particles with 100 ml of deionized water, and repeat the centrifugation and washing;

[0086] (8) Discard the supernatant and add 100 ml of working solution (HEPES buffer containing 0.5% BSA and 0.05% NaN3, pH 8.5). The solution obtained after ultrasonic resuspension is reagent R2.

[0087] Vitamin D calibrators are as follows: add 20, 50, 100, and 150 ng / ml of vitamin D to the working solution, respectively, and sterilize and filter.

[0088] Example 3

[0089] Vitamin D Testing Equipment and Process

[0090] Detection tools: Mindray BS480 fully automatic biochemical analyzer;

[0091] Wavelength: 700nm;

[0092] Sample volume: 3.0ul;

[0093] R1: 160ul, R1 includes dissociation agent

[0094] R2: 40ul, R2 is the detection reagent in Example 2

[0095] Detection conditions: 37°C; after mixing the sample with the dissociation agent for 5 minutes, add R2, mix for 15 seconds and read the absorbance A1, continue the reaction for 5 minutes and read the absorbance A2. The calculation of the reaction absorbance is the difference between A2 and A1.

[0096] Calculation method: With the concentration of the calibrator as the horizontal axis and the corresponding ΔOD700 as the vertical axis, a dose / response curve is made based on the absorbance and the value of the calibrator. The sample content can be calculated on the dose / response curve based on its absorbance value.

[0097] Example 4

[0098] The fixed value sample was used as the detection object, and alkaline dissociation agents (T1-T5) of different pH were used as R1. The configuration of the detection reagents was referred to Example 2, and the detection parameters and steps were referred to Example 3. The results of the sample concentration (ng / ml) were shown in Table 2 below:

[0099] Table 2

[0100]

[0101]

[0102] From the above results, it can be seen that vitamin D can be effectively detected when an alkaline dissociator is added.

[0103] Example 5

[0104] The vitamin D detection reagent of the alkaline dissociator of the present invention is used to detect the fixed value sample, and the fixed value sample concentration is used as a reference value to evaluate the detection performance of the scheme of the present invention for vitamin D.

[0105] The preparation of the detection reagent in this example refers to Example 2, and the detection parameters and steps refer to Example 3.

[0106] Vitamin D was detected using R1 prepared with T1, T2, T3, T4, and T5 of different pH values. The correlations between the test results of clinical samples and the reference values ​​are shown in the following table. Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 As shown, the linear correlation function and correlation coefficient are as shown in Table 3 below:

[0107] Table 3

[0108] Dissociative Agents Linear correlation function Correlation coefficient T1(pH7.5) y=1.05x-0.0699 <![CDATA[R 2 =0.9606]]> T2(pH7.5) y=1.0207x-0.188 <![CDATA[R 2 =0.9535]]> T3(pH7.0) y=0.9924x+1.3057 <![CDATA[R 2 =0.9609]]> T4 (pH 8.6) y=1.0093x+0.1363 <![CDATA[R 2 =0.969]]> T5 (pH 11.9) y=0.9996x-0.534 <![CDATA[R 2 =0.9556]]>

[0109] From the above results, it can be concluded that the reagent scheme of the alkaline treatment solution of the present invention is highly consistent with the reference value of the fixed value sample, and the linear correlation R 2 Both are greater than 0.95.

[0110] The pH of the dissociator is adjusted to prepare a vitamin D detection reagent containing an acidic dissociator. The same fixed value sample is tested using a vitamin D detection reagent containing an acidic dissociator and a vitamin D detection reagent containing an alkaline dissociator of the present invention. The test results of the two are as follows: Figure 6 , Figure 7 As shown, the linear correlation function and correlation coefficient are as shown in Table 4 below:

[0111] Table 4

[0112] Dissociative Agents Linear correlation function Correlation coefficient T3(pH8.0) y=0.7091x-3.6194 R2＝0.9625 T3(pH3.8) y=0.7435x+0.3247 R2＝0.9031

[0113] From the above data, it can be seen that the test results obtained using the vitamin D detection reagent containing alkaline dissociator have a better correlation with the reference value of the fixed value sample.

[0114] Example 6

[0115] In this embodiment, the following R1 reagent is prepared: 0.1% DTT is added to T1, and the pH is adjusted to 8.5; 2% GC is added to T1, and the pH is adjusted to 8.5 and 7.5 respectively; 2% GC and 0.1% DTT are added to T2, and the pH is adjusted to 7.5; 0.5% perfluorooctanoic acid is added to T5, and the pH is adjusted to 11.9; 0.5% perfluorooctanoic acid and 0.1% DTT are added to T5, and the pH is adjusted to 11.9. The above-prepared R1 reagent is used to detect and analyze vitamin D samples including abnormal detection. The preparation of the detection reagent refers to Example 2, and the detection parameters and steps refer to Example 3. The specific detection results are shown in Table 5 below:

[0116] Table 5

[0117]

[0118] From the above data, it can be seen that by adding DTT, GC and perfluorooctanoic acid to the alkaline dissociator, vitamins can be detected more effectively, and at low concentrations, the addition of DTT, GC and perfluorooctanoic acid has higher detection accuracy.

[0119] Example 7

[0120] In this embodiment, vitamin D dissociators (R1) with different pH values ​​were prepared, and the dissociators contained 0.14% S9 (m / v), 0.15% sodium perfluorooctanoate (m / v), 4.5% guanidine hydrochloride (m / v) and a base solution, wherein the base solution included 50 mM Tris, 450 mM NaCl, 2% dextran (m / v) and 1% P300 (v / v).

[0121] According to the method in Implementation 2 and Example 3, the fixed value sample was used as the detection object, and the alkaline dissociators of different pH were used as the dissociators of vitamin D in the detection process. The concentration (ng / ml) results were shown in Table 6 below:

[0122] Table 6

[0123]

[0124] From the above results, it can be seen that alkaline dissociation agents can effectively detect vitamin D.

[0125] Example 8

[0126] In this example, the preparation of the detection reagent refers to Example 2, and the detection parameters and detection steps refer to Example 3.

[0127] First, the concentration of dextran was fixed at 1% in an alkaline dissociator, and the final concentration of S9 was gradually increased. The other components and concentrations were the same as those in Example 7. Vitamin D in clinical samples (serum) was detected and analyzed. The measured concentrations (ng / ml) are shown in Table 7:

[0128] Table 7

[0129]

[0130] Next, the final concentration of S9 was fixed at 0.16% in the alkaline dissociation agent, and the final concentration of dextran was gradually increased. The other components and concentrations were the same as in Example 7. Vitamin D in clinical samples (serum) was detected and analyzed. The measured concentrations (ng / ml) are shown in Table 8:

[0131] Table 8

[0132]

[0133] From the above results, it can be seen that alkaline dissociation agents can effectively detect vitamin D.

[0134] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for homogeneous immunoassay of vitamin D, characterized in that: The following steps are involved: a) contacting the sample to be tested with an alkaline dissociation agent, wherein the pH of the dissociation agent is in the range of 7.0-12.0; b) Detecting the presence and / or amount of vitamin D in the sample.

2. The method according to claim 1, characterized in that The method uses at least two antibodies: Optionally, the method is a non-competitive method; Optionally, the method is a sandwich method; Optionally, the first antibody specifically binds to vitamin D, and the second antibody specifically binds to a complex formed by the first antibody and vitamin D; Optionally, the primary antibody and the secondary antibody are added simultaneously.

3. The method according to claim 2, characterized in that The method is double latex immunoturbidimetry; Optionally, the two antibodies are separately coupled to latex.

4. The method according to any one of claims 1 to 3, characterized in that: The method does not include the step of acid-base neutralization.

5. The method according to any one of claims 1 to 3, characterized in that: The volume ratio of the dissociation agent to the sample to be tested is: 100:1-10:1; Preferably, the volume ratio of the dissociation agent to the sample to be tested is 80:1-20:

1.

6. The method according to any one of claims 1 to 3, characterized in that: The contact time between the dissociation agent and the sample is: 1 min-10 min; Preferably, the dissociation agent is in contact with the sample for 1 min to 5 min.

7. The method according to any one of claims 1 to 3, characterized in that: The correlation coefficient R of the method 2 Greater than 0.

95.

8. The method according to any one of claims 1 to 3, characterized in that: The pH range of the dissociation agent is 7.5-12.0; Preferably, the pH range of the dissociation agent is 7.5-10.5; Preferably, the pH range of the dissociation agent is 7.5-9.5; Preferably, the pH range of the dissociation agent is 7.5-8.5; Preferably, the pH range of the dissociation agent is 7.0-8.6; Preferably, the pH range of the dissociation agent is 7.2-8.0; Preferably, the pH range of the detection system comprising the sample, alkaline dissociation agent, and antibody in the method is between 7.0 and 13.0; Preferably, the pH range of the detection system is 7.5-12.0; Preferably, the pH range of the detection system is 7.5-10.5; Preferably, the pH range of the detection system is 7.5-9.5; Preferably, the pH range of the detection system is 7.5-8.5 Preferably, the pH range of the detection system is 7.0-9.

0.

9. A reagent for detecting vitamin D, characterized in that: The reagent comprises the dissociation agent according to any one of claims 1 to 8; Preferably, the reagent comprises at least two antibodies; Optionally, the first antibody specifically binds to vitamin D, and the second antibody specifically binds to a complex formed by the first antibody and vitamin D; Preferably, the first antibody and / or the second antibody is coupled to nanoparticles; Preferably, the reagent further comprises a protective agent, an antibacterial agent, and an electrolyte.

10. The reagent according to claim 9, characterized in that The components of the dissociating agent include one or more of a surfactant and guanidine; Preferably, the dissociating agent comprises a perfluoroalkyl surfactant and guanidine hydrochloride; Preferably, the surfactant is S9; Preferably, the dissociation agent further comprises dithiothreitol; Preferably, the reagent further comprises one or more of C1-4 alcohol, EDTA, and dextran.

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