Small molecule hapten detection reagent and detection method

Abstract

The invention discloses a small molecule hapten detection reagent and a detection method, and relates to the technical field of immunodiagnosis. The detection reagent disclosed by the invention comprises a small-molecule hapten, an anti-small-molecule hapten antibody, a compound antibody and a signal marker, the detection method includes detecting at least a first signal and a second signal. According to the small-molecule hapten detection scheme, a more accurate detection reagent is provided for small-molecule hapten detection, and meanwhile, a novel more accurate detection method is provided for small-molecule hapten detection.

Description

Technical Field

[0001] The present invention relates to the field of antibody technology, and in particular to a small molecule hapten detection reagent and a detection method. Background Art

[0002] Immunoassay is a trace analysis method based on the specific recognition and reversible binding reaction between antigen and antibody. It is not only suitable for the detection of macromolecular compounds (such as proteins, nucleic acids, bacteria), but also for the determination of small molecular compounds (such as mycotoxins, pesticides, veterinary drugs, environmental hormones, prohibited food additives and physiologically active chemicals). Immunoassay has the following advantages: strong specificity, high sensitivity, simple operation, rapidity, low cost and suitable for on-site large-scale sample screening. Therefore, it is widely used in environmental monitoring, food safety testing, clinical diagnosis and biological analysis.

[0003] At present, the commonly used immunoassay methods are mainly divided into two categories: one is the double antibody sandwich method. This method has been widely used to detect macromolecular antigens containing multiple antigenic determinants, such as proteins, microorganisms and cells. However, for small molecule compounds (molecular weight less than 6000) with only a single antigenic determinant, the existing double antibody sandwich immunoassay for detecting macromolecules is not suitable for small molecule compounds with a single antigenic determinant. Therefore, the competition method has become a common method for detecting small molecule haptens. Common competition methods mainly have two detection modes, namely direct competition method and indirect competition method. The direct competition method plays an important role in the detection of small molecule haptens due to its simple and rapid operation. However, the traditional direct competition method has obvious disadvantages. Due to the relatively high binding force between the competing antigen and the antibody, it is difficult for the competing antigen to be competed by the target analyte, thereby affecting the sensitivity.

[0004] Therefore, there is a strong demand in the art for immunoassay reagents and methods that can sensitively and accurately identify small molecule haptens. In view of this, the present invention is proposed. Summary of the invention

[0005] The present application provides a small molecule hapten detection reagent and detection method, which provide important reagent sources and methodological support for the detection of small molecules.

[0006] In order to achieve the above-mentioned object, according to one aspect of the present invention, a special double antibody sandwich reagent for detecting small molecule haptens is provided, the reagent comprising a first antigen, an anti-small molecule hapten antibody, a complex antibody and a signal marker; the small molecule hapten and the anti-small molecule hapten antibody can combine to form a complex, and the complex antibody can bind to the complex; the anti-small molecule hapten antibody is labeled with a signal marker; the first antigen and the anti-small molecule hapten antibody are coupled to different solid phase carriers; or, the first antigen and the anti-small molecule hapten antibody are coupled to different regions of the same solid phase carrier; the first antigen can bind to the anti-small molecule hapten antibody.

[0007] In an optional embodiment, the binding force K1 between the complex antibody and the small molecule hapten and the binding force K2 of the anti-small molecule hapten antibody are both lower than the binding force K3 between the complex antibody and the complex, for example, K1 is only 10%, 5%, 1%, 0.1% or lower of K3.

[0008] In order to achieve the above-mentioned object, according to the second aspect of the present invention, a small molecule hapten detection reagent is provided, and the small molecule hapten detection reagent is an immunochromatography reagent, and the immunochromatography reagent comprises a chromatographic membrane, and the chromatographic membrane is sequentially provided with a marking pad, a first detection zone and a second detection zone; the marking pad is provided with an anti-small molecule hapten antibody marked with a signal marker, the first detection is provided with the complex antibody, and the second detection zone is provided with the first antigen; the first antigen can bind to the anti-small molecule hapten antibody.

[0009] In an alternative embodiment, the first detection zone and the second detection zone of the reagent generate the same type of detectable signal.

[0010] In an alternative embodiment, the solid support includes microspheres, plates and membranes.

[0011] In an optional embodiment, the first antigen and the small molecule hapten are the same substance, or are derivatives or analogs of the small molecule hapten; the small molecule hapten or the first antigen includes hormones, vitamins, drugs, and narcotics.

[0012] In an optional embodiment, the signal marker is selected from colloidal gold, fluorescent microspheres, fluorescent dyes, enzymes, radioactive isotopes, chemiluminescent markers, electrochemiluminescent markers, latex, and nanoparticle markers.

[0013] In an optional embodiment, the first antigen is coupled to a bridging protein; preferably, the bridging protein includes chicken ovalbumin, gelatin, keyhole limpet hemocyanin, bovine serum albumin, human serum albumin, rabbit serum albumin, poly-lysine or an artificially synthesized non-functional linear polypeptide.

[0014] In an optional embodiment, the first antigen is coupled to the solid phase carrier via a bridging protein.

[0015] In order to achieve the above object, according to the third aspect of the present invention, a method for detecting small molecule haptens is provided, comprising at least the following steps:

[0016] A first signal is generated after a small molecule hapten in a detection sample binds to an anti-small molecule hapten antibody labeled with a signal marker to form a complex and the complex antibody binds;

[0017] Detecting a second signal generated by the binding of an anti-small molecule hapten antibody labeled with a signal marker to the first antigen;

[0018] In an optional embodiment, the concentration of the small molecule hapten in the sample is calculated based on the first signal intensity and the second signal intensity.

[0019] In an optional embodiment, the concentration of the small molecule hapten in the sample is calculated by the ratio of the first signal intensity to the second signal intensity.

[0020] In an optional embodiment, the sample is blood or urine; optionally, the small molecule hapten or the first antigen includes hormones, vitamins, drugs, and narcotics; optionally, the first antigen is coupled with a bridging protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] 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.

[0022] Figure 1 Signal ratios were obtained for sample detection for both the example and comparative example protocols. DETAILED DESCRIPTION

[0023] In the present invention, the term "hapten" is also called incomplete antigen, which refers to a class of antigens that only have the ability to bind to corresponding immune substances (e.g., antibodies). Due to their small molecular weight, they cannot induce immune responses when they exist alone and lack immunogenicity. Haptens need to be combined with other substances (e.g., proteins, polylysine) to be immunogenic. Haptens include but are not limited to hormones, vitamins, polysaccharides, lipids, nucleic acids, small molecule compounds, drugs and narcotics. Hormones include but are not limited to progesterone, aldosterone, estradiol, and testosterone; vitamins include but are not limited to vitamin D, vitamin B, vitamin C, and folic acid; and narcotics include but are not limited to heroin, methamphetamine, morphine, barbiturates, methylenedioxymethamphetamine, lysergic acid diethylamide, and phencyclidine.

[0024] In the present invention, the term "antibody" is used in the broadest sense, which may include full-length monoclonal antibodies or antigen-binding fragments, bispecific or multispecific antibodies, and chimeric antibodies, as long as they exhibit the desired biological activity. In an optional embodiment, the antigen-binding fragment is selected from any one of F(ab')2, Fab', Fab, Fv and scFv of the antibody. Antigen-binding fragments generally have the same binding specificity as the antibody from which they are derived. It is easy for those skilled in the art to understand based on the contents described in the present invention that the above-mentioned antigen-binding fragments can be obtained by methods such as enzymatic digestion (including pepsin or papain) and / or by chemical reduction splitting disulfide bonds. Based on the structure of the complete antibody disclosed in the present invention, it is easy for those skilled in the art to obtain the above-mentioned antigen-binding fragments.

[0025] The above antigen-binding fragments can also be synthesized by recombinant genetic techniques also known to those skilled in the art or by, for example, an automatic peptide synthesizer, such as those sold by Applied BioSystems and the like.

[0026] In an alternative embodiment, the antibody is coated onto a solid support; alternatively, the complex antibody is coated onto a solid support.

[0027] In an alternative embodiment, the antibody is labeled with a signal marker; alternatively, the anti-small molecule hapten antibody is labeled with a signal marker.

[0028] In an optional embodiment, the above-mentioned signal marker refers to a class of substances having signals that can be directly observed by the naked eye or detected or detected by instruments, such as luminescence, color development, radioactivity, etc., and the detectable signal can be used to achieve qualitative or quantitative detection of the corresponding target.

[0029] In alternative embodiments, signal markers include, but are not limited to, fluorescent dyes, enzymes, radioisotopes, chemiluminescent agents, and nanoparticle-based markers.

[0030] In actual use, those skilled in the art can select a suitable signal marker according to the detection conditions or actual needs. No matter which signal marker is used, it belongs to the protection scope of the present invention.

[0031] In an optional embodiment, the fluorescent dye is not limited to fluorescein dyes and their derivatives (for example, including but not limited to fluorescein isothiocyanate (FITC), hydroxyfluorescein (FAM), tetrachlorofluorescein (TET), etc. or their analogs), rhodamine dyes and their derivatives (for example, including but not limited to red rhodamine (RBITC), tetramethylrhodamine (TAMRA), rhodamine B (TRITC), etc. or their analogs), Cy series dyes and their derivatives (for example, including but not limited to Cy2, Cy3, Cy3B, Cy3.5, Cy3.7, Cy3.8, Cy3.9, Cy3.10, Cy3.11, Cy3.12, Cy3.13, Cy3.14, Cy3.15, Cy3.16, Cy3.17, Cy3.18, Cy3.19, Cy3.20, Cy3.21, Cy3.22, Cy3.23, Cy3.24, Cy3.25, Cy3.26, Cy3.27, Cy3.28, Cy3.29, Cy3.30, Cy3.31, Cy3.32, Cy3.33, Cy3.34, Cy3.35, Cy3.36, Cy3.37, Cy3.38, Cy3.39, Cy3.40, Cy3.41, Cy3.42, Cy3.43, Cy3.44, Cy3.45, Cy3.46, Cy3.47, Cy3.48, Cy3.49, Cy3.50, Cy3.51, Cy3.52, Cy3.53, Cy3.54, Cy3.55, Cy3.56, Cy3.57, Cy3.58, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.59, Cy3.5 y5, Cy5.5, Cy3, etc. or their analogs), Alexa series dyes and their derivatives (for example, including but not limited to AlexaFluor350, 405, 430, 488, 532, 546, 555, 568, 594, 610, 33, 647, 680, 700, 750, etc. or their analogs) and protein dyes and their derivatives (for example, including but not limited to phycoerythrin (PE), phycocyanin (PC), allophycocyanin (APC), pre-chlorophyll protein (preCP), etc.).

[0032] In alternative embodiments, the enzyme includes, but is not limited to, horseradish peroxidase, alkaline phosphatase, β-galactosidase, glucose oxidase, carbonic anhydrase, acetylcholinesterase, and 6-phosphoglucose deoxygenase.

[0033] In an optional embodiment, the chemiluminescent reagent includes but is not limited to luminol and its derivatives, lucigenin, crustacean fluorescein and its derivatives, bipyridine ruthenium and its derivatives, acridinium esters and their derivatives, dioxetanes and their derivatives, lophanes and their derivatives, and peroxyoxalates and their derivatives.

[0034] In an optional embodiment, the nanoparticle markers include, but are not limited to, nanoparticles, colloids, magnetic nanoparticles, quantum dot nanoparticles, and rare earth complex nanoparticles.

[0035] In alternative embodiments, the colloid includes, but is not limited to, colloidal metals, dye-labeled microspheres, and latex.

[0036] In an alternative embodiment, the colloidal metal includes, but is not limited to, colloidal gold, colloidal silver, colloidal selenium and colloidal carbon.

[0037] In an optional embodiment, the dye-labeled microspheres and latex include colored latex and fluorescent microspheres. Optionally, the fluorescent microspheres are polystyrene fluorescent microspheres.

[0038] In an optional embodiment, the rare earth element in the rare earth complex nanoparticles is a lanthanide element, and preferably the lanthanide element is Eu3+, Tb3+, Sm3+, or Dy3+.

[0039] In an optional embodiment, the solid phase carrier includes but is not limited to magnetic microspheres, plastic microspheres, plastic microparticles, microwell plates, glass, capillaries, nylon and nitrocellulose membranes.

[0040] As used herein, the term "sample" refers to a composition obtained from or derived from a subject and / or individual of interest, comprising, for example, cells and / or other molecular entities to be characterized and / or identified based on physical, biochemical, chemical, and / or physiological characteristics. In some aspects, the sample is a blood sample (e.g., a whole blood sample, a serum sample, or a plasma sample). Other samples include, but are not limited to, primary or cultured cells or cell lines, cell supernatants, cell lysates, platelets, vitreous fluid, synovial fluid, follicular fluid, semen, amniotic fluid, breast milk, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, feces, tumor lysates, and tissue culture media, tissue extracts such as homogenized tissues, cell extracts, and combinations thereof.

[0041] 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.

[0042] 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.

[0043] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of cell biology, molecular biology (including recombinant techniques), microbiology, biochemistry, and immunology, which are within the capabilities of a skilled artisan.

[0044] The features and properties of the present invention are further described in detail below in conjunction with the vitamin D embodiment.

[0045] Example 1 Preparation of Vitamin D Detection Reagent

[0046] (1) Anti-vitamin D antibody (PhiPeng Biotechnology, catalog number VD-REAB-E1-007) labeling: Take 0.1 ml of 1% solid content fluorescent microspheres, add 900 ul activation buffer, centrifuge to remove the supernatant, add 1 ml activation buffer and ultrasonically mix, then add activator, shake and mix in the dark for 20 min, centrifuge to remove the supernatant, add 1 ml coupling buffer (MES buffer, pH = 6.0), ultrasonically mix, add 0.1 mg of anti-vitamin D antibody, shake and mix in the dark for 3 h, finally add blocking buffer for blocking, shake and mix in the dark for 1 h, terminate labeling, centrifuge to remove the supernatant, re-dissolve the microspheres with microsphere preservation solution, ultrasonically mix and store at 4°C before use.

[0047] (2) Preparation of label pad: After the labeled anti-vitamin D antibody is diluted to 10% with microsphere diluent, the signal marker is sprayed on the glass fiber using a spray pad apparatus. The sprayed label pad is placed in a 50°C oven and dried for more than 2 hours.

[0048] (3) Test line preparation process: Anti-vitamin D complex monoclonal antibody (PhiPeng Biotechnology, catalog number VD-REAB-E1-006) was diluted to 1.0-1.5 mg / ml using coating diluent and then coated on the first test line; anti-vitamin D complex monoclonal antibody only binds to the complex formed by the combination of anti-vitamin D antibody and vitamin D, but does not bind to unbound anti-vitamin D antibody monomers or vitamin D monomers; bovine serum albumin-coupled vitamin D (PhiPeng Biotechnology, catalog number VD-AG-E3-002) was diluted to 0.8-1.2 mg / ml using coating diluent and then coated on the second test line and dried in a 50°C oven overnight.

[0049] (4) Vitamin D testing

[0050] Add 5 μL of sample to a 1.5 ml EP tube, add 200 μL of dissociation agent, and shake to mix. Take 75 μL of the dissociated reaction solution and add it to the 3.5 mm wide test strip marking pad, let it stand at room temperature for 15 minutes, and then test it on the machine.

[0051]

[0052]

[0053] Comparative Example 1

[0054] (1) Labeling of anti-vitamin D antibody (PhiPeng Biotechnology, catalog number VD-REAB-E1-007) and goat anti-chicken IgY antibody: Take 0.1 ml of 1% solid content fluorescent microspheres, add 900 ul activation buffer, centrifuge to remove the supernatant, add 1 ml activation buffer and mix by sonication, then add activator, shake and mix in the dark for 20 min, centrifuge to remove the supernatant, add 1 ml coupling buffer (MES buffer, pH = 6.0), mix by sonication, add 0.1 mg antibody, shake and mix in the dark for 3 h, finally add blocking buffer for blocking, shake and mix in the dark for 1 h, terminate labeling, centrifuge to remove the supernatant, re-dissolve the microspheres with microsphere preservation solution, mix by sonication, and store at 4°C before use.

[0055] (2) Preparation of label pad: dilute the labeled anti-vitamin D antibody and goat anti-chicken IgY antibody with microsphere diluent, and use a spray pad instrument to spray the signal marker on the glass fiber. Place the sprayed label pad in a 50°C oven and dry for more than 2 hours.

[0056] (3) Preparation process of test line and quality control line: dilute the anti-vitamin D complex antibody (Phipeng Biotechnology, catalog number VD-REAB-E1-006) to 1.0-1.5 mg / ml with coating diluent and then coat it on the test line; dilute the chicken IgY antibody to 1.0-1.5 mg / ml with coating diluent and then coat it on the quality control line, and dry it in a 50°C oven overnight.

[0057] (4) Vitamin D testing

[0058] Add 5 μL of sample to a 1.5 ml EP tube, add 200 μL of dissociation agent, and shake to mix. Take 75 μL of the dissociated reaction solution and add it to the 3.5 mm wide test strip marking pad, let it stand at room temperature for 15 minutes, and then test it on the machine.

[0059]

[0060]

[0061] Comparative Example 2

[0062] (1) Labeling with anti-vitamin D antibody (PhiPeng Biotechnology, catalog number VD-REAB-E1-007) and goat anti-chicken IgY antibody: Take 0.1 ml of 1% solid content fluorescent microspheres, add 900 ul activation buffer, centrifuge to remove the supernatant, add 1 ml activation buffer and ultrasonically mix, then add activator, shake and mix in the dark for 20 min, centrifuge to remove the supernatant, add 1 ml coupling buffer (MES buffer, pH = 6.0), ultrasonically mix, add 0.1 mg of anti-vitamin D antibody, shake and mix in the dark for 3 h, finally add blocking buffer for blocking, shake and mix in the dark for 1 h, terminate labeling, centrifuge to remove the supernatant, re-dissolve the microspheres with microsphere preservation solution, ultrasonically mix and store at 4°C before use.

[0063] (2) Preparation of label pad: dilute the labeled anti-vitamin D antibody and goat anti-chicken IgY antibody with microsphere diluent, and use a spray pad instrument to spray the signal marker on the glass fiber. Place the sprayed label pad in a 50°C oven and dry for more than 2 hours.

[0064] (3) Preparation process of test line and quality control line: Vitamin D and bovine serum albumin were coupled, diluted to 1.0 mg / ml with coating diluent, and then coated on the test line; chicken IgY antibody was diluted to 1.0 mg / ml with coating diluent, and then coated on the quality control line, and dried in a 50°C oven overnight.

[0065] (4) Vitamin D testing

[0066] Add 5 μL of sample to a 1.5 ml EP tube, add 200 μL of dissociation agent, and shake to mix. Take 75 μL of the dissociated reaction solution and add it to the 3.5 mm wide test strip marking pad, let it stand at room temperature for 15 minutes, and then test it on the machine.

[0067] Test results:

[0068]

[0069]

[0070] In the field of fluorescent immunoassay, the faster the signal ratio changes, the better the gradient and the more sensitive the detection. Figure 1 It can be clearly seen from the results shown in that the present application scheme has the advantage of high sensitivity.

[0071] 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. Small molecule hapten detection reagent, It is characterized in that The reagents include a first antigen, an anti-small molecule hapten antibody, a complex antibody and a signal marker; The small molecule hapten can bind to the anti-small molecule hapten antibody to form a complex, and the complex antibody can bind to the complex; Anti-small molecule hapten antibodies are labeled with a signal marker; The first antigen and the anti-small molecule hapten antibody are coupled to different solid phase carriers; or, the first antigen and the anti-small molecule hapten antibody are coupled to different regions of the same solid phase carrier; the first antigen can bind to the anti-small molecule hapten antibody.

2. Small molecule hapten detection reagents, It is characterized in that The small molecule hapten detection reagent is an immunochromatography reagent, which includes a chromatographic membrane, on which a marking pad, a first detection zone and a second detection zone are sequentially arranged; the marking pad is provided with an anti-small molecule hapten antibody marked with a signal marker, the first detection zone is provided with the complex antibody, and the second detection zone is provided with the first antigen; the first antigen can bind to the anti-small molecule hapten antibody.

3. The small molecule hapten detection reagent according to claim 2, It is characterized in that The first detection zone and the second detection zone of the reagent generate the same type of detectable signal.

4. The small molecule hapten detection reagent according to any one of claim 1, It is characterized in that The solid supports include microspheres, plates and membranes.

5. The small molecule hapten detection reagent according to any one of claims 1 to 3, It is characterized in that The first antigen and the small molecule hapten are the same substance, or are derivatives or analogs of the small molecule hapten; The small molecule hapten or first antigen includes hormones, vitamins, drugs, and narcotics.

6. The small molecule hapten detection reagent according to any one of claims 1 to 3, It is characterized in that The signal marker is selected from colloidal gold, fluorescent dye, enzyme, chemiluminescent marker, electrochemiluminescent marker and nanoparticle marker.

7. The small molecule hapten detection reagent according to any one of claims 1 to 3, It is characterized in that The first antigen is coupled with a bridging protein; Preferably, the bridging protein comprises chicken ovalbumin, gelatin, keyhole limpet hemocyanin, bovine serum albumin, human serum albumin, rabbit serum albumin, poly-lysine or an artificially synthesized non-functional linear polypeptide.

8. Method for detecting small molecule haptens, It is characterized in that Includes at least the following steps: a) detecting a first signal generated by the combination of a small molecule hapten in a sample and an anti-small molecule hapten antibody labeled with a signal marker to form a complex and the complex antibody; b) detecting a second signal generated by the binding of an anti-small molecule hapten antibody labeled with a signal marker to the first antigen; c) calculating the concentration of the small molecule hapten in the sample according to the first signal intensity and the second signal intensity.

9. The method for detecting small molecule haptens according to claim 8, It is characterized in that The concentration of small molecule hapten in the sample is calculated by the ratio of the first signal intensity to the second signal intensity.

10. The method for detecting small molecule haptens according to claim 8, It is characterized in that The sample is blood or urine; optionally, the small molecule hapten or the first antigen includes hormones, vitamins, drugs, and narcotics; optionally, the first antigen is coupled with a bridging protein.

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