Method for using sandwich method to test target molecule in sample

Abstract

The present invention relates to the technical field of in-vitro testing, and in particular to a method for using a sandwich method to test a target molecule in a sample. The present invention provides a method for testing a target molecule in a sample under test, and testing is mainly performed by mean of a sandwich method, greatly improving the accuracy of testing of small molecules in serum. According to the method in the present invention, a complex-specific antibody specifically binding to a target molecule-target molecule binding protein is utilized to form a target molecule-target molecule binding protein-antibody ternary complex for testing. When a blood / serum sample containing the target molecule-target molecule binding protein is tested, in the method in the present invention, firstly, a specific dissociation solution is utilized to treat the sample under test so as to dissociate the target molecule from the target molecule-target molecule binding protein complex in the sample under test, so that the accuracy of testing of the target molecule can be further improved.

Description

Sandwich method for detecting target molecules in samples

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202311690319.7 filed on December 7, 2023, and the contents of the above-mentioned Chinese patent application disclosure are hereby incorporated by reference in their entirety as a part of this application. Technical Field

[0003] The present invention relates to the technical field of in vitro detection, and in particular to a method for detecting target molecules in a sample using a sandwich method. Background Art

[0004] Some small molecules in blood or serum samples usually exist in a bound form (for example, small molecules will bind to some binding proteins to form complexes). This results in some bound small molecules not being detected during clinical testing, thus affecting the accuracy of clinical small molecule testing. For example, FA (folic acid) usually exists in the human body in a bound form. Three types of folate-binding proteins have been discovered: high-affinity folate-binding proteins (soluble FRs present in the blood), membrane-associated binding proteins, and cytoplasmic binding proteins. High-affinity folate-binding proteins (FRs) protect the stable presence of folic acid in the blood and may also control the specificity of folate distribution in plasma.

[0005] The main analytical methods for small molecule organic compounds in serum samples include liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS). While highly sensitive, these methods also require high levels of expertise and are labor-intensive, making them unsuitable for grassroots use. Most critically, their analysis time is long. Clinical testing typically utilizes a combination of chemiluminescence and immunoassays. However, due to the small size of these small molecule epitopes, conventional double-antibody sandwich assays are difficult to detect.

[0006] Competition is the main methodology for small molecule detection, but it has the following defects compared to the sandwich method: ① poor sensitivity, poor detection rate or repeatability of low-value samples; ② insufficient accuracy, the binding affinity between natural small molecules and small molecule analogs and antibodies may differ, affecting accuracy.

[0007] Therefore, there is an urgent need to develop a method that can accurately and cost-effectively detect small molecules in blood or serum samples. Summary of the Invention

[0008] The present invention aims to solve one of the technical problems in the related art to at least a certain extent. To this end, one purpose of the present invention is to propose a method for detecting a target molecule in a sample to be tested, which is mainly detected by a sandwich method, greatly improving the accuracy of small molecule detection in serum. The method of the present invention utilizes a complex antibody that specifically binds to a target molecule-target molecule binding protein to form a target molecule-target molecule binding protein-antibody ternary complex for detection. When detecting a biological sample containing a target molecule-target molecule binding protein, the method of the present invention first utilizes a specific reaction system to treat the sample to be tested to dissociate the target molecule in the target molecule-target molecule binding protein complex in the sample to be tested, so that the accuracy of target molecule detection can be further improved.

[0009] To this end, the first aspect of the present invention provides a method for detecting a target molecule in a sample to be tested. According to an embodiment of the present invention, the above-mentioned detection method includes:

[0010] S1: Providing a detection reagent containing a target molecule binding protein and a detection reagent containing an antibody, wherein the target molecule binding protein can bind to a target molecule in a sample to be tested to form a target molecule-target molecule binding protein complex, and the target molecule in the complex induces the binding protein to form at least one new conformational epitope, and the antibody can specifically bind to the at least one new conformational epitope;

[0011] S2: contacting the sample to be tested with a detection reagent containing the target molecule binding protein and a detection reagent containing an antibody to obtain a target molecule-target molecule binding protein-antibody complex;

[0012] S3: The target molecule-target molecule binding protein-antibody complex obtained by detecting S2 is used to detect the target molecule contained in the sample.

[0013] The method of the present invention mainly utilizes a complex antibody that specifically binds to a target molecule-target molecule binding protein to form a target molecule-target molecule binding protein-antibody ternary complex for detection, thereby improving the accuracy of target molecule detection.

[0014] According to an embodiment of the present invention, the target molecule binding protein is not an antibody.

[0015] According to an embodiment of the present invention, the sample to be tested includes any one selected from a calibrator, a quality control product, a standard, a serum sample or a plasma sample.

[0016] According to an embodiment of the present invention, when the sample to be tested contains a target molecule-target molecule binding protein complex, the above detection method further includes, before S2, treating the sample to be tested with a dissociation solution to dissociate the target molecule from the target molecule-target molecule binding protein complex in the sample to be tested.

[0017] According to an embodiment of the present invention, the dissociation solution includes an alkaline reagent with a pH of 12-14 and a reducing agent.

[0018] By providing an alkaline environment, the target molecule binding protein (e.g., FA-binding protein FABP) in the blood is denatured, breaking the disulfide bonds to generate sulfhydryl groups. Further addition of a reducing agent further reduces the disulfide bonds, causing the target molecule binding protein to lose its ability to bind to the target molecule, thereby obtaining a free target small molecule.

[0019] According to an embodiment of the present invention, the alkaline agent includes NaOH and / or KOH.

[0020] According to an embodiment of the present invention, the reducing agent includes at least one selected from DTT, TCEP, BME or DTE.

[0021] According to an embodiment of the present invention, the reducing agent is DTT.

[0022] According to an embodiment of the present invention, step S2 further comprises:

[0023] The above-mentioned detection reagent containing the antibody and the above-mentioned detection reagent containing the target molecule binding protein are added to the sample to be tested treated with the above-mentioned dissociation solution to obtain a reaction system 1.

[0024] The amount of the antibody-containing detection reagent added ensures that the pH of the reaction system 1 is 7-10, and the target molecule in the sample after dissociation does not bind to the target molecule binding protein contained in the sample to be tested, but binds to the target molecule binding protein in the detection reagent.

[0025] Adding the above-mentioned detection reagent containing the antibody to the sample to be tested that has been treated with the above-mentioned dissociation solution can, on the one hand, neutralize the strong alkaline environment of the dissociation solution, and the dissociation solution is further diluted. At this time, the dissociation solution cannot play the role of dissociating the target molecule-target molecule binding protein, and will not affect the formation of the ternary complex in the next step; on the other hand, it can also ensure that the detection reagent containing the antibody is not affected during the detection (for example, the luminescence signal of some signal generators will be affected in a strong alkaline environment).

[0026] According to an embodiment of the present invention, the pH of the antibody-containing detection reagent is 5-7.

[0027] According to an embodiment of the present invention, the pH of the detection reagent containing the target molecule binding protein is 4-10.

[0028] According to an embodiment of the present invention, the above-mentioned detection reagent containing the target molecule binding protein includes a carrier coated with the target molecule binding protein.

[0029] According to an embodiment of the present invention, the carrier comprises magnetic beads and / or microspheres.

[0030] According to an embodiment of the present invention, the antibody-containing detection reagent includes a signal generator linked to the antibody.

[0031] According to an embodiment of the present invention, the signal generator comprises a chemiluminescent compound.

[0032] According to an embodiment of the present invention, the signal generator includes at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, lucigenin, horseradish peroxidase or alkaline phosphatase.

[0033] According to an embodiment of the present invention, the signal generator is ABEI.

[0034] According to an embodiment of the present invention, the affinity constant KD value of the above antibody and the target molecule-target molecule binding protein complex is 1×10 -10 ~9×10 -9 .

[0035] According to an embodiment of the present invention, the affinity constant KD value of the above-mentioned antibody and the above-mentioned target molecule binding protein alone is 1×10 -5 ~9×10 -4 .

[0036] A second aspect of the present invention provides a target molecule detection kit. According to an embodiment of the present invention, the target molecule detection kit comprises:

[0037] 1) Detection reagent containing target molecule binding protein;

[0038] 2) detection reagents containing antibodies; and

[0039] Optionally, 3) dissociation solution;

[0040] The target molecule binding protein can bind to the target molecule in the sample to be tested to form a target molecule-target molecule binding protein complex, and the target molecule in the complex induces the binding protein to form at least one new conformational epitope, and the antibody can specifically bind to the at least one new conformational epitope;

[0041] The above-mentioned detection reagent containing an antibody includes a signal generator linked to the above-mentioned antibody;

[0042] The dissociation solution is used to dissociate the target molecule from the target molecule-target molecule binding protein complex in the blood sample;

[0043] The dissociation solution includes an alkaline reagent with a pH of 12-14 and a reducing agent.

[0044] According to an embodiment of the present invention, the above-mentioned detection reagent containing the target molecule binding protein includes a carrier coated with the target molecule binding protein.

[0045] According to an embodiment of the present invention, the carrier comprises magnetic beads and / or microspheres.

[0046] According to an embodiment of the present invention, the signal generator comprises a chemiluminescent compound.

[0047] According to an embodiment of the present invention, the signal generator includes at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, lucigenin, horseradish peroxidase or alkaline phosphatase.

[0048] According to an embodiment of the present invention, the signal generator is ABEI.

[0049] According to an embodiment of the present invention, the pH of the detection reagent containing the target molecule binding protein is 4-10.

[0050] According to an embodiment of the present invention, the pH of the antibody-containing detection reagent is 5-7.

[0051] According to an embodiment of the present invention, the alkaline agent includes NaOH and / or KOH.

[0052] According to an embodiment of the present invention, the reducing agent includes at least one selected from DTT, TCEP, BME or DTE.

[0053] According to an embodiment of the present invention, the reducing agent is DTT.

[0054] The third aspect of the present invention provides use of the target molecule detection kit described in the second aspect in detecting target molecules in a sample using a chemiluminescence method.

[0055] The present invention provides a method for detecting target molecules in a sample. Using antibodies that specifically bind to a complex of a target molecule (e.g., FA) and a target binding protein (e.g., FABP), this method enables linear detection of the target molecule. Furthermore, by adjusting the reaction system, the consistency between sample detection results and mass spectrometry results can be improved during sample testing.

[0056] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The accompanying drawings, which constitute part of the present invention, are intended to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are intended to explain the present invention and do not constitute an undue limitation of the present invention. In the accompanying drawings:

[0058] FIG1 shows the standard curve obtained by detecting 10 standards using the method of the present invention in Example 1.

[0059] FIG2 shows the curves obtained by expanding the evaluation of 50 samples at different levels using the optimal solution of the present invention. DETAILED DESCRIPTION

[0060] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be understood as limiting the present invention.

[0061] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be understood to indicate or imply relative importance or implicitly specify the number of the technical features indicated. Therefore, features defined as "first" or "second" may explicitly or implicitly include one or more of such features. Furthermore, in the description of the present invention, unless otherwise specified, "plurality" means two or more.

[0062] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0063] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In this article, the terms "comprise" or "include" are open-ended expressions, that is, including the contents specified in the present invention, but not excluding other aspects.

[0064] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0065] As used herein, the term "and / or" encompasses all combinations of items connected by the term, and should be treated as if each combination had been individually listed herein. For example, "A and / or B" encompasses "A," "A and B," and "B." For example, "A, B, and / or C" encompasses "A," "B," "C," "A and B," "A and C," "B and C," and "A and B and C."

[0066] According to a specific embodiment of the present invention, the present invention provides a method for detecting a target molecule in a sample to be tested, comprising:

[0067] S1: Providing a detection reagent containing a target molecule binding protein and a detection reagent containing an antibody, wherein the target molecule binding protein can bind to a target molecule in a sample to be tested to form a target molecule-target molecule binding protein complex, and the target molecule in the complex induces the binding protein to form at least one new conformational epitope, and the antibody can specifically bind to the at least one new conformational epitope;

[0068] S2: contacting the sample to be tested with a detection reagent containing the target molecule binding protein and a detection reagent containing an antibody to obtain a target molecule-target molecule binding protein-antibody complex;

[0069] S3: The target molecule-target molecule binding protein-antibody complex obtained by detecting S2 is used to detect the target molecule contained in the sample.

[0070] It should be noted that the target molecule can be any small molecule clinically detected. Preferably, the target molecule has a molecular weight of less than 1 kDa and is present in the test sample in a bound state, such as folic acid, E2, testosterone, T3, rT3, T4, cortisol, etc. In other words, the method for detecting a target molecule in a test sample provided by the present invention can be used to accurately detect the target molecule in any clinical sample.

[0071] It should be noted that the source of the above-mentioned target molecule binding protein can be the target molecule binding protein extracted from an animal, or the target molecule binding protein obtained by in vitro expression or direct synthesis. The target molecule binding protein contained in the detection reagent and the target molecule binding protein contained in the sample can be the same protein or different proteins.

[0072] The "induction" mentioned above in this article refers to the fact that after the target molecule in the sample to be tested binds to the target molecule binding protein in the detection reagent containing the target molecule binding protein, the conformation of the target molecule binding protein in the binding complex changes, forming a new conformational epitope, and the antibody in the detection reagent containing the antibody can specifically bind to the new conformational epitope.

[0073] The "new conformational epitope" mentioned above in this article refers to a new conformational epitope formed when the target molecule in the sample to be tested binds to the target molecule binding protein in the detection reagent containing the target molecule binding protein, causing the conformation of the target molecule binding protein in the binding complex to change, or a new conformational epitope formed between the target molecule and the target molecule binding protein at the binding site after the target molecule and the target molecule binding protein bind.

[0074] According to a specific embodiment of the present invention, the target molecule binding protein can be any protein. All types of binding proteins that can bind to the target molecule are included in the scope of the target molecule binding protein. However, it should be noted that the target molecule binding protein is not an antibody.

[0075] According to a specific embodiment of the present invention, when the sample to be tested contains a target molecule-target molecule binding protein complex, the above-mentioned detection method further includes, before S2, treating the sample to be tested with a dissociation solution to dissociate the target molecule from the target molecule-target molecule binding protein complex in the sample to be tested.

[0076] According to a specific embodiment of the present invention, the dissociation solution includes an alkaline reagent with a pH of 12-14 and a reducing agent.

[0077] It should be noted that there is no particular restriction on the composition of the dissociation solution. On the one hand, the pH of the dissociation solution needs to be ensured to be 12-14. On the other hand, it needs to contain a reducing agent. The above-mentioned reducing agent can enable the target molecule denatured under alkaline conditions to further react with some chemical groups in the binding protein, thereby obtaining free target small molecules.

[0078] According to a specific embodiment of the present invention, the sample to be tested may be a biological sample such as blood, serum or plasma, or may be a standard, quality control or calibration product.

[0079] Target molecules (e.g., FA) are typically present in a bound state in biological samples. When using the aforementioned detection methods to detect bound target molecules in samples, the challenge is how to dissociate the target molecules from the binding proteins in the blood and then bind the free target molecules to the binding proteins provided by the reagents, enabling recognition by the complex antibodies. The detection methods provided by the present invention address this problem.

[0080] According to a specific embodiment of the present invention, the alkaline reagent includes but is not limited to at least one of NaOH and KOH. The alkaline reagent may also be other strong base chemical reagents, which are also covered by the protection scope of the present invention.

[0081] According to a specific embodiment of the present invention, the reducing agent includes at least one selected from DTT, TCEP, BME or DTE.

[0082] According to a preferred embodiment of the present invention, the reducing agent is DTT.

[0083] It should be noted that there are no specific restrictions on the concentration or amount of reducing agent added to the system. Based on the generally known range of target molecule content in the test sample, a slight excess of reducing agent can be added to ensure complete dissociation of the target molecule from the target molecule-target molecule binding protein complex in the test sample. For example, when the reducing agent is DTT or TCEP, the working concentration is 0.75 mg / μL to 1 mg / μL.

[0084] According to a specific embodiment of the present invention, step S2 further comprises:

[0085] The above-mentioned detection reagent containing the antibody and the above-mentioned detection reagent containing the target molecule binding protein are added to the sample to be tested treated with the above-mentioned dissociation solution to obtain a reaction system 1.

[0086] The amount of the antibody-containing detection reagent added ensures that the pH of the reaction system 1 is 7-10, and the target molecule in the sample after dissociation does not bind to the target molecule binding protein contained in the sample to be tested, but binds to the target molecule binding protein in the detection reagent.

[0087] In this step, the strongly alkaline liquid environment after sample treatment is neutralized by the detection reagent containing the antibody, and the reducing agent is further diluted. At this point, when the detection reagent containing the target molecule binding protein is added to the sample, because the strongly alkaline environment is no longer present and the concentration of the reducing agent is diluted, the sample treatment agent (dissociation solution) is unable to exert its dissociation effect. As a result, the target molecule contained in reaction system 1 binds to the target molecule binding protein in the detection reagent containing the target molecule binding protein and is recognized by the antibody in reaction system 1, thereby forming a target molecule-target molecule binding protein-antibody ternary complex.

[0088] The reason why the target molecule in the sample after dissociation does not bind to the target molecule binding protein contained in the above-mentioned sample to be tested may be that the strong alkaline environment of the dissociation solution denatures the target molecule binding protein contained in the above-mentioned sample to be tested, and the disulfide bonds are opened to generate sulfhydryl groups. The reducing agent in the dissociation solution further reduces the disulfide bonds, causing the target molecule binding protein to lose the ability to bind to the target molecule.

[0089] According to a specific embodiment of the present invention, the pH of the antibody-containing detection reagent is 5-7.

[0090] The detection reagent containing antibodies at a pH of 5-7 is added to the sample to be tested that has been treated with the above dissociation solution to neutralize the strong alkaline environment and further dilute the reducing agent.

[0091] According to a specific embodiment of the present invention, the pH of the detection reagent containing the target molecule binding protein is 4-10.

[0092] According to a specific embodiment of the present invention, the structure of the antibody in the above-mentioned detection reagent containing an antibody is not particularly limited. As long as the above-mentioned antibody can specifically bind to at least one new conformational epitope induced by the target molecule in the complex (target molecule-target molecule binding protein complex), all antibody types are included in the scope of protection of the present invention. Preferably, the affinity constant KD value of the above-mentioned antibody and the target molecule-target molecule binding protein complex is 1×10 -10 ~9×10 -9 .

[0093] Furthermore, the affinity constant KD value of the above antibody and the above target molecule binding protein alone is 1×10 -5 ~9×10 -4 .

[0094] Furthermore, the above-mentioned antibody does not bind to the above-mentioned target molecule binding protein alone.

[0095] According to a specific embodiment of the present invention, the above-mentioned detection reagent containing the target molecule binding protein includes a carrier coated with the target molecule binding protein.

[0096] According to a specific embodiment of the present invention, the carrier comprises magnetic beads and / or microspheres.

[0097] It should be noted that there are no special restrictions on the specifications of magnetic beads and microspheres (such as particle size), the feed ratio of target molecule binding protein and microspheres, and the concentration of the target molecule binding protein-microsphere solution. Depending on the different target molecules to be detected, more appropriate specifications, feed ratios, and concentrations can be explored and determined.

[0098] According to a specific embodiment of the present invention, the detection reagent containing the target molecule binding protein and the detection reagent containing the antibody generally contain a commonly used buffer, such as PBS buffer, Tris buffer, MES buffer, etc., and of course other types of buffers for in vitro diagnosis can also be used. In addition, the detection reagent containing the target molecule binding protein and the detection reagent containing the antibody can also contain other ingredients. For example, the detection reagent containing the target molecule binding protein can also contain at least one of mannitol, BSA, NaCl or MgCl2 in addition to the buffer to improve the stability of the protein in solution; the detection reagent containing the antibody can also contain at least one of dipotassium ethylenediaminetetraacetic acid, PEG6000 or BSA in addition to the buffer.

[0099] According to a specific embodiment of the present invention, the above-mentioned detection reagent containing an antibody includes a signal generator connected to the above-mentioned antibody.

[0100] According to a specific embodiment of the present invention, the signal generator includes a chemiluminescent compound.

[0101] According to a specific embodiment of the present invention, the signal generator includes at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, lucigenin, horseradish peroxidase or alkaline phosphatase.

[0102] According to a specific embodiment of the present invention, the signal generator is ABEI.

[0103] It should be noted that the signal generator linked to the above-mentioned antibody is typically provided in solution. There are no specific restrictions on the ratio of antibody to signal generator in the signal generator linked to the antibody, and this ratio can be adjusted based on the target molecule being detected. There are also no specific restrictions on the concentration of the signal generator linked to the antibody, and this ratio can be adjusted as needed, provided that the stability of the connection between the antibody and signal generator is maintained.

[0104] It should be noted that there are no special restrictions on the incubation time and reaction temperature in each binding reaction step. On the one hand, different target molecule types have different suitable binding temperatures and reaction times. On the other hand, different signal generators have different suitable conditions for different specifications of magnetic beads and microspheres, and adaptive adjustments can be made as needed.

[0105] According to a specific embodiment of the present invention, when the sample to be tested contains a target molecule-target molecule binding protein complex, the above detection method includes:

[0106] S1': providing a dissociation solution, wherein the dissociation solution is used to dissociate the target molecule from the target molecule-target molecule binding protein complex contained in the sample to be tested, wherein the dissociation solution comprises an alkaline reagent with a pH of 12-14 and a reducing agent;

[0107] S2': providing a carrier coated with a target molecule binding protein and a signal detection reagent, wherein the signal detection reagent comprises a chemiluminescent compound linked to a complex antibody, and the complex antibody is an antibody that can specifically bind to a new conformational epitope formed by the target molecule-target molecule binding protein complex but does not bind to the target molecule binding protein alone;

[0108] S3′: contacting the sample to be tested with a dissociation solution to dissociate the target molecule from the target molecule-target molecule binding protein complex, thereby obtaining a pre-treated sample to be tested;

[0109] S4': mixing the pre-treated sample to be tested with the signal detection reagent and the carrier coated with the target molecule binding protein to obtain a complex of target molecule-carrier coated with the target molecule binding protein-complex antibody-chemiluminescent compound,

[0110] Wherein, the pH of the above signal detection reagent is 4-8,

[0111] The carrier coated with the target molecule binding protein is placed in a first solution, wherein the pH of the first solution is 5-7;

[0112] S5′: Using the luminescence mechanism of the chemiluminescent compound, the light intensity of the target molecule-target molecule binding protein-complex antibody-chemiluminescent compound complex obtained in S4′ is detected to detect the target molecule contained in the sample to be tested.

[0113] According to a specific embodiment of the present invention, the target molecule is FA, and the target molecule binding protein is FABP.

[0114] Taking FA detection as an example, the detection scheme is as follows:

[0115] Step 1: Use an alkaline environment with a pH range of 12-14 and an appropriate amount of reducing agent in the sample treatment agent to obtain free FA molecules.

[0116] In step 1, the substance for adjusting pH can be: NaOH, KOH; the reducing agent can be DTT, TCEP.

[0117] Step 2: The mixture obtained in step 1 is added to a magnetic ball reagent (pH 5-7) connected to FABP and an ABEI reagent (pH 4-8) coated with a complex antibody. The reaction pH range after mixing is 8-10. The complex antibody specifically recognizes the FABP-FA complex. After the strong alkaline liquid environment after sample treatment in step 2 is neutralized by the ABEI reagent and the magnetic ball reagent, the reducing agent is further diluted. At this time, when the magnetic ball reagent is added to the sample, because it no longer has a strong alkaline environment and the concentration of the reducing agent is diluted, it is difficult for the sample treatment agent to play the dissociation effect of the previous step. Therefore, the small molecule analyte obtained in step 1 can bind to the magnetic ball reagent connected to FABP to form a FA-FABP complex and be recognized by the complex antibody to form a FA-FABP-complex antibody ternary complex. And because ABEI itself is a substance that emits light in a strong alkaline environment, it acts as a neutralizing agent in this scheme. After mixing with the strong alkaline reagent, it can still play the role of a signal generator and does not affect the detection of the signal. Therefore, in this scheme, other signal generators that can emit light in a strong alkaline environment are all alternatives to this scheme, such as acridinium ester, luminol, etc.

[0118] In step 2, the source of FABP can be extracted from milk.

[0119] The scheme of the present disclosure will be explained below in conjunction with the examples. Those skilled in the art will understand that the following examples are only used to illustrate the present disclosure and should not be considered to limit the scope of the present disclosure. Where specific techniques or conditions are not specified in the examples, they are carried out according to the techniques or conditions described in the literature in this area or according to the product instructions. Where the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be obtained commercially.

[0120] Antibody preparation process:

[0121] Preparation process of anti-FA-FABP complex antibody (Ab2)

[0122] 1. Immunization of Mice

[0123] The folic acid standard 5-MTHF (TRC) and the folate-binding protein FABP were dialyzed into 0.01M PBS and incubated at 37°C for 2 hours at a molar ratio of ≥10:1. Excess antigen was removed by dialyzation into 0.01M PBS and used as the immunogen. The immunogen was then emulsified with an equal volume of Freund's complete adjuvant (Sigma) and injected subcutaneously at multiple sites in 6-8 week-old SPF-grade Balb / c mice at 200 μg / mouse. Three weeks later, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at multiple sites at 150 μg / mouse. Two booster immunizations were performed, with a shock injection intraperitoneally three days prior to fusion.

[0124] 2. Cell fusion and subclone screening

[0125] The spleen of the immunized mouse was taken, ground and separated to obtain dispersed single spleen cells, and the spleen cells and myeloma cells were mixed using PEG. After the culture medium was terminated, the cells were centrifuged and re-dissolved and plated into a 96-well plate. After one week, the medium was changed and the supernatant was taken for indirect enzyme immunoassay detection.

[0126] The detection method is as follows: the odd-numbered columns of the ELISA plate are coated with FABP, and the even-numbered columns are coated with a complex formed by incubating FABP with 5-MTHF at a molar ratio of ≥10:1. After blocking, cell supernatant is added and incubated at 37°C for 0.5 h. After washing, goat anti-mouse IgG secondary antibody-HRP (Jackson) is added and incubated at 37°C for 0.5 h. After washing, TMB is added for color development, and then 2 M H2SO4 is added for termination.

[0127] The wells with almost no titer or low titer in the odd-numbered columns and high titer in the even-numbered columns were picked as positive wells, and further subcloned by the limiting dilution method. After one week of culture, enzyme immunoassay was continued. The method was the same as above. The best positive wells were selected for subcloning. This was repeated 3-4 times until all sub-wells were positive. Positive single colonies were picked for expansion culture to obtain the specific cell line 3D10F1.

[0128] 3. Preparation of Ascites from Positive Cell Lines

[0129] Single colony cells were expanded and cultured, and injected into mice that had been pre-immunized with IFA to prepare ascites. The ascites was collected to obtain the complex antibody Ab2, which was affinity purified by the SPA method.

[0130] Antibody binding affinity verification

[0131] BIAcore technology was used to measure the binding affinity of folate antibodies, folate-folate binding protein complexes, and folate complex antibody (Ab2) (as shown in Table 1). Experiments were performed according to the BiacoreR 3000 and Biacore AB specifications. The equilibrium dissociation constant, Kd (M), represents the degree of dissociation of binding affinity at equilibrium; a smaller Kd (M) indicates a stronger affinity.

[0132] Table 1 Summary of equilibrium dissociation constants Kd (M) of folic acid project antibodies against complexes

[0133] Example 1 verifies that the detection method provided by the present invention can realize the linear detection of FA

[0134] 1. Preparation of reagents contained in the detection kit:

[0135] Reagent 1: 0.5 M NaOH solution;

[0136] Reagent 2: DTT solution (main components are 598 mM disodium hydrogen phosphate-sodium dihydrogen phosphate, 10% mannitol), concentration is 2 mg / ml;

[0137] Reagent 3: Magnetic sphere reagent, coated with FABP-linked magnetic spheres, pH 7.0, with a FABP:magnetic sphere ratio of 1 mg:12 μg, and a 20 mg / mL magnetic sphere suspension concentration. Buffer: 20 mM potassium dihydrogen phosphate-sodium dihydrogen phosphate (other components include 10% mannitol, 1% BSA, 0.8% NaCl, and 0.013% MgCl2).

[0138] Reagent 4: ABEI reagent conjugated to the antibody complex (Ab2), pH 5.5, with a ratio of 0.5 mg of antibody complex to 50 μg of ABEI; ABEI suspension concentration: 50 μg / ml. Buffer: 598 mM sodium dihydrogen phosphate-disodium hydrogen phosphate (other components include 0.1% dipotassium ethylenediaminetetraacetic acid, 0.5% PEG6000, and 0.5% BSA).

[0139] Reagent 5: The concentration of the calibrator is 5ug / ml.

[0140] 2. Detection method:

[0141] Test solution: dilute a series of calibrators with concentration gradients to 24 ng / ml, 16.132 ng / ml, 10.843 ng / ml, 7.289 ng / ml, 4.899 ng / ml, 3.293 ng / ml, 2.213 ng / ml, 1.488 ng / ml, 1 ng / ml, and 0 ng / ml;

[0142] Testing process:

[0143] Add 40 μL of reagent 1 and 40 μL of reagent 2 to 10 μL of sample, incubate at 37°C for 10 min, then add 40 μL of ABEI + 20 μL of magnetic balls to the system, incubate at 37°C for 10 min, wash and rinse three times, and detect with Maglumi 4000P. The pH during detection is 9.0, incubate for 10 min, wash three times, and detect.

[0144] The verification results are as follows:

[0145] The linear results are shown in Figure 1, which shows the linear function y=ax+b and R 2 Table 2 below shows the theoretical concentrations of the 10 standards, samples 1-10, the concentrations actually measured using the above method, and the difference between the actual concentrations and the theoretical concentrations.

[0146] Table 2

[0147] Figure 1 and Table 2 Data Description: Determination of standard curve, correlation between measured concentration and theoretical concentration R 2 The detection method provided by the present invention can achieve accurate detection of FA items.

[0148] Example 2 Detection of serum samples

[0149] Sample source: Nileke County Maternal and Child Health Hospital, a total of 50 samples.

[0150] The steps and reagents of the detection method provided by the present invention are as follows:

[0151] ① Add 55 μL of mixed internal standard (folic acid-[ 13 C5], 5-methyltetrahydrofolate-[ 13 C5], 5-formyltetrahydrofolate-[ 13 C5]) and 770 μL SPE sample buffer (10 g / L ammonium formate, 1 g / L ascorbic acid, pH 3.2);

[0152] ② Mix thoroughly and equilibrate at 4°C for at least 20 minutes;

[0153] ③Solid-phase extraction: Use 1mL phenyl SPE Cartridges (100mg BondElut) for the small column. Activate the chromatographic column with 2mL acetonitrile, methanol, and SPE sample buffer respectively. Then add 1mL of sample and balance for 1min to allow full contact between the sample and the small column packing. Then add 3mL of SPE eluent (0.5g / L ammonium formate, 0.05g / L ascorbic acid, pH 3.4) to elute impurities. Add 1mL of SPE eluent (400mL / L methanol, 100mL / L acetonitrile, 10mL / L acetic acid, 1g / L ascorbic acid) for elution and collect the eluate, mix and filter it before preparing for testing.

[0154] ID LC-MS / MS testing:

[0155] ①Chromatographic column: Luna C-8 (3 mm × 150 mm, 5-μm particle size);

[0156] ②Injection volume: 20 μL;

[0157] ③Mobile phase: 400 mL / L methanol, 100 mL / L acetonitrile, 10 mL / L acetic acid;

[0158] ④ Gradient elution mode: isocratic; flow rate: 0.25 mL / min;

[0159] ⑤Mass spectrometry ionization mode: ESI+;

[0160] ⑥ FA quantitative monitoring ion pair: 442→295; 5-methyl THF quantitative monitoring ion pair: 460→313; 5-formyl THF quantitative monitoring ion pair: 474→327

[0161] ⑦ 13 C5-FA internal standard quantitative monitoring ion pair: 447→295; 5-methyltetrahydrofolate-[ 13 C5] internal standard quantitative monitoring ion pair: 465→313; 5-formyltetrahydrofolate-[ 13 C5] Internal standard quantitative monitoring ion pair: 479→327.

[0162] Table 3 below shows the reagent dosages, components, and system pH settings used in the detection methods of Examples 2.1-2.6, with the Abbott competitive method used as a comparative example.

[0163] Table 3

[0164] Each example was prepared and added with each component as shown in Table 3, and the specific concentration comparison results are shown in Tables 4 and 5.

[0165] Table 4

[0166] Table 5

[0167] The results in Tables 3-5 show that, in the first step, 40 μL of DTT and 40 μL of NaOH were used to treat the sample. When the DTT loading volume was adjusted to 30 μL, the average deviation between the sample concentration and the mass spectrum was 11.3%. Further adjustments to 40 μL and 50 μL achieved comparable sample concentrations and mass spectra. The optimal DTT loading volume for this system was 40 μL, at which point the average deviation between the sample concentration and the mass spectrum was 3.1-4.1%. This demonstrates that Examples 2.1-2.3 outperform Comparative Example 1, which had an average deviation of 14.5% between the sample concentration and the mass spectrum.

[0168] Effect of pH of verification system on detection discrimination

[0169] Samples to be tested: The hospital provides gradient serum samples

[0170] The reagent preparation and detection process differ from Example 1 in that the pH value of the system is optimized by adjusting the amount of ABEI added, thereby improving the discrimination. The results of the effect of system pH on discrimination are shown in Table 6 below:

[0171] Table 6

[0172] Each example was prepared and added according to the ingredients shown in Table 3, and the specific discrimination results are shown in Table 6 above. The results show that after the first step of sample treatment, ABEI with a pH of 6.5 and magnetic balls were added in the second step to adjust the pH of the reaction system to 9.0. The discrimination was improved by adjusting the pH of the system. When the pH of the system reached 9.0, J / A, J / I, and B / A were 84, 1.38, and 2.14, respectively (A refers to sample 16, B refers to sample 17, I refers to sample 18, and J refers to sample 19), and the discrimination was optimal. Adjusting the pH of the system did not affect the sample concentration and mass spectrometry results, with an average deviation of about 3%. The above-mentioned supporting detection system was used for evaluation, and the remaining examples can be prepared according to this process.

[0173] 50 samples were tested using the method of the present invention, and the mass spectrometry results were used as a positive control.

[0174] Sample source: Nileke County Maternal and Child Health Hospital. The detection method of the present invention was carried out according to the method of Example 1 using the reagents of Example 2.5. Mass spectrometry detection method:

[0175] Sample pre-treatment process:

[0176] ① Add 55 μL of mixed internal standard (folic acid-[ 13 C5], 5-methyltetrahydrofolate-[ 13 C5], 5-formyltetrahydrofolate-[ 13 C5]) and 770 μL SPE sample buffer (10 g / L ammonium formate, 1 g / L ascorbic acid, pH 3.2);

[0177] ② Mix thoroughly and equilibrate at 4°C for at least 20 minutes;

[0178] ③Solid-phase extraction: Use 1mL phenyl SPE Cartridges (100mg BondElut) for the small column. Activate the chromatographic column with 2mL acetonitrile, methanol, and SPE sample buffer respectively. Then add 1mL of sample and balance for 1min to allow full contact between the sample and the small column packing. Then add 3mL of SPE eluent (0.5g / L ammonium formate, 0.05g / L ascorbic acid, pH 3.4) to elute impurities. Add 1mL of SPE eluent (400mL / L methanol, 100mL / L acetonitrile, 10mL / L acetic acid, 1g / L ascorbic acid) for elution and collect the eluate, mix and filter it before preparing for testing.

[0179] ID LC-MS / MS testing:

[0180] ①Chromatographic column: Luna C-8 (3 mm × 150 mm, 5-μm particle size);

[0181] ②Injection volume: 20 μL;

[0182] ③Mobile phase: 400 mL / L methanol, 100 mL / L acetonitrile, 10 mL / L acetic acid;

[0183] ④ Gradient elution mode: isocratic; flow rate: 0.25 mL / min;

[0184] ⑤Mass spectrometry ionization mode: ESI+;

[0185] ⑥ FA quantitative monitoring ion pair: 442→295; 5-methyl THF quantitative monitoring ion pair: 460→313; 5-formyl THF quantitative monitoring ion pair: 474→327

[0186] ⑦ 13 C5-FA internal standard quantitative monitoring ion pair: 447→295; 5-methyltetrahydrofolate-[ 13 C5] internal standard quantitative monitoring ion pair: 465→313; 5-formyltetrahydrofolate-[ 13 C5] Internal standard quantitative monitoring ion pair: 479→327.

[0187] Test indicator introduction: y = ax + b, where a is the slope, representing the overall deviation 1 between the sandwich method and the mass spectrometry detection results. The closer it is to 1, the smaller the deviation. b is the intercept, representing the overall deviation 2 between the sandwich method and the mass spectrometry detection results. The closer it is to 0, the smaller the deviation.

[0188] FIG2 shows the correlation between the FA concentrations of 50 blood samples (C1-C50) detected by the method of the present invention and mass spectrometry and the detection results of mass spectrometry.

[0189] The results in Figure 2 show that the correlation R 2 The results showed that the method of the present invention can accurately detect small molecules (FA) in serum samples, and the detection results are highly consistent with the mass spectrometry detection results.

[0190] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", "some implementation plans" or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification and features of different embodiments or examples without contradiction.

[0191] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for detecting a target molecule in a sample to be tested, characterized in that: include: S1: providing a detection reagent containing a target molecule binding protein and a detection reagent containing an antibody, wherein the target molecule binding protein can bind to a target molecule in a sample to be tested to form a target molecule-target molecule binding protein complex, and the target molecule in the complex induces the binding protein to form at least one new conformational epitope, and the antibody can specifically bind to the at least one new conformational epitope; S2: contacting the sample to be tested with a detection reagent containing the target molecule binding protein and a detection reagent containing an antibody to obtain a target molecule-target molecule binding protein-antibody complex; S3: The target molecule-target molecule binding protein-antibody complex obtained by detecting S2 is used to detect the target molecule contained in the sample.

2. The detection method according to claim 1, characterized in that: The target molecule binding protein is not an antibody.

3. The detection method according to claim 1, characterized in that: The sample to be tested includes any one selected from calibrators, quality control products, standard products, serum samples or plasma samples.

4. The detection method according to claim 1, characterized in that: When the sample to be tested contains a target molecule-target molecule binding protein complex, the detection method further includes, before S2, treating the sample to be tested with a dissociation solution to dissociate the target molecule from the target molecule-target molecule binding protein complex in the sample to be tested.

5. The detection method according to claim 4, characterized in that: The dissociation solution includes an alkaline reagent with a pH of 12-14 and a reducing agent; Optionally, the alkaline reagent comprises NaOH and / or KOH; Optionally, the reducing agent comprises at least one selected from DTT, TCEP, BME or DTE; Optionally, the reducing agent is DTT.

6. The detection method according to claim 5, characterized in that: Step S2 further comprises: Adding the detection reagent containing the antibody and the detection reagent containing the target molecule binding protein to the sample to be tested treated with the dissociation solution to obtain a reaction system 1, The amount of the antibody-containing detection reagent added ensures that the pH of the reaction system 1 is 7-10, and the target molecule in the sample after dissociation does not bind to the target molecule binding protein contained in the sample to be tested, but binds to the target molecule binding protein in the detection reagent.

7. The detection method according to any one of claims 1 to 6, characterized in that: The pH of the antibody-containing detection reagent is 5-7.

8. The detection method according to any one of claims 1 to 6, characterized in that: The pH of the detection reagent containing the target molecule binding protein is 4-10.

9. The detection method according to claim 1, characterized in that: The detection reagent containing the target molecule binding protein includes a carrier coated with the target molecule binding protein; Optionally, the carrier comprises magnetic beads and / or microspheres.

10. The detection method according to claim 1, characterized in that: The antibody-containing detection reagent includes a signal generator connected to the antibody; Optionally, the signal generator comprises a chemiluminescent compound; Optionally, the signal generator comprises at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, lucigenin, horseradish peroxidase or alkaline phosphatase; Optionally, the signal generator is ABEI.

11. The detection method according to claim 1, characterized in that: The affinity constant KD value of the antibody and the target molecule-target molecule binding protein complex is 1×10 -10 ~9×10 -9 ; Optionally, the affinity constant KD value of the antibody and the target molecule binding protein alone is 1×10 -5 ~9×10 -4 .

12. A target molecule detection kit, characterized in that: include: 1) Detection reagent containing target molecule binding protein; 2) Detection reagents containing antibodies; as well as Optionally, 3) a dissociation solution; Wherein, the target molecule binding protein can bind to the target molecule in the sample to be tested to form a target molecule-target molecule binding protein complex, and the target molecule in the complex induces the binding protein to form at least one new conformational epitope, and the antibody can specifically bind to the at least one new conformational epitope; The antibody-containing detection reagent includes a signal generator connected to the antibody; Wherein, the dissociation solution is used to dissociate the target molecule from the target molecule-target molecule binding protein complex in the blood sample; The dissociation solution includes an alkaline reagent with a pH of 12-14 and a reducing agent.

13. The kit according to claim 12, characterized in that The detection reagent containing the target molecule binding protein includes a carrier coated with the target molecule binding protein; Optionally, the carrier comprises magnetic beads and / or microspheres.

14. The kit according to claim 12, characterized in that The signal generator includes a chemiluminescent compound; Optionally, the signal generator comprises at least one selected from ABEI, acridinium ester, luminol, isoluminol, AHEI, ITCI, lucigenin, horseradish peroxidase or alkaline phosphatase; Optionally, the signal generator is ABEI.

15. The kit according to claim 12, characterized in that The pH of the detection reagent containing the target molecule binding protein is 4-10; Optionally, the pH of the antibody-containing detection reagent is 5-7.

16. The kit according to claim 12, characterized in that The alkaline reagent includes NaOH and / or KOH; Optionally, the reducing agent comprises at least one selected from DTT, TCEP, BME or DTE; Optionally, the reducing agent is DTT.

17. Use of the target molecule detection kit according to any one of claims 12 to 16 in detecting target molecules in a sample to be tested by chemiluminescence.

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