A homogeneous chemiluminescent detection method

Abstract

This application relates to a homogeneous chemiluminescence detection method. The method involves contacting a sample to be tested with a reaction system comprising a first composition and a second composition, and then detecting the light signal intensity of the reaction product. The biomolecules in the first composition and / or the second composition are directionally bound to the target substance via a Catcher-Tag system, and these biomolecules can interact with the target molecule. This technical solution reduces the loss of biomolecule activity in the detection reagent, improves the utilization rate of biomolecules, and enhances the specific reaction rate between biomolecules and the target molecule, thereby improving detection sensitivity and accuracy.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and more particularly to a homogeneous chemiluminescence detection method. Background Technology

[0002] Photochemiluminescence analysis is a new generation of homogeneous immunoassay technology based on nanoscale polymer particles. It has advantages such as no need for separation and washing, high precision, high analytical sensitivity, and fast detection speed, and is widely used in clinical research and diagnosis.

[0003] Photochemiluminescence analysis utilizes photosensitive microspheres loaded with photosensitizers and luminescent microspheres loaded with luminescent compositions. In the presence of the analyte antigen, antibodies modified on the surfaces of the two types of microspheres undergo an antigen-antibody specific binding reaction with the antigen, forming an immune complex and shortening the spatial distance between the two types of microspheres. Therefore, reactive oxygen species generated by the photosensitizer in the photosensitive microspheres under excitation light can diffuse to the luminescent microspheres, reacting with the luminescent composition within the microspheres to emit a light signal, thereby enabling qualitative or quantitative analysis of the analyte antigen.

[0004] In the detection of antigens using a photocatalytic chemiluminescence (PDC) analysis platform, antibodies modified on the surface of microspheres serve as the core raw material for the immunoreaction. Their activity, specificity, affinity, and batch-to-batch variation directly affect detection performance, such as repeatability, sensitivity, and the stability and reliability of the results. Furthermore, because PDC is a completely homogeneous immunoassay method, complex sample matrices can easily interfere with the binding performance of antibodies modified on the microsphere surface, affecting detection sensitivity. Therefore, there is an urgent need to establish a rapid, accurate, highly repeatable, and highly sensitive homogeneous immunoassay method. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a homogeneous chemiluminescence detection method that can reduce the loss of biomolecule activity in detection reagents, improve the utilization rate of biomolecules, and enhance the specific reaction rate between biomolecules and target molecules, thereby improving detection sensitivity and accuracy of detection results.

[0006] The first aspect of this application provides a homogeneous chemiluminescence detection method, which involves contacting a sample to be tested with a reaction system containing a first composition and a second composition, and then detecting the light signal intensity of the reaction product after the reaction; wherein, the biomolecules in the first composition and / or the biomolecules in the second composition are directionally bound to the target substance through a Catcher-Tag system, and the biomolecules are capable of interacting with the target molecule to be tested.

[0007] In some embodiments, the non-specific binding region of the biomolecule is directionally bound to the target substance via a Catcher-Tag system.

[0008] In some embodiments, the biomolecule is an antigen or an antibody.

[0009] In some embodiments, the Catcher-Tag system is selected from the Spy Catcher-Tag system or the Snoop Catcher-Tag system, which are capable of forming isopeptide bonds.

[0010] In some implementations, the Catcher-Tag system includes a tag protein.

[0011] In some embodiments, the molecular weight of the tag protein is 15kDa to 50kDa.

[0012] In some embodiments, the tag protein contains lysine residues.

[0013] In some embodiments, the lysine residues in the tagged protein account for 0.05 to 5 wt%.

[0014] In some embodiments, the tag protein contains 3 to 50 lysine residues.

[0015] In some embodiments, the isoelectric point of the tag protein is between 4 and 7.5.

[0016] In some implementations, the tag protein is co-expressed with the Catcher.

[0017] In some implementations, the tag protein binds to the N-terminus and / or C-terminus of the catcher.

[0018] In some embodiments, the tag protein binds to the catcher via (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) at one end.

[0019] In some embodiments, the first composition includes luminescent microspheres and a first antibody, the first antibody being directionally coated onto the luminescent microspheres via a first Catcher-Tag system.

[0020] In some embodiments, the first antibody binds to the luminescent microspheres via its Fc terminal or Fab-connected hinge region.

[0021] In some embodiments, in the first Catcher-Tag system, the Catcher is coated on luminescent microspheres, and the Tag is co-expressed and linked with the first antibody to form a first fusion protein.

[0022] In some embodiments, the mass ratio of the luminescent microspheres to the Catcher is 10:(0.05~1). In some embodiments, the mass ratio of the luminescent microspheres coated with Catcher to the first fusion protein is 10:(0.25~5).

[0023] In some embodiments, the luminescent microspheres are modified with active groups, which bind to the Catcher; preferably, the active groups are at least one of carboxyl, amino, aldehyde, and thiol groups.

[0024] In some embodiments, the second composition includes biotin and a second antibody, wherein the biotin is labeled with the second antibody via a second Catcher-Tag system.

[0025] In some embodiments, the second antibody binds to biotin via its Fc terminal or Fab-connected hinge region.

[0026] In some embodiments, in the second Catcher-Tag system, the Catcher binds to biotin, and the Tag is co-expressed and linked with the second antibody to form a second fusion protein.

[0027] In some embodiments, the molar ratio of the Catcher to biotin is 1:(10~45).

[0028] In some embodiments, the molar ratio of the biotin-labeled Catcher to the second fusion protein is 1:(1~4).

[0029] In some embodiments, both the first composition and the second composition employ a Catcher-Tag system containing a tag protein.

[0030] The technical solution provided in this application can include the following beneficial effects: by introducing directional coupling in photo-induced chemiluminescence detection, the effective amount of protein in the detection reagent that can bind to the target molecule is increased, and protein activity is preserved to the maximum extent, thereby improving detection performance such as detection sensitivity, as well as the consistency and reproducibility of detection results. This solution can provide high sensitivity and a wide detection range, and is suitable for the detection of both low-value and high-value samples, and has extremely high clinical application value.

[0031] 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

[0032] 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.

[0033] 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.

[0034] 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.

[0035] I. Terminology The term "sample to be tested" as used herein refers to a mixture that may contain an analyte, including but not limited to proteins, hormones, antibodies, or antigens. Typical samples to be tested that can be used in the methods disclosed in this invention include bodily fluids such as blood, blood derivatives, serum, plasma, urine, cerebrospinal fluid, saliva, synovial fluid, and emphysema effusion. The sample to be tested can be diluted with a diluent or buffer solution as needed before use. For example, to avoid the hook effect, the analyte can be diluted with a sample diluent before detection on the instrument; in this case, the diluted solution that may contain the analyte is collectively referred to as the sample to be tested.

[0036] The term "target molecules" as used in this article refers to all detectable substances present in biological fluids, including both macromolecules and small molecules. Target molecules include, but are not limited to, proteins, hormones, antibodies, or antigens.

[0037] 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.

[0038] The target substance described herein refers to a substance used in homogeneous chemiluminescence detection to carry or label biomolecules (e.g., antibodies), enabling them to be recognized by a specific detection method. The target substance typically binds to the biomolecule via chemical coupling. The target substance can be a substance capable of generating a detectable signal, such as a light signal or a radioactive signal, under certain conditions; it can also be a substance capable of amplifying the detection signal by assembling a substance-biomolecule complex capable of generating a detectable signal; or it can be a carrier containing a substance capable of generating a detectable signal or a substance capable of amplifying a detection signal. Typical examples applicable to this application include luminescent microspheres and biotin.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The Catcher-Tag system described in this article is a protein covalent linking technology, comprising two parts: a Tag and a Catcher, each consisting of multiple amino acid residues. Covalent linking of proteins is achieved through a specific reaction between the Tag and the Catcher. The Tag-Catcher system may include, but is not limited to, the Spy Catcher-Tag system and the Snoop Catcher-Tag system.

[0043] 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.).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The luminescent microspheres 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 microparticles. 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-alkylacridinium, arylate ethers, diethylene oxide, dimethylthiophene, aromatic imidazoles, or gloss enhancers. In other specific embodiments of the invention, the luminescent composition may further comprise europium complexes; more preferably, the europium complex is MTTA-EU. 3+ .

[0051] The photosensitive microspheres 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 microparticles. Solutions containing such photosensitive microspheres 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 microspheres 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] The Spy Catcher described in this article is the Spy Catcher protein without a tag protein. The SpyCatcher Pro described in this article is the Spy Catcher protein with a tag protein, wherein the tag protein can be linked to the C-terminus or N-terminus of the Catcher protein via a linker peptide (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5).

[0056] II. Specific Implementation Plan This application will now be described in more detail.

[0057] The inventors of this application discovered in their research that in a photoluminescence detection system, when the luminescent microspheres are coated with specific antigens / antibodies that can specifically bind to the target antibody / antigen, and when the specific antigens / antibodies are labeled, the active regions of the specific antigens / antibodies may be occupied or folded, and cannot be effectively displayed, thereby affecting their activity and consequently affecting the detection sensitivity and the accuracy of the detection results.

[0058] The homogeneous chemiluminescence detection method disclosed in this application determines whether the sample contains a target molecule and / or the content of the target molecule in the sample by contacting the sample with a reaction system comprising a first composition and a second composition, and then detecting the light signal intensity of the reaction product after the reaction. Specifically, the biomolecules in the first composition and / or the second composition are directionally bound to the target substance via a Catcher-Tag system, and these biomolecules are capable of interacting with the target molecule.

[0059] The biomolecules suitable for the methods described in this application may be, for example, antigens or antibodies. The target substances suitable for the methods described in this application may be, for example, luminescent microspheres or biotin.

[0060] In this application, the ability of a biomolecule to interact with a target molecule means that the two molecules can recognize and bind to each other, and this interaction has a high degree of specificity, such as the specific recognition and binding performance of antigen-antibody.

[0061] Using the Catcher-Tag system as a carrier for biomolecules to stably exist in the detection reagent, its directional immobilization function ensures that biomolecules are directionally bound to the target substance, thereby retaining the effective affinity ends of the biomolecules that can specifically react with the target molecule, improving the utilization rate of the biomolecules, and ensuring the specific reaction between the biomolecules and the target molecule after mixing with the sample, ensuring the transfer efficiency of singlet oxygen, so that the light signal intensity emitted by the luminescent microspheres meets the needs of photo-induced chemiluminescence clinical detection, and the detection results are consistent and reproducible in clinical applications, with clear discrimination, high precision and significant sensitivity.

[0062] Specifically, the non-specific binding regions of biomolecules can be directionally bound to the target substance through the Catcher-Tag system.

[0063] The Catcher-Tag system can be selected from tag systems composed of two substances capable of forming isopeptide bonds; preferably SpyCatcher-Tag or Snoop Catcher-Tag; further, it can be selected from Spy Catcher-Tag or Snoop Catcher-Tag containing tag proteins.

[0064] In some embodiments of this application, the Catcher-Tag system is a system with a tagged protein, wherein the tagged protein binds to the Catcher protein. Furthermore, the tagged protein and the Catcher protein can be co-expressed and linked through chemical coupling or gene editing. For example, genetic engineering techniques can be used to insert the sequence of the tagged protein into the beginning or end of the Catcher protein sequence to construct a fusion expression vector, and then the Catcher protein fused with the tagged protein can be obtained through induced expression and purification, enabling simultaneous expression.

[0065] Specifically, the tag protein can bind to the N-terminus and / or C-terminus of the Catcher protein. Further, the tag protein can bind to the Catcher 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.

[0066] In some embodiments of this application, the molecular weight of the tag protein can be 15 kDa to 50 kDa; preferably 15 kDa to 30 kDa.

[0067] In some embodiments of this application, the tag protein contains lysine residues. Increasing the lysine content on the catcher carrier protein via the tag protein can improve the efficiency of biomolecule coating or labeling, and also increase the number of binding sites on the catcher that can connect to luminescent microspheres or biotin, thereby enhancing the stability of the biomolecules after coating or labeling, improving the stability of the detection reagent, and ultimately increasing the sensitivity of the detection reagent.

[0068] To further improve the stability and sensitivity of the detection reagent, the proportion of lysine residues in the tag protein is 0.05-5 wt%; and / or, the number of lysine residues in the tag protein is 3-50; the isoelectric point of the tag protein is between 4 and 7.5; preferably 6.5-7.5.

[0069] The tag protein can be attached to the N-terminus and / or C-terminus of the catcher protein to facilitate binding between the tag protein and the catcher. When a tag protein is introduced to both the N-terminus and C-terminus of the catcher protein, the introduced tag proteins may be the same or different.

[0070] 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; preferably Flag-tag, Avi-tag, Myc-tag, Strep-tag II, or Arg-tag.

[0071] In other embodiments of this application, the tag protein can also be constructed by gene editing into a polypeptide sequence containing a specific amino acid, such as a polypeptide sequence containing lysine or arginine.

[0072] When the biomolecule in this method is an antibody molecule and the target substance is a luminescent microsphere, in some specific embodiments of this application, the first composition may include luminescent microspheres and a first antibody, with the first antibody being directionally coated onto the luminescent microspheres via a first Catcher-Tag system; in other embodiments of this application, the second composition may include biotin and a second antibody, with the biotin being directionally coated onto the second antibody via a second Catcher-Tag system.

[0073] When both the first antibody and the second antibody have a Y-shaped structure, they each include two Fab segments and one Fc segment. The non-specific binding region of the first antibody and / or the second antibody is the Fc terminus of the first antibody and / or the second antibody.

[0074] When both the first and second antibodies have a V-shaped structure, they include two Fab segments. The non-specific binding regions of the first and / or second antibodies are the Fab-linked hinge regions of the first and / or second antibodies.

[0075] The complementarity-determining region (CDR), located in the Fab segment, is the region where a specific binding reaction occurs with the target molecule. Therefore, in this application, the Fab segment of the antibody is referred to as the specific binding region, and the opposite segment as the non-specific binding region. When the CDR region is occupied or the Fab segment is folded during coating, labeling, or other steps of the first or second antibody, the Fab segment may not be effectively displayed, affecting the amount of antibody that can bind to the target molecule and the antibody activity in the reaction system, thus impacting the photochemical detection performance, such as sensitivity.

[0076] To further improve detection sensitivity, both the first composition at the coating end and the second composition at the labeling end are directionally coupled to the target substance using a Cater-Tag system; preferably, both the first and second compositions use a Cater-Tag system containing a tag protein. That is, the first antibody is directionally coated onto the luminescent microspheres using the first Cater-Tag system, and the second antibody and biotin are directionally bound using the second Cater-Tag system. The first and second Cater-Tag systems can be the same or different; preferably, they are different. Using different types of Cater-Tag systems at the coating and labeling ends can reduce cross-interference between the two systems in the detection reagent, avoid generating additional background signals that affect the accuracy of the detection results, and further improve detection performance.

[0077] Furthermore, in the first Catcher-Tag system, the Catcher is coated onto the luminescent microspheres, and the Tag is co-expressed and linked with the first antibody to form the first fusion protein. In some embodiments of this application, when the Catcher binds to the luminescent microspheres, the mass ratio of the luminescent microspheres to the Catcher is 10:(0.05~1). In some embodiments of this application, the mass ratio of the luminescent microparticles coated with the Catcher to the first fusion protein is 10:(0.25~5).

[0078] In some embodiments of this application, the luminescent microspheres are modified with active groups, which bind to the Catcher protein. These active groups can be carboxyl, amino, aldehyde, thiol, etc. The Catcher protein can bind to the luminescent microspheres modified with the active groups through its terminal lysine residues, resulting in luminescent microspheres coated with Catcher.

[0079] In the second Catcher-Tag system, the Catcher binds to biotin, and the Tag is co-expressed and linked with the second antibody to form the second fusion protein. In some embodiments of this application, the molar ratio of Catcher to biotin is 1:(10~45). In some embodiments of this application, the molar ratio of biotin-bound Catcher to the second fusion protein is 1:(1~4).

[0080] The luminescent microspheres or biotin-labeled catchers that have completed directional coating can be coupled to any protein containing a tag, enabling directional coating or labeling of proteins from different projects.

[0081] 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.

[0082] The homogeneous chemiluminescence detection method of this application is applicable to photo-induced chemiluminescence detection of both high-value and low-value samples. It can provide high sensitivity and a wide detection range, meeting the performance requirements of both high-value and low-value samples for chemiluminescence detection.

[0083] 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.

[0084] The Spy Catcher-Tag system is used as an example. The Catcher, which has the ability to specifically recognize the Fc end of the antibody, is coated onto the receptor microspheres (luminescent microspheres) or labeled with biotin. Then, it is coupled with the Fc end of the antibody to achieve directional coating and directional labeling. By comparing the detection discrimination between different experimental groups, its improvement effect on homogeneous luminescence detection performance is judged.

[0085] Example 1: Preparation of Spy Catcher, Spy Catcher Pro and Spy Tag fusion proteins 1. Preparation and purification of Spy Catcher and Spy 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).

[0086] (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.

[0087] (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.

[0088] (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.

[0089] Prepare Spy Catcher Pro containing the tagged proteins in Table 1 following the steps described above: Table 1

[0090] 2. Preparation and purification of Spy Tag antibodies The Spy Tag (sequence AHIVMVDAYKPTK) was ligated to the Fc region of the antibody using pClick technology to obtain the Spy Tag-linked antibody. The protein concentration of the collected fraction was determined using the BCA method, and the protein-containing fraction was then subjected to electrophoresis. Fractions matching the molecular weight of the Tag-antibody were collected and dialyzed into the buffer required for subsequent conjugation reactions to obtain the co-expressed Spy Tag-antibody.

[0091] Example 2: Preparation of Detection Reagents 1. Preparation of the first composition and receptor reagent R1 1.1 Preparation of directionally coated luminescent microparticles (1) Binding of luminescent microparticles (target material) to Catcher: ① Dialyze the purified Spy Catcher and SpyCatcher Pro to 0.05M CB, pH 9.6 buffer and determine the protein concentration. ② Dialyze 10 mg of luminescent microparticles to 0.05M CB, pH 9.6 buffer by centrifugation. ③ Add a certain amount of Tween-20 to the luminescent microparticle solution and sonicate to disperse (add 0.8 mg of Tween-20 per 10 mg of luminescent microparticles). ④ After sonication, add 0.05~1 mg of Spy Catcher or Spy Catcher Pro protein per 10 mg of microparticles according to the coating ratio, vortex to mix, and react overnight at room temperature. ⑤ Remove the reaction tube, add 10 μL of 8 mg / mL NaBH4, vortex to mix, and react for 2 h. ⑥ After the reaction is complete, centrifuge and wash with luminescent buffer. ⑦ Adjust the volume to 20 mg / mL using buffers such as 10 mM PBS, 50 mM Tris-HCl, and 50 mM HEPES (pH 6-8).

[0092] (2) Screening of luminescent microparticles FG microparticles coated with Spy Catcher and Spy Catcher Pro were reacted with an antibody against the tag protein purified by Catcher (Biotin Anti-6X His tag® antibody, purchased from Abcam) in a photochemiluminescence platform. The reaction system was: 25 μL FG-Catcher (concentration 50 μg / mL) + 25 μL Biotin Anti-6X His tag® antibody (2 μg / mL) + 25 μL 10 mM PBS. After incubation for 17 min, 175 μL of R3 universal photosensitive solution (containing photosensitive microparticles) was added and incubated for 10 min. The readings were then taken.

[0093] Screening for FG-catchers with strong reaction signals indicates that SpyCatcher / Spy Catcher Pro was successfully coated onto the luminescent microparticles, and they were used for subsequent coupling reactions.

[0094] (3) Conjugation of luminescent microparticles with Spy Tag-antibody: ① Mix FG microparticles coated with Spy Catcher or Spy Catcher Pro with Spy tag-antibody 1 (hereinafter collectively referred to as Ab1-tag) at a mass ratio of FG:Ab1-tag=10:(0.25~5), with a microparticle concentration of 10 mg / mL, and react at room temperature for 1 h; ② Wash with luminescent buffer and remove any possible free antibody by centrifugation, repeating the centrifugation and washing 3 times; finally, resuspend in luminescent buffer and adjust the volume to 10 mg / mL to obtain luminescent microparticles with directional conjugated antibody. The directionally coated microparticles are referred to as FG-Ab1-tag (first composition).

[0095] 1.2 Preparation of directly coated luminescent microparticles The process of directly coating antibody Ab1 is the same as the process of targeted antibody coating using Catcher-Tag. Hereinafter, the directly coated microparticles will be referred to as FG-Ab1.

[0096] 1.3 The antibody-directly coated particles FG-Ab1-tag and the antibody-directly coated particles FG-Ab1 were prepared with luminescent buffer to obtain R1 reagent containing 50 μg / mL FG-Ab1-tag or FG-Ab1.

[0097] 2. Preparation of the second composition and biotin reagent R2 2.1 Biotin-directed labeled antibody (1) Catcher-labeled biotin (target substance): Purified Spy Catcher and Spy Catcher Pro were dialyzed into 0.1M NaHCO3, pH=8.3 buffer. NHs-LC-LC-Biotin (purchased from Thermo Fisher Scientific) was added at a molar ratio of Catcher to biotin of 1:(10~45) and reacted overnight at 4°C. Free biotin was removed by dialysis, desalting or other similar methods, and finally stored in 0.01M NaHCO3, pH=8.3 buffer, and the protein concentration was determined.

[0098] (2) Cater-antibody conjugation reaction: The labeled Spy Catcher or Spy Catcher Pro was mixed with Spy Tag-antibody 2 (hereinafter collectively referred to as Ab2-tag) at a molar ratio of 1:(1~4). After the reaction, a second purification was performed using Protein A chromatography to remove free (i.e., uncovalently bonded to the antibody) Caterpillar, yielding the directionally conjugated biotinylated antibody (Ab2-tag biotin). The protein concentration of the collected fraction was determined by 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 (second composition).

[0099] 2.2 Biotin-labeled antibodies The process of directly labeling biotin with antibodies is the same as the process of targeted labeling with Catcher-Tag. Hereinafter, the directly labeled antibody will be referred to as Ab2 biotin.

[0100] 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.

[0101] Example 3: Comparison of detection performance of coated end reagents Taking the glial fibrillary acidic protein (GFAP) detection reagent as an example, the effective antibody coating amount of the directionally coated antibody FG-Ab1-tag and the directly coated antibody FG-Ab1 was qualitatively compared using the secondary antibody. Reagents R1 and R2, prepared using the method described in Example 2, were combined with photosensitive microparticles (purchased from PerkinElmer) universal solution and reacted in a photoluminescence platform. The reaction signals of the directionally coated antibody luminescent microparticles and the directly coated antibody luminescent microparticles with the biotinylated antibody were compared; a higher signal indicated better antibody activity after coating. Specifically, in reagent R1, Ab1 in both the directionally coated antibody FG-Ab1-tag and the directly coated antibody FG-Ab1 microparticles was mouse IgG antibody; in reagent R2, Ab2 in the biotinylated antibody Ab2 biotin was mouse IgG secondary antibody, both purchased from Invitrogen.

[0102] 1. Experimental Procedure 25 μL of R1 reagent + 25 μL of R2 reagent + 25 μL of 10 mM PBS were added and incubated for 17 min. Then, 175 μL of universal photosensitive solution was added and incubated for 10 min. The photoluminescence instrument readings were then taken. The detection results are shown in the table below.

[0103] 2. Experimental Results Table 2

[0104] Note: The Catcher in FG-I-Ab1-tag is Spy Catcher Pro fused with tag protein I; the Catcher in FG-II-Ab1-tag is Spy Catcher Pro fused with tag protein II; the Catcher in FG-III-Ab1-tag is Spy Catcher Pro fused with tag protein III; and the Catcher in FG-IV-Ab1-tag is Spy Catcher Pro fused with tag protein IV.

[0105] 3. Experimental Data Analysis Combinations ①-⑤ showed good linear correlation with the calibrators, exceeding 0.99. Different proteins fused to Catcher exhibited varying reactivity.

[0106] In combinations ①-④, the coating end all employed isopeptide-coupled antibody technology, but the proteins fused to the Catcher differed. Results showed that the S Catcher Pro fused with tag protein II exhibited better reactivity, with higher signal abundance, correlation, and slope. Therefore, the fused tag protein should contain lysine residues, ideally with a molecular weight between 15kDa and 30kDa, and an isoelectric point (PI) range of 6.5 to 7.5.

[0107] Compared with ⑤, the discrimination of groups ①-④ using targeted conjugated antibodies was significantly improved, especially in both low and high value samples, indicating improved detection accuracy. This suggests that the targeted coating method preserves the antibody activity to the greatest extent.

[0108] Example 4: Comparison of the stability of coated end reagents 1. Experimental Procedure 1.1 Accelerated test at 37℃ using reagent R1 Reagent R1 was placed in an incubator at 37°C for a total of 7 days. On day 0 (D0, before accelerated experiment), day 1 (D1), day 3 (D3), day 5 (D5), and day 7 (D7), reagent R1 was reacted with reagent R2 (directly labeled antibody) in a photoluminescence platform with a universal photosensitive solution for detection. The stability of the luminescent microparticle reagent with directional antibody coating and the luminescent microparticle reagent with direct antibody coating were compared.

[0109] 1.2 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. The photoluminescence instrument readings were then taken. The detection results are shown in the table below.

[0110] 2. Experimental Results (1) Stability test results of luminescent microparticle reagents for directional coating of antibodies.

[0111] Table 3

[0112] (2) Stability test results of luminescent microparticle reagents directly coated with antibodies.

[0113] Table 4

[0114] 3. Experimental Data Analysis In the accelerated experiment at 37℃, the correlation of the luminescent microparticles with the directional coated antibody showed almost no significant fluctuation, and the average decrease in signal value was less than 10%; while the antibody directly labeled at the coated end showed large signal fluctuations, with the signal value decreasing by up to 23%.

[0115] The stability of the coated end is significantly improved compared to reagents that are directly coated with antibodies after using directional coupling technology.

[0116] Example 5: Comparison of detection performance of labeled reagents The antibody activities of biotin-directed Ab2-tag biotin and biotin-directed Ab2 biotin were qualitatively analyzed using secondary antibodies.

[0117] 1. Experimental Procedure (1) The luminescent microparticle reagent (R1 reagent) for direct-coated antibody prepared as in Example 2, together with the biotin-directed coated antibody and biotin-direct coated antibody reagent (R2 reagent) prepared in Example 3, and photosensitive microparticles (purchased from PerkinElmer) universal solution, were reacted on a photoluminescence platform, and the intensity of the reaction signal was compared. The higher the signal, the better the activity of the labeled antibody. In the R1 reagent, the Ab1 of the luminescent microparticle FG-Ab1 is a mouse IgG secondary antibody; in the R2 reagent, the Ab2 of the biotinylated antibody Ab2-tag biotin and Ab2 biotin is a mouse IgG antibody. (2) Reaction system: 25 μL R1 reagent + 25 μL R1 reagent + 25 μL 10 mM PBS, incubated for 17 min, then 175 μL R3 reagent (universal photosensitive solution) was added and incubated for 10 min, and the reading was taken. The detection results are shown in the table below.

[0118] 2. Experimental Results Table 5

[0119] 3. Experimental Data Analysis Biotin-labeled reagents prepared by direct labeling and antibody labeling using directional conjugation technology showed significant differences in signal abundance and correlation. Compared with direct labeling, directional labeling significantly improved both the detection signal value and correlation, with the correlation reaching 0.999.

[0120] In addition, the signal differentiation of the directionally coupled label is also higher, indicating that the antibody activity is better after directional labeling compared with direct labeling.

[0121] Example 6: Comparison of the stability properties of labeled reagents 1. Experimental Procedure Both directionally labeled Ab2-tag biotin and directly labeled Ab2 biotin were prepared into R2 reagent (containing 2 μg / mL Bio-mouse IgG antibody) with biotin-buffered saline solution at a concentration of 2 μg / mL. The R2 reagent was incubated at 37°C for a total of 7 days. On days 0 (D0, before accelerated experiments), 1 (D1), 3 (D3), 5 (D5), and 7 (D7), the R2 reagent was reacted with the directly antibody-coated luminescent microparticles (R1 reagent) using a universal photosensitive solution in a photo-induced chemiluminescence platform for detection. The stability of the biotin-directly labeled antibody reagent and the biotin-directly labeled antibody reagent was compared. The detection results are shown in the table below.

[0122] 2. Experimental Results (1) Stability test results of biotin-directed labeled antibody reagent.

[0123] Table 6

[0124] (2) Stability test results of biotin-labeled antibody reagent.

[0125] Table 7

[0126] 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.

[0127] The reagent stability was good after the labeling end adopted the directional coupling technology, which was significantly improved compared with direct labeling.

[0128] Based on the above experimental results, using isopeptide-coupled antibody technology for targeted antibody labeling or coating in a photo-induced chemiluminescence platform can reduce the impact of labeling and coating methods on antibody activity and achieve high detection accuracy. Fusing a II-tagged protein to the catcher followed by biotin labeling or coating it onto an FG can further enhance the activity of the labeled and coated antibodies.

[0129] Example 7: Preparation of CEA reagent The following uses the CEA (carcinoembryonic antigen, purchased from Fitzgerald, USA) photochemiluminescence detection reagent as an example to compare the preparation of detection reagents coated or labeled with anti-CEA (carcinoembryonic antigen antibody) using the method in Example 2.

[0130] CEA detection kit assembly: 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).

[0131] The specific differences between CEA reagents are shown in Table 8.

[0132] Table 8

[0133] Example 8: Comparison of the anti-hooking ability of coated end reagents 1. Experimental Procedure (1) CEA antigen (purchased from Fitzgerald, USA) was diluted with physiological saline to multiple concentration gradients between 10,000 and 200,000 ng / mL. The information on the CEA detection reagents used is shown in the table below: Table 9

[0134] The CEA antigen dilutions at the above concentration gradients were tested using experimental groups A, B, and C respectively. The concentration at which the chemiluminescence value decreased with increasing concentration was taken as the lowest concentration of antigen at which the hook effect occurred, and this concentration was taken as the HOOK concentration.

[0135] (2) Detection method: On an automated chemiluminescence detection instrument, add 25 μL of the antigen or sample to be tested, 25 μL of reagent 1, and 25 μL of reagent 2 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.

[0136] 2. Experimental Results Table 10

[0137] 3. Experimental Data Analysis Compared to R2 reagent, which directly coats antibodies, Spy Catcher (Pro)-Tag-mediated coating can enhance anti-HOOK ability, especially Spy Catcher Pro-Tag, which shows a more significant enhancement effect.

[0138] Example 9: Comparison of the anti-hooking ability of labeled reagents 1. Experimental Procedure CEA antigen was diluted with physiological saline to multiple concentration gradients between 10,000 and 200,000 ng / mL. Information on the CEA detection reagents used is shown in the table below. Table 11

[0139] Following the method in Example 8, the CEA antigen dilutions at the aforementioned concentration gradients were tested using experimental groups A, B, and C. The test results are recorded in the table below.

[0140] 2. Experimental Results Table 12

[0141] 3. Experimental Data Analysis Spy Catcher (Pro)-Tag-mediated labeling of R2 reagents can enhance anti-HOOK ability compared to R2 reagents that directly label biotin, especially Spy Catcher Pro-Tag, which has a more significant enhancement effect.

[0142] Example 10: Comparison of the anti-hooking ability of coated and labeled reagents 1. Experimental Procedure CEA antigen was diluted with physiological saline to multiple concentration gradients between 10,000 and 200,000 ng / mL. Information on the CEA detection reagents used is shown in the table below. Table 13

[0143] Following the method in Example 8, the CEA antigen dilutions at the aforementioned concentration gradients were tested using experimental groups A, B, and C. The test results are recorded in the table below.

[0144] 2. Experimental Results Table 14

[0145] 3. Experimental Data Analysis The combined use of Spy Catcher (Pro)-Tag-mediated coating of R1 reagent and Spy Catcher (Pro)-Tag-mediated labeling of R2 reagent can further enhance anti-HOOK ability, especially the enhancement effect of Spy Catcher Pro-Tag is more significant.

[0146] Based on the above experimental results, using isopeptide-coupled antibody technology for targeted labeling or coating of antibodies in a photo-induced chemiluminescence platform can reduce the impact of labeling and coating methods on antibody activity and improve the activity of labeled and coated antibodies. Reagents prepared using targeted coupling technology exhibit good reaction correlation, good anti-hooking ability, and high detection accuracy.

[0147] 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 homogeneous chemiluminescent detection method characterized in that, The sample to be tested is brought into contact with a reaction system containing the first composition and the second composition, and the light signal intensity of the reaction product is detected after the reaction. In this embodiment, the biomolecules in the first composition and / or the biomolecules in the second composition are directionally bound to the target substance via a Catcher-Tag system, and the biomolecules are capable of interacting with the target molecule to be tested.

2. The method according to claim 1, characterized in that, The non-specific binding region of the biomolecule is directionally bound to the target substance via the Catcher-Tag system; Preferably, the biomolecule is an antigen or an antibody.

3. The method according to claim 1, characterized in that, The Catcher-Tag system is selected from the Spy Catcher-Tag system and the Snoop Catcher-Tag system; Preferably, the Catcher-Tag system comprises a tag protein; More preferably, the molecular weight of the tag protein is 15kDa to 50kDa; and / or, the tag protein contains lysine residues; and / or, the proportion of lysine residues in the tag protein is 0.05 to 5wt%; and / or, the number of lysine residues in the tag protein is 3 to 50; and / or, the isoelectric point of the tag protein is between 4 and 7.

5.

4. The method according to claim 3, characterized in that, The tag protein is co-expressed with the Catcher; Preferably, the tag protein binds to the N-terminus and / or C-terminus of the catcher; Preferably, the tag protein binds to the Catcher via (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) at one end.

5. The method according to any one of claims 1 to 4, characterized in that, The first composition comprises luminescent microspheres and a first antibody, wherein the first antibody is directionally coated onto the luminescent microspheres via a first Catcher-Tag system; Preferably, the first antibody binds to the luminescent microspheres via its Fc terminal or Fab-connected hinge region.

6. The method according to claim 5, characterized in that, In the first Catcher-Tag system, the Catcher is coated on luminescent microspheres, and the Tag is co-expressed and linked with the first antibody to form a first fusion protein; Preferably, the mass ratio of the luminescent microspheres to the Catcher is 10:(0.05~1); Preferably, the mass ratio of the luminescent microspheres coated with Catcher to the first fusion protein is 10:(0.25~5).

7. The method according to claim 5, characterized in that, The luminescent microspheres are modified with active groups, which bind to the Catcher. Preferably, the active group is at least one selected from carboxyl, amino, aldehyde, and thiol groups.

8. The method according to any one of claims 1 to 7, characterized in that, The second composition comprises biotin and a second antibody, wherein the biotin is labeled with the second antibody via a second Catcher-Tag system; Preferably, the second antibody binds to biotin via its Fc terminal or Fab-linked hinge region.

9. The method according to claim 8, characterized in that, In the second Catcher-Tag system, the Catcher binds to the biotin, and the Tag is co-expressed and linked with the second antibody to form a second fusion protein; Preferably, the molar ratio of the Catcher to biotin is 1:(10~45); Preferably, the molar ratio of the biotin-labeled Catcher to the second fusion protein is 1:(1~4).

10. The method according to any one of claims 1 to 9, characterized in that, Both the first and second compositions employ a Catcher-Tag system containing a tag protein.

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