Reagent for photo-induced chemiluminescence detection, kit and preparation method thereof

Abstract

This application relates to a reagent, kit, and preparation method for photochemiluminescence detection. The reagent comprises an acceptor capable of reacting with singlet oxygen to generate a detectable signal, a first biomolecule binding to the acceptor, and a biotin-labeled second biomolecule. The biomolecule specifically binds to the analyte. The biotin is directionally labeled onto the biomolecule using a Catcher-Tag system. The technical solution of this application provides a detection reagent with high sensitivity and strong anti-hooking ability, which can improve the abundance of photochemiluminescence detection signals and the accuracy of detection results.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to a reagent, kit, and preparation method for photo-induced chemiluminescence detection. Background Technology

[0002] Chemiluminescence immunoassay is an analytical technique that combines highly sensitive chemiluminescence assay with highly specific immunoreaction to detect various antigens, haptens, small molecules, antibodies, and drugs.

[0003] Based on the presence or absence of a separation and washing step, chemiluminescence is divided into heterogeneous chemiluminescence and homogeneous chemiluminescence. Homogeneous chemiluminescence, such as photo-induced chemiluminescence (PET) detection, uses two special microspheres—donor microspheres and acceptor microspheres. The donor microspheres generate singlet oxygen under excitation light. When an antibody labeled on the donor microsphere binds to an antigen in the sample, and simultaneously another antibody labeled on the acceptor microsphere binds to the antigen, the singlet oxygen generated by the donor microsphere is transferred to the acceptor microsphere via an antigen bridge, thus initiating a chemiluminescence reaction on the acceptor microsphere. The luminescence intensity is related to the concentration of the antigen in the sample. PET detection enables highly sensitive immunoassays without the need for a separation and washing step.

[0004] When using diagnostic reagents on a photochemiluminescence platform for testing, due to the complexity of antigen-antibody reactions, if the antigen concentration in the sample is too high, a hook effect may occur, meaning that the test results may actually be lower or false negatives may occur, affecting the accuracy of the test results. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a reagent, kit, and preparation method for photochemiluminescence detection. The detection reagent has high sensitivity and strong anti-hooking ability, which can improve the abundance of photochemiluminescence detection signal and the accuracy of detection results.

[0006] The first aspect of this application provides a photo-induced chemiluminescence detection reagent, comprising a receptor capable of reacting with singlet oxygen to generate a detectable signal and a first biomolecule bound thereto, and a biotin-labeled second biomolecule, wherein the biomolecule is capable of specifically binding to the analyte; and the biotin is directionally labeled onto the second biomolecule using a Catcher-Tag system.

[0007] In some embodiments, the Catcher in the Catcher-Tag system is linked to biotin to form a marker Catcher, and the Tag is co-expressed with the second biomolecule to form a fusion biomolecule; the marker Catcher and the fusion biomolecule achieve targeted labeling of biotin on the second biomolecule through isopeptide bonds between the Catcher-Tag system.

[0008] In some embodiments, the molar ratio of the Catcher to biotin is 1:(10~45).

[0009] In some embodiments, the molar ratio of the labeled Catcher to the fused biomolecule is 1:(1~6).

[0010] In some embodiments, the Tag in the fused biomolecule is co-expressed with the N-terminus or C-terminus of the second biomolecule.

[0011] In some embodiments, the tag is linked to the second biomolecule via a linker peptide at one end; preferably, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n; more preferably, n is a natural number from 1 to 5.

[0012] In some embodiments, the Catcher-Tag system is selected from the Spy Catcher-Tag system and the SnoopCatcher-Tag system; preferably, the Catcher-Tag system contains a tag protein.

[0013] In some embodiments, in the Catcher-Tag system, the tag protein is co-expressed with the Catcher; preferably, the tag protein is linked to the C-terminus or N-terminus of the Catcher; more preferably, the tag protein is linked to the Catcher via (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) at one end.

[0014] In some embodiments, the tagged protein is selected from at least one of a solubilization tag, a molecular chaperone, an enzyme tag, and a purification tag.

[0015] In some embodiments, the tag protein is selected from at least one of 6xHIS, Flag, Strep, Arg, Avi, VSV-G, GST, MBP, NusA, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, and SUMO.

[0016] In some embodiments, the tag protein is selected from polypeptide sequences containing (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) constructed through gene editing.

[0017] In some embodiments, the biomolecule is selected from antigens or antibodies.

[0018] A second aspect of this application provides a photo-induced chemiluminescence detection kit, comprising the reagents described above.

[0019] A third aspect of this application provides a method for preparing the above-mentioned reagent, comprising: biotin labeling a Catcher to obtain a labeled Catcher, then mixing it with a fusion-expressed Tag-biomolecule, and forming an isopeptide bond through the interaction between the Catcher and the Tag to obtain a biotin-directed labeled biomolecule.

[0020] The technical solution provided in this application can include the following beneficial effects: by introducing the Catcher-Tag system into the photo-induced chemiluminescence detection reagent for targeted labeling of biomolecules such as antigens / antibodies, the activity of specific antigens / antibodies in the detection reagent can be preserved to the maximum extent, significantly improving the anti-hooking ability of the detection reagent, improving detection sensitivity and accuracy of detection results, while also broadening the detection range of the reagent and improving the stability of the reagent, which can be widely used in clinical testing.

[0021] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Detailed Implementation

[0022] To facilitate understanding of the present invention, it will be described in detail below. However, before describing the present invention in detail, it should be understood that the present invention is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for describing specific embodiments only and is not intended to be restrictive.

[0023] Where numerical ranges are provided, it should be understood that every intermediate value between the upper and lower limits of the range and any other specified or intermediate value within the specified range is covered by this invention. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered by this invention, subject to any explicitly excluded limits within the specified range. Where a specified range includes one or two limits, the range excluding any or both of those included limits is also included by this invention.

[0024] Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While any methods and materials, or equivalents thereof, may be used in the practice or testing of this invention, preferred methods and materials are now described.

[0025] I. Terminology The term "analytes" as used in this article refers to all detectable substances present in biological fluids, including both macromolecules and small molecules. Analytes include, but are not limited to, proteins, hormones, antibodies, or antigens. Examples of biological fluids include blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion.

[0026] The term "sample to be tested" as used in this article refers to a mixture that may contain the target analyte. Before use, the sample to be tested can be diluted with a diluent or buffer solution as needed. For example, to avoid the hook effect, the target analyte can be diluted with a sample diluent before testing on the instrument. In this case, any diluted solution that may contain the target analyte is referred to as the sample to be tested.

[0027] The term "biomolecules" as used herein refers broadly to all types of molecules unique to living organisms, all of which are organic compounds. Based on molecular weight, they are divided into two main categories: biological macromolecules and biological small molecules. Biological macromolecules generally have a molecular weight of over 10,000 and include proteins, nucleic acids, and polysaccharides. Biological small molecules generally have a molecular weight below 1,000. The basic building blocks of biological macromolecules with biological activity, such as amino acids, small peptides, oligopeptides, oligosaccharides, and oligonucleotides, are biological small molecules. Vitamins (such as biotin and biotin derivatives), minerals, plant secondary metabolites and their degradation products (such as aglycones, flavonoids, glycosides, and alkaloids) are also biological small molecules. The biomolecules used in this application can be antigens or antibodies.

[0028] The terms “combination,” “connection,” and “coupling” as used in this article refer to the union between two substances caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, or interactions including but not limited to salt bridges and water bridges.

[0029] The specific binding described in this article refers to the mutual recognition and selective binding reaction between two substances, which, from a stereostructural perspective, is the conformational correspondence between the reactants in the response.

[0030] The directional binding described in this article refers to the phenomenon where two or more molecules bind to each other in a specific direction and at specific sites. This binding is not random, but rather based on the molecular structure, chemical properties, and the specificity of the interaction. For example, the antigen-binding site on an antibody molecule can recognize and bind to specific epitopes on an antigen molecule; this binding exhibits high specificity and directionality.

[0031] The Cater-Tag system described in this article is a protein covalent linking technology, comprising two parts: a Tag consisting of multiple amino acid residues and a Cater. Covalent linking of proteins is achieved through a specific reaction between the Tag and the Cater. The Tag-Catcher system may include, but is not limited to, the Spy Catcher-Tag system, the Snoop Catcher-Tag system, or other Cater proteins that can form isopeptide bonds with the corresponding Tag.

[0032] The Spy Catcher-Tag system described in this paper is developed based on the CnaB2 domain of the fibronectin FbaB from Streptococcus pyogenes. The Spy Tag is a short peptide containing 13 amino acid residues, and the Spy Catcher is a protein containing 138 amino acid residues. During the linkage process, the aspartic acid in the Spy Tag and the lysine in the Spy Catcher spontaneously react to form a heteropeptide covalent bond, catalyzed by the glutamate adjacent to the lysine. The Catcher protein in the Tag-Catcher system can exist as a monomer or a multimeric protein. A multimeric protein is a protein composed of two or more polypeptide chains, which can be identical or different, linked together by covalent or non-covalent bonds (such as hydrogen bonds, hydrophobic interactions, van der Waals forces, etc.).

[0033] The Snoop Catcher-Tag system described in this article was developed from the fimbriae protein RrgA of Streptococcus pneumoniae. The D4 domain of RrgA generates a stable isopeptide bond through an E803 catalytic reaction between a lysine (K742) and an asparagine (N854) that are spatially adjacent.

[0034] Biotin, as described in this article, is widely found in animal and plant tissues. Its molecule has two ring structures: an imidazoline ring and a thiophene ring. The imidazoline ring is the primary site for binding to avidin. Activated biotin can couple with almost all known biomolecules, including proteins, nucleic acids, polysaccharides, and lipids, mediated by protein cross-linking agents.

[0035] The avidin described herein is a protein secreted by Streptomyces. The streptavidin molecule consists of four identical peptide chains, each capable of binding one biotin, with a molecular weight of 65 kDa. Each antigen or antibody can simultaneously couple multiple biotin molecules, thereby creating a "tentacle effect" with avidin to enhance analytical sensitivity. Where necessary, any reagent used in this invention, including antigens, antibodies, receptors, or donors, can be conjugated to any member of the biotin-streptavidin specific binding pair, as required.

[0036] Co-expression, as described in this article, refers to fusing proteins from two or more genes that require co-expression, enabling simultaneous expression. The co-expression linkage can be achieved by linking the coding sequences of two or more genes together using gene recombination technology to form a fusion gene. This fusion gene, upon expression, produces a fusion protein containing different functional domains encoded by multiple genes. Alternatively, the co-expression linkage can be achieved using technologies such as pClick (see "Synthesis of precision antibody conjugates using proximity-induced chemistry" (Theranostics. 2021 Aug 27; 11(18): 9107-9117. doi: 10.7150 / thno.62444.) to link two or more peptides, proteins, etc., with different or identical gene sequences, forming a fusion protein. Multiple genes in the fusion protein can be expressed simultaneously.

[0037] The S Catcher described in this article is a Spy Catcher, Snoop Catcher, or other Cater protein that does not contain a tag protein and can form an isopeptide bond with the corresponding tag. The Cater Pro described in this article is a SCatcher protein that contains a tag protein, which can be linked to the C-terminus or N-terminus of the SCatcher via a linker peptide (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5).

[0038] The tag protein described in this article refers to a polypeptide or protein molecule that is fused with a target protein (such as a Catcher in a Cater-Tag system) using in vitro DNA recombination technology for expression, detection, purification, labeling, and ligation of the target protein. Tag proteins can be selected from commonly used protein tags, such as 6xHIS, Flag, Strep, Arg, Avi, VSV-G, GST, MBP, NusA, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, SUMO, etc.; or they can be constructed through gene editing to create polypeptide sequences containing specific amino acids.

[0039] The S Tag in the S Tag fusion antigen / antibody described in this article can be a Spy Tag, Snoop Tag, or other Tag protein that can form an isopeptide bond with the corresponding Caterer.

[0040] The term "antibody" as used herein is used in the broadest sense, including any isotype of antibody, antibody fragments that retain specific binding to antigens, including but not limited to Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, bispecific antibodies, and fusion proteins comprising the antigen-binding portion of an antibody and non-antibody proteins. Where desired, antibodies may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin.

[0041] The antigens described herein refer to substances capable of inducing antibody production, and can be classified into complete antigens and incomplete antigens (haptens). These antigens can be natural antigens extracted from pathogens or animal tissues, or recombinant antigens with specific antigenic properties prepared through genetic engineering techniques. Where necessary, antigens can be further conjugated to other components, such as specific binding pairing members, for example, biotin or avidin.

[0042] The term "reactive oxygen species" as used in this article refers to a general term for oxygen-containing and reactive substances in the body or natural environment. It primarily refers to excited-state oxygen molecules, including the one-electron reduction product of oxygen (superoxide anion (O2·-), the two-electron reduction product of oxygen (hydrogen peroxide (H2O2), the three-electron reduction product of oxygen (hydroxyl radical (·OH), as well as nitric oxide and reactive oxygen species). 1 O2), etc.

[0043] The luminescent microparticles described herein refer to polymeric microparticles filled with a luminescent composition, capable of reacting with reactive oxygen species to generate detectable light signals. Luminescent microspheres may also be called acceptor microspheres or luminescent microspheres. In some specific embodiments of the invention, the luminescent composition undergoes a chemical reaction with reactive oxygen species to form an unstable metastable intermediate, which can decompose and emit light simultaneously or subsequently. Typical examples of such substances include, but are not limited to: enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acrylamide, aryl vinyl ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. In other specific embodiments of the invention, the luminescent composition may further include europium complexes; more preferably, the europium complex is MTTA-EU. 3+ .

[0044] The photosensitive microparticles described herein refer to polymeric microparticles filled with photosensitizers that can generate reactive oxygen species upon photoexcitation. These can also be called donor microspheres or photosensitive microspheres. Solutions containing such photosensitive microparticles can be called photosensitive solutions or universal solutions. The photosensitizers can be those known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, but are not limited to these. The photosensitive microparticles can also be filled with other sensitizers; non-limiting examples include certain compounds that catalyze the conversion of hydrogen peroxide to singlet oxygen and water. Other examples of sensitizers include 1,4-dicarboxyethyl-1,4-naphthalene endoperoxide, 9,10-diphenylanthracene-9,10-endoperoxide, etc. Heating these compounds or direct light absorption by these compounds releases reactive oxygen species.

[0045] The microparticles described herein can be of any size and shape, expandable or non-expandable, porous or non-porous, and have any density, but preferably close to that of water. They are preferably buoyant in water and are composed of transparent, partially transparent, or opaque materials. The microparticles can be solids (such as polymers, metals, glass, organic or inorganic substances such as minerals, salts, and diatoms), small oil droplets (such as hydrocarbons, fluorocarbons, and siliceous fluids), vesicles (such as synthetic phospholipids, or natural substances such as cells and organelles). A non-limiting example of microparticles suitable for use in this invention is carboxylated polystyrene latex microspheres.

[0046] II. Specific Implementation Plan This application will now be described in more detail.

[0047] In photoluminescence detection systems, two specific antigens / antibodies are typically included to detect the target antibody / antigen in the sample. These two specific antigens / antibodies are coated on luminescent microspheres and labeled with one of their specific binding partners, such as biotin. When the target antibody / antigen is present in the sample, it binds to both the specific antigen / antibody on the luminescent microspheres and the biotin-labeled specific antigen / antibody. The biotin binds to the avidin-coated, highly photosensitive microspheres, forming a "sandwich" structure. Upon excitation by light, the excited microspheres undergo reactive oxygen species transfer with the luminescent microspheres, inducing a chemiluminescence reaction that generates a detectable light signal. The intensity of this light signal is directly proportional to the concentration of the target antibody / antigen in the sample. The inventors of this application discovered in their research that the activity and affinity of specific antigens / antibodies in the detection reagents can affect their ability to specifically recognize and bind to the antibody / antigen to be tested. This results in a large number of antibody / antigen to be tested that fail to produce an immune response, or a large number of immune products that can produce an immune response but fail to trigger a chemiluminescent reaction, leading to a hook effect and affecting the accuracy of the detection results.

[0048] The photo-induced chemiluminescence detection reagent involved in this application includes an acceptor capable of reacting with singlet oxygen to generate a detectable signal, a first biomolecule bound thereto, and a biotin-labeled second biomolecule. The biomolecule is capable of specifically binding to the target analyte, and the biotin is directionally labeled onto the second biomolecule using a Catcher-Tag system.

[0049] Introducing the Catcher-Tag system into the biotin labeling process of the second biomolecule enables biotin to bind directionally to the non-specific binding end of the second biomolecule, thereby fully exposing the end with specific recognition and binding capabilities, preserving the activity of the biomolecule, and fully leveraging its specific recognition and binding capabilities with the analyte in the sample, reducing the occurrence rate of the hook effect, and improving detection sensitivity.

[0050] The receptor is referred to as receptor microspheres, or luminescent microspheres or luminescent particles. Receptor microspheres include a carrier and a luminescent composition coated on or filled within the carrier, enabling them to react with reactive oxygen species to generate a detectable light signal. The carrier can be a polymer microparticle; suitable polymer microparticles for this application are polystyrene microspheres; however, other detectable microspheres made of other materials are also possible and are not limited thereto. The luminescent composition may include, for example, enol ethers, enamines, 9-alkylxanthan gum, 9-alkyl-N-alkylacridinium, arylate ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. Furthermore, the luminescent composition may also include europium complexes; preferably, the europium complex is MTTA-EU. 3+ .

[0051] In some embodiments of this application, biotin may be an activated biotin derivative, such as amino or carboxyl activated biotin.

[0052] In some embodiments of this application, the biomolecule can be an antigen or an antibody. When the biomolecule is an antigen, biotin can be directionally bound to the N-terminus or C-terminus of the antigen via the Catcher-Tag system; when the biomolecule is an antibody, biotin can be directionally bound to the heavy chain or light chain of the antibody via the Catcher-Tag system.

[0053] The first and second biomolecules can specifically bind to different binding sites of the target analyte (antigen / antibody) to form a "first biomolecule-target analyte-second biomolecule" complex.

[0054] When biotin is labeled on biomolecules, the Catcher in the Catcher-Tag system is linked to biotin to form a labeled Catcher. The Tag is co-expressed and linked with the biomolecule to form a fused biomolecule. The labeled Catcher and the fused biomolecule achieve directional labeling of biotin on the biomolecule through the spontaneously formed isopeptide bonds between the Catcher-Tag system.

[0055] This application utilizes isopeptide bonds in the labeling of antigens / antibodies to form an irreversible link between biotin and biomolecules, enabling both antigens and antibodies to be effectively and stably labeled. This avoids the presence of a large number of free specific antigens / antibodies in the detection reagents, which could lead to ineffective recognition and binding with the antibody / antigen to be tested, thus affecting the sensitivity and accuracy of the detection results.

[0056] In some embodiments of this application, when Catcher is directionally linked to biotin, the molar ratio of the two is 1:(10~45).

[0057] In some embodiments of this application, the molar ratio of the labeled Catcher to the fused biomolecule is 1:(1~6).

[0058] In some embodiments of this application, the Catcher-Tag system may be selected from the Spy Catcher-Tag system, Snoop Catcher-Tag system, or other Catcher-Tag systems that can form isopeptide bonds, with or without a tag protein; preferably, it is the S Catcher-Tag system that includes a tag protein.

[0059] In some embodiments of this application, the tag protein can be attached to any position in the Catcher-Tag system; preferably, it is attached to the Catcher protein in the Catcher-Tag system; more preferably, it is attached to the N-terminus and / or C-terminus of the Catcher protein. When the tag protein is introduced into both the N-terminus and C-terminus of the Catcher protein, the introduced tag proteins may be the same or different.

[0060] In some embodiments of this application, the tag protein can bind to the Catcher protein through co-expression or chemical coupling; co-expression is preferred.

[0061] In some embodiments of this application, the tag protein binds to the catcher via a linker peptide at one end. More preferably, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n; even more preferably, n is a natural number from 1 to 5.

[0062] In some embodiments of this application, the tag protein may be selected from at least one of commonly used protein tags, such as solubilization tags, molecular chaperones, enzyme tags, and purification tags. Specifically, the tag protein may be selected from at least one of Flag, Strep, Arg, Avi, VSV-G, GST, MBP, NusA, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, and SUMO.

[0063] In some other embodiments of this application, the tag protein is selected from polypeptide sequences containing (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) constructed through gene editing.

[0064] For example, genetic engineering techniques can be used to insert the sequence of the tag protein into the beginning or end of the Catcher protein sequence to construct a fusion expression vector. Then, through methods such as induced expression and purification, the Catcher protein fused with the tag protein can be obtained, enabling it to be expressed simultaneously.

[0065] When the catcher is linked to biotin, biotin can be attached to either the C-terminus or the N-terminus of the catcher. Alternatively, biotin can be attached to a tag protein, which in turn attaches to the catcher, thus enabling biotin labeling of the catcher protein.

[0066] The biotin-directed ligation Catcher can be coupled to any protein containing a tag, enabling targeted labeling of proteins from different projects.

[0067] When a tag is co-expressed with a biomolecule, the tag can be linked by co-expression with the N-terminus or C-terminus of the biomolecule. Further, the tag can be linked to the biomolecule via a linker peptide at one end; preferably, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5). The tag can be fused to the N-terminus or C-terminus of an antigen or the heavy or light chain of an antibody via its linker peptide.

[0068] This application also relates to a photo-induced chemiluminescence detection kit, which includes the detection reagents described above. Specifically, it includes a biotin reagent, which achieves directional labeling of biotin on biomolecules using a Catcher-Tag system.

[0069] In addition, the kit includes a luminescent reagent and a photosensitive reagent. The luminescent reagent includes a receptor and a first biomolecule coated on the surface of the receptor; the biotinylate reagent includes a second biomolecule and biotin labeled to the second biomolecule via a Catcher-Tag system; the photosensitive reagent includes a donor and avidin coated on the surface of the donor; wherein the first and second biomolecules are capable of undergoing specific immune responses with the target substance to be tested, respectively.

[0070] The receptor, or receptor microsphere, or luminescent microsphere, is filled with a luminescent composition and can react with reactive oxygen species to generate a detectable light signal; the donor, or donor microsphere, or photosensitive microsphere, is filled with a photosensitizer and can generate reactive oxygen species when excited by light of a certain wavelength.

[0071] The preparation method of the photo-induced chemiluminescence detection reagent described in this application includes biotin labeling a Catcher to obtain a labeled Catcher, which is then mixed with a fused-expressed Tag-biomolecule. Through the interaction between the Catcher and the Tag, an isopeptide bond is formed to obtain a biotin-directed labeled biomolecule.

[0072] Specifically, this may include: 1) Inserting the tag sequence into the biomolecule sequence and co-expressing it in cells yields a fusion biomolecule; 2) Mix Catcher with biotin to conjugate Catcher and obtain labeled Catcher; 3) Mix the labeled Catcher with the fused biomolecule, purify, and obtain the biotin-labeled biomolecule.

[0073] In the reagents and kits of this application, biotin is directionally labeled in biomolecules, which allows the active end of the biomolecules to be fully exposed, thereby improving the utilization rate of biomolecules. This avoids the presence of a large number of free unlabeled biomolecules or a large number of target substances that fail to specifically react with biomolecules in the photo-induced chemiluminescence detection system, enabling rapid and effective immune reactions and chemiluminescence reactions between antigens and antibodies, improving the anti-hooking ability of the detection system, and enhancing detection sensitivity and accuracy.

[0074] III. Specific Implementation Examples To make the present invention easier to understand, the present application will be further described in detail below with reference to embodiments. These embodiments are for illustrative purposes only and are not limited to the scope of application of the present application. Unless otherwise specified, the raw materials or components used in the present application can be obtained commercially or by conventional methods.

[0075] The following example uses antibodies as biomolecules. Biotin reagents prepared by biotin-directed labeling of antibodies using the Catcher-Tag system are compared with biotin reagents prepared by direct labeling of antibodies using a photo-induced chemiluminescence platform to detect the test samples. Qualitative comparison is performed using the light signal values ​​to determine the improvement effect of the photo-induced chemiluminescence detection performance of the directed labeling antibodies.

[0076] Example 1: Preparation of Detection Reagent 1. Preparation of reagent R1 (receptor reagent) 10 mg of luminescent microparticles were dialyzed into 0.05 M CB, pH 9.6 buffer solution by centrifugation; a certain amount of Tween-20 was added to the luminescent microparticle solution, and the microparticles were dispersed by sonication (0.8 mg of Tween-20 was added per 10 mg of luminescent microparticles); they were mixed at a mass ratio of FG:Ab1 = 10:(0.25~5) to a microparticle concentration of 10 mg / mL, and reacted at room temperature for 1 h; ② the microparticles were washed with luminescent buffer and centrifuged to remove any possible free antibodies, repeating the centrifugation and washing process 3 times; finally, the microparticles were resuspended in luminescent buffer and brought to a final volume of 10 mg / mL to obtain directly antibody-coated luminescent microparticles. These are hereinafter referred to as FG-Ab1.

[0077] The R1 reagent, containing 50 μg / mL FG-Ab1, was prepared using a luminescent buffer solution.

[0078] 2. Preparation of reagent R2 (biotin reagent) 2.1 Preparation of Biotin-Directed Labeled Antibodies 2.1.1 Preparation and purification of S Catcher (Pro) (1) Construction of Catcher plasmid expression vector: The expression vector of Catcher (E. coli vector-pet series expression plasmid) was constructed based on the sequence of Spy Catcher (GenBank accession number: JQ478411.1). (If Catcher containing the tag protein is used, the expression vector of Catcher is constructed based on the sequence of Spy Catcher and the sequence of the tag protein.) (2) Catcher expression induced by E. coli: The E. coli expression vector was used to induce the expression of Catcher. The culture conditions were as follows: ① Self-induction medium (10g peptone, 5g yeast extract, 1x NPS, 1mM MgCl2, 1x 5052, pH=7.4), with 100nM ampicillin added; ② Induction conditions: overnight culture at 30℃ and 200rpm in a shaker.

[0079] (3) Catcher purification: ① After overnight reaction, centrifuge at 8000 rpm and collect the bacterial pellet; ② After culture, centrifuge the bacterial solution at 8000 rpm for 2 minutes, discard the supernatant, and retain the bacterial cells; ③ Resuspend the bacterial cells in 100 mL of purification loading buffer A (50 mM PBS, 150 mM NaCl, 20 mM ID, pH=7.4), and after the resuspended bacterial solution is homogenized by high pressure homogenizer, centrifuge at 18000 rpm for 40 minutes to separate the supernatant and inclusion bodies; ④ Collect the supernatant, sonicate for 1 minute (sonication conditions: 2s sonication, 3s interval, 60% power) to break the nucleic acid, and filter with a 0.22 μm needle filter after sonication; ⑤ Obtain Catcher protein using conventional Ni-NTA affinity chromatography gravity column purification method, purification buffer: supernatant (soluble protein) of solution A, loading and equilibration buffer (50 mM PBS, 150 mM NaCl, 25 mM imidazole, pH=7.4). 7.4) + B solution supernatant (soluble protein) elution buffer (50mM PBS, 150mM NaCl, 500mM imidazole, pH 7.4), the target protein is obtained by adjusting the imidazole concentration; ⑥ Elution conditions are: 5%, 15%, 50% and 100% of B solution for fractional elution.

[0080] (4) The protein concentration of the collected portion was determined by the BCA method. Then, the portion containing protein was subjected to reduction and non-reduction electrophoresis. A collection tube that matches the molecular weight of the Catcher protein was selected and dialyzed into the subsequently labeled buffer.

[0081] 2.1.2 Expression and purification of S Tag fusion antibody The Spy Tag (sequence AHIVMVDAYKPTK) was linked to the antibody using pClick technology to obtain the STag-linked antibody. The protein concentration of the collected fraction was determined by the BCA method, and the fraction containing protein was then subjected to electrophoresis. Fractions matching the molecular weight of the Tag-antibody were collected and dialyzed into the buffer required for subsequent coupling reactions to obtain the co-expressed STag-antibody.

[0082] 2.1.3 Biotin-directed labeled antibody (1) Biotin-labeled Catcher: The purified Spy Catcher was dialyzed into a 0.1 M NaHCO3, pH 8.3 buffer solution. NHs-LC-LC-Biotin (purchased from Thermo Fisher Scientific) was added at a Catcher to biotin molar ratio of 1:(10~45) and reacted overnight at 4°C. Free biotin was removed by dialysis, desalting or other similar methods, and the final product was stored in a 0.01 M NaHCO3, pH 8.3 buffer solution, and the protein concentration was determined.

[0083] (2) Labeled Catcher and Antibody Coupling Reaction: Biotin-labeled Spy Catcher and Spy Tag fusion antibody (hereinafter collectively referred to as Ab2-tag) were mixed at a molar ratio of 1:(1~6). After the reaction, a second purification was performed using Protein A chromatography to remove free (i.e., uncovalently bonded to the antibody) Catcher, yielding the directionally conjugated biotinylated antibody (Ab2-tag biotin). The protein concentration of the collected fraction was determined using the BCA method, and the fraction containing protein was then subjected to electrophoresis. Collection tubes matching the molecular weight after conjugation were selected and dialyzed into biotinylate storage buffer. The directionally labeled antibody is referred to as Ab2-tag biotin.

[0084] 2.2 Biotin-labeled antibodies The process of directly labeling antibodies with biotin is the same as the process of targeted labeling with Catcher-Tag. Hereinafter, the directly labeled antibody will be referred to as Ab2 biotin.

[0085] 2.3 The directionally labeled biotinylated antibody Ab2-tag biotin and the directly labeled biotinylated antibody Ab2biotin were prepared with biotin buffer to obtain R2 reagent containing 2 μg / mL Ab2-tag biotin or Ab2 biotin.

[0086] Example 2: Performance testing of S Catcher-Tag-labeled biotin reagent 1. Experimental Procedure (1) Both S Catcher-Tag-labeled Ab2-tag biotin and directly labeled antibody Ab2 biotin were reacted with antibody FG-Ab1 coated on luminescent microparticles FG on a photo-induced chemiluminescence platform. Twenty serum samples were tested simultaneously to compare the reactivity of the directionally labeled Ab2-tag biotin and the directly labeled antibody Ab2 biotin with FG-Ab1. The antibodies Ab1 and Ab2 were both CA72-4 antibodies, purchased from Invitrogen.

[0087] (2) The reaction system was as follows: 25 μL R1 reagent (containing 50 μg / mL FG-Ab1) + 25 μL R2 reagent (containing 5 μg / mL Ab2-tag biotin or Ab2 biotin) + 25 μL 10 mM PBS. After incubation for 17 min, 175 μL R3 universal photosensitive solution (containing photosensitive microparticles coated with streptavidin, purchased from PerkinElmer) was added and incubated for 10 min. The readings were then taken. The test data were compared with the results of Roche reagent detection, and the correlation between the two results was calculated. The test data are shown in the table below.

[0088] 2. Experimental Results 2.1 Sample test results.

[0089] Table 1

[0090] 3. Experimental Data Analysis Directed biotin reagent outperformed directly labeled biotin reagent in all aspects: ①Signal abundance: Within the linear range, the signal value of directional labeling can reach about 26w, which is about twice the highest signal value of direct labeling. ②Relevance: The correlation of targeted labels can reach over 0.98, which is much higher than that of direct labels. Therefore, based on experimental data, it is shown that when directional labeling is used at the labeling end in photo-induced chemiluminescence detection reagents, it is superior to direct labeling in terms of signal abundance and correlation.

[0091] Example 3: Anti-HOOK ability test of S Catcher-Tag directionally labeled biotin reagent 1. Experimental Procedure As shown in Example 2, reagent R2, prepared by directionally labeling Ab2-tag biotin with directed-labeled antibodies and Ab2 biotin with directly labeled antibodies, was used in conjunction with reagent R1, prepared by FG-Ab1 directly coated with antibodies, to detect serum samples. The results were then compared with those obtained using Roche reagents. The test data are shown in the table below.

[0092] 2. Experimental Results Table 2

[0093] 3. Experimental Data Analysis When the sample concentration reaches about 40,000 U / mL, the signal value of the directly labeled antibody reagent reaches its highest value. As the sample concentration increases thereafter, the signal value drops instead. That is, the anti-HOOK ability of the directly labeled antibody detection reagent is about 40,000 U / mL.

[0094] The detection reagents that use isopeptide-coupled antibody technology to directionally label antibodies have an anti-HOOK ability of about 150,000 U / mL, and the signal value discrimination and abundance are higher.

[0095] Example 4: Anti-HOOK ability test of S Catcher Pro-Tag directionally labeled biotin reagent The following uses the CEA (carcinoembryonic antigen, purchased from Fitzgerald, USA) photochemiluminescence detection reagent as an example to conduct a comparative experiment on the preparation of a detection reagent labeled with anti-CEA (carcinoembryonic antigen antibody) using the method in Example 1.

[0096] 1. Experimental Procedure (1) Assembly of CEA detection kit: R1 reagent: luminescent microspheres and anti-CEA bound to the luminescent microspheres; R2 reagent: Biotin-labeled anti-CEA; Pair with R3 reagent: photosensitive reagent (streptavidin-photosensitive microsphere solution).

[0097] The specific CEA reagents are shown in Table 3.

[0098] Table 3

[0099] Note: Spy Catcher Pro is Spy Catcher containing a tagged protein, the molecular weight of which is approximately 16.5 kDa.

[0100] (2) CEA antigen was diluted with physiological saline to multiple concentration gradients between 10,000 and 200,000 ng / mL. The CEA antigen dilution solutions of multiple concentration gradients were detected using the above CEA detection reagent combinations A, B and C respectively. The concentration at which the chemiluminescence value decreased with increasing concentration was taken as the lowest concentration of antigen when the hook effect appeared as the HOOK concentration.

[0101] (3) Detection method On an automated chemiluminescence detection instrument, add 25 μL of the antigen or sample to be tested, 25 μL of R1 reagent, and 25 μL of R2 reagent sequentially to one reaction well, and incubate at 37°C for 15 min. Then add 175 μL of the universal solution (photosensitive reagent) for the photo-induced chemiluminescence analysis system to the above reaction well, and incubate at 37°C for 10 min. Use the detection instrument to read the values ​​and obtain the chemiluminescence signal values ​​(RLU). The detection results are recorded in the table below.

[0102] 2. Experimental Results Table 4

[0103] 3. Experimental Data Analysis Compared to R2 reagents that directly label antibodies, Spy Catcher (Pro)-Tag-mediated labeling can enhance anti-HOOK ability, especially Spy Catcher Pro-Tag, which shows a more significant enhancement effect.

[0104] Example 5: Sensitivity Comparison of Biotin Reagents with Different Labeling Methods Taking the glial fibrillary acidic protein (GFAP) detection reagent as an example, the detection performance of the directionally labeled antibody Ab2-tag biotin and the directly labeled antibody Ab2 biotin was compared and tested using GFAP calibrators.

[0105] 1. Experimental Procedure The labeled Ab2-tag biotin and Ab2 biotin were diluted to 5 μg / mL with luminescent diluent, and the coated FG-Ab1 was diluted to 50 μg / mL with diluent. These were then placed in the corresponding reagent racks of the photochemiluminescence instrument. GFAP calibrators C1, C2, C3, C4, C5, and C6 were placed in the corresponding sample racks of the photochemiluminescence instrument.

[0106] The reaction pattern was as follows: 25 μL each of reagents R1 and R2, 10 μL of calibrator, 15 μL of sample diluent (10 mM PBS), and 25 μL of universal photosensitive solution R3. After incubation for 10 min, readings were taken, and the correlation and slope were calculated. The test data are shown in the table below.

[0107] 2. Experimental Results Table 5

[0108] 3. Experimental Data Analysis The correlation of the detection reagents labeled by the method of antibody conjugation by isopeptide bond can reach above 0.99; the slope can reflect the sensitivity of the reagent detection, and the slope value can be used to judge that the sensitivity of the directionally labeled reagents is much higher than that of the directly labeled reagents.

[0109] Example 6: Comparison of the stability of biotin reagents with different labeling methods 1. Experimental Procedure Reagent R2 was incubated at 37°C for a total of 7 days. On days 0 (D0, before the accelerated experiment), 1 (D1), 3 (D3), 5 (D5), and 7 (D7), reagent R2 was reacted with luminescent microparticles R1 directly coated with antibody, along with a universal photosensitive solution, in a photo-induced chemiluminescence platform for detection. The stability of biotin-directed labeled antibody reagents and biotin-direct labeled antibody reagents was compared. Specifically, the antibody Ab1 coated with luminescent microparticles in reagent R1 was a secondary antibody against mouse IgG (purchased from Invitrogen), and the biotin-labeled antibody Ab2 in reagent R2 was a mouse IgG antibody (purchased from Invitrogen).

[0110] (2) Reaction system: 25 μL R1 reagent + 25 μL R2 reagent + 25 μL 10 mM PBS, incubated for 17 min, then 175 μL universal photosensitive solution was added and incubated for 10 min before reading the values. The detection results are shown in the table below.

[0111] 2. Experimental Results (1) Stability test results of biotin-directed labeled antibody reagent.

[0112] Table 6

[0113] (2) Stability test results of biotin-labeled antibody reagent.

[0114] Table 7

[0115] 3. Experimental Data Analysis In the accelerated experiment at 37℃, the correlation of the biotin-directed labeling antibody reagent showed almost no significant fluctuation, and the average decrease in signal value remained within 10%, while the decrease in signal value of the biotin-directed labeling antibody reagent reached 21%, which was much higher than that of the directed labeling reagent.

[0116] The reagent stability was good after the labeling end adopted the directional coupling technology, which was significantly improved compared with direct labeling.

[0117] Therefore, in photo-induced chemiluminescence platforms, labeling with isopeptide-coupled antibodies can significantly improve the signal abundance, sensitivity, stability, correlation, and anti-hooking ability of the reagents.

[0118] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A reagent for photo-induced chemiluminescence detection, characterized in that, The reagent includes a receptor capable of reacting with singlet oxygen to generate a detectable signal, a first biomolecule that binds to the receptor, and a biotin-labeled second biomolecule; the biomolecule is capable of specifically binding to the analyte; the biotin is directionally labeled onto the second biomolecule using a Catcher-Tag system.

2. The reagent according to claim 1, characterized in that, In the Catcher-Tag system, the Catcher is linked to biotin to form a labeled Catcher, and the Tag is co-expressed and linked with the second biomolecule to form a fusion biomolecule. The labeled Catcher and the fusion biomolecule achieve targeted labeling of biotin on the second biomolecule through isopeptide bonds between the Catcher-Tag system.

3. The reagent according to claim 2, characterized in that, The molar ratio of the catcher to biotin is 1:(10~45); and / or, The molar ratio of the labeled Catcher to the fusion biomolecule is 1:(1~6).

4. The reagent according to claim 2, characterized in that, In the fused biomolecule, the Tag is co-expressed and linked with the N-terminus or C-terminus of the second biomolecule; Preferably, the tag is linked to the second biomolecule via a linker peptide at one end; preferably, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n; more preferably, n is a natural number from 1 to 5.

5. The reagent according to any one of claims 1 to 4, characterized in that, The Catcher-Tag system is selected from SpyCatcher-Tag system and Snoop Catcher-Tag system; Preferably, the Catcher-Tag system contains a tag protein.

6. The reagent according to claim 5, characterized in that, The tag protein is co-expressed with the Catcher; Preferably, the tag protein is attached to the C-terminus or N-terminus of the catcher; Preferably, the tag protein is connected to the Catcher via (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) at one end.

7. The reagent according to claim 5, characterized in that, The tag protein is selected from at least one of the following: a solubilization tag, a molecular chaperone, an enzyme tag, and a purification tag; Alternatively, the tag protein is selected from a polypeptide sequence containing (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) constructed through gene editing.

8. The reagent according to any one of claims 1 to 7, characterized in that, The biomolecules are selected from antigens or antibodies.

9. A photo-induced chemiluminescence detection kit comprising the reagents described in any one of claims 1 to 8.

10. A method for preparing the reagent according to any one of claims 1 to 8, comprising: The Catcher is biotin-labeled to obtain a labeled Catcher, which is then mixed with the fusion-expressed Tag-biomolecule. Through the interaction between the Catcher and the Tag, an isopeptide bond is formed, resulting in a biotin-directed labeled biomolecule.

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