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

Abstract

This application relates to a photoluminescence detection reagent, kit, and preparation method thereof. The detection reagent comprises luminescent microparticles and biomolecules directionally coated on the luminescent microparticles; the luminescent microparticles can react with singlet oxygen to generate a detectable signal, and the biomolecules can specifically bind to the analyte; the biomolecules are directionally coated onto the luminescent microparticles using a Catcher-Tag system. The technical solution of this application features highly stable and active specific biomolecules coated on the luminescent microspheres, which can effectively bind to the analyte in the sample, improve the anti-hooking ability of the detection reagent, enhance the sensitivity, signal abundance, and accuracy of the detection results, and broaden the detection range.

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 a technique that combines highly sensitive chemiluminescence assay with highly specific immunoreaction to analyze 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 antibody I, labeled on the acceptor microsphere, binds to the antigen in the sample, another antibody II, also labeled, also binds to the antigen. Simultaneously, the singlet oxygen generated by the donor microsphere can be 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 immunoassay without the need for a separation and washing step.

[0004] When using diagnostic reagents from a photochemiluminescence detection platform to test samples, due to the complexity of antigen-antibody reactions, a high antigen concentration in the sample may lead to a hook effect, resulting in false negatives and 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 photo-induced chemiluminescence detection reagent, kit, and preparation method thereof. The specific biomolecules coated on the luminescent microspheres are stable and highly active, effectively binding to the target analytes in the sample, enhancing the anti-hooking ability of the detection reagent, and improving the sensitivity, signal abundance, and accuracy of the detection results.

[0006] The first aspect of this application provides a photo-induced chemiluminescence detection reagent, comprising luminescent microparticles and a biomolecule directionally coated on the luminescent microparticles; the luminescent microparticles are capable of reacting with singlet oxygen to generate a detectable signal, and the biomolecules are capable of specifically binding to a target analyte; the biomolecules are directionally coated on the luminescent microparticles via a Catcher-Tag system.

[0007] In some embodiments, in the Cater-Tag system, the Cater is coated on the luminescent microparticles, and the Tag is co-expressed and linked with the biomolecule to form a fused biomolecule; the directional coating of the biomolecule on the luminescent microparticles is achieved through the isopeptide bonds between the Cater and the Tag on the luminescent microparticles.

[0008] In some embodiments, the mass ratio of the luminescent particles to the Catcher is 10:(0.05~1).

[0009] In some embodiments, the mass ratio of the luminescent microparticles coated with Catcher to the fused biomolecule is 1:(0.25~5).

[0010] In some embodiments, the Tag in the fused biomolecule is bound to the N-terminus and / or C-terminus of the biomolecule.

[0011] In some embodiments, the tag is connected to the biomolecule via (Gly-Gly-Gly-Gly-Ser)n at one end; preferably, n is selected from natural numbers 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, the molecular weight of the tag protein is 15-50 kDa.

[0014] In some embodiments, the tag protein contains lysine residues; more preferably, the proportion of lysine residues in the tag protein is 0.05~5wt%; even more preferably, the number of lysine residues in the tag protein is 3~50. In some embodiments, the isoelectric point of the tag protein is between 4 and 7.5.

[0015] In some embodiments, the tag protein is co-expressed with the catcher protein.

[0016] In some implementations, the tag protein is attached to the C-terminus and / or N-terminus of the catcher.

[0017] In some embodiments, the tag protein is connected to the Catcher via (Gly-Gly-Gly-Gly-Ser)n at one end; preferably, n is selected from natural numbers from 1 to 5.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] In some embodiments, the biomolecule is selected from antigens or antibodies.

[0022] A second aspect of this application provides a photo-induced chemiluminescence detection kit, comprising the above-mentioned reagents.

[0023] A third aspect of this application provides a method for preparing the above-mentioned reagent, comprising: coating a Catcher onto luminescent microparticles, then mixing them with fused-expressed Tag-biomolecules, forming heteropeptide bonds through the interaction between the Catcher and the Tag, thereby obtaining luminescent microparticles oriented with biomolecules.

[0024] 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 coating 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, and at the same time, it can also broaden the detection range of the reagent, which can be widely used in clinical testing.

[0025] 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

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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+ The luminescent microparticles can introduce active groups onto their surface through chemiluminescence. These active groups include, for example, carboxyl (-COOH), amino (-NH2), thiol (-SH), and aldehyde (-CHO). The active groups can be introduced onto the surface of the luminescent microparticles through chemical grafting, where a compound containing the active group reacts with the microparticles to attach the active groups to the surface of the luminescent microparticles.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.).

[0041] 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.

[0042] 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).

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] The Bland-Altman (BA) statistical analysis method described in this article is a method for evaluating the agreement limit between two measurement results. It visually reflects this agreement limit graphically, typically by plotting a scatter plot with the difference in measurement results on the vertical axis and the mean of the measurement results on the horizontal axis, and marking the 95% agreement limit.

[0051] The Plackett-Burman (PB) statistical analysis method described in this article is a two-level experimental design method, mainly used to quickly and effectively screen out the most important factors from a large number of factors under consideration. It uses a graphical method to reflect the relationship between two variables.

[0052] The Spearman correlation coefficient, described in this article, is a nonparametric statistic used to measure the strength and direction of a monotonic relationship (not necessarily a linear one) between two variables. It calculates the correlation based on the variable's rank (ranked position) rather than its actual value. The value ranges from -1 to 1. When the Spearman correlation coefficient is 1, the data points in the PB plot generally show a trend from the lower left to the upper right, indicating a perfectly positive correlation between the ranks of the two variables; that is, an increase in the rank of one variable leads to an increase in the rank of the other. When the coefficient is -1, the data points in the PB plot generally show a trend from the upper left to the lower right, indicating a perfectly negative correlation; that is, an increase in the rank of one variable leads to a decrease in the rank of the other. When the coefficient is 0, the distribution of data points in the PB plot is rather chaotic, without a clear monotonic trend, indicating that there is no monotonic relationship between the variables.

[0053] II. Specific Implementation Plan This application will now be described in more detail.

[0054] 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.

[0055] The photo-induced chemiluminescence detection reagent involved in this application includes luminescent microparticles and biomolecules directionally coated on the luminescent microparticles. The luminescent microparticles can react with singlet oxygen to generate a detectable signal, and the biomolecules can specifically bind to the target analyte. The biomolecules are directionally coated on the luminescent microparticles through a Catcher-Tag system.

[0056] Introducing the Catcher-Tag system during the coating of luminescent microparticles with antigens / antibodies allows one end of the antigen / antibody to bind directionally to the luminescent microparticle. This ensures that the end of the antigen / antibody with specific recognition and binding capabilities is fully exposed, preserving its activity and increasing the effective amount of antigen / antibody on the surface of the luminescent microparticle. This allows for sufficient contact and binding with the target antigen / antibody, avoiding the presence of a large number of unresponsive target antigens / antibodies in the detection system. Simultaneously, the spontaneously formed heteropeptide bonds in the Catcher-Tag system ensure irreversible and stable binding of the antigen / antibody to the surface of the luminescent microparticle, preventing the presence of a large number of free specific antigens / antibodies in the detection system. This overall reduces the occurrence rate of the hook effect and improves detection sensitivity.

[0057] In some embodiments of this application, the biomolecule can be an antigen or an antibody. When the biomolecule is an antigen, the N-terminus or C-terminus of the antigen can be directionally bound to the luminescent microparticle using a Catcher-Tag system; when the biomolecule is an antibody, the heavy chain or light chain of the antibody can be directionally bound to the luminescent microparticle using a Catcher-Tag system.

[0058] The luminescent microparticles include a carrier and a luminescent composition coated on or filled within the carrier, enabling it to react with reactive oxygen species to generate a detectable light signal. The carrier can be a polymer microparticle; suitable for this application, the polymer microparticle can be polystyrene microparticles; however, other detectable microparticles are also possible and are not limited thereto. The luminescent composition can be, for example, enol ethers, enamines, 9-alkylidene xanthan gum, 9-alkylidene-N-alkyl acrylamide, aryl vinyl 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+ .

[0059] In some embodiments of this application, the Catcher-Tag system may be selected from the Spy Catcher-Tag system, the Snoop Catcher-Tag system, or other Catcher-Tag systems that can form isopeptide bonds.

[0060] When biomolecules are coated onto luminescent microparticles, in the Catcher-Tag system, the Catcher is coated onto the luminescent microparticles, and the Tag is co-expressed and linked with the biomolecules to form a fused biomolecule; the directional coating of biomolecules on the luminescent microparticles is achieved through the isopeptide bonds between the Catcher and the Tag on the luminescent microparticles.

[0061] This application utilizes isopeptide bonds in the coating of antigens / antibodies to create an irreversible and stable link between biomolecules and luminescent microparticles. This allows for the effective and stable immobilization of specific antigens / antibodies, avoiding the presence of a large number of free specific antigens / antibodies in the detection reagents, thereby improving detection sensitivity and the accuracy of detection results.

[0062] In some embodiments of this application, when the Catcher is combined with the luminescent microparticles, the mass ratio of the luminescent microparticles to the Catcher is 10:(0.05~1).

[0063] In some embodiments of this application, the mass ratio of the luminescent microparticles coated with Catcher to the fused biomolecule is 1:(0.25~5).

[0064] Furthermore, the Catcher-Tag system may or may not contain a tag protein, but it is preferred to contain a tag protein.

[0065] 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.

[0066] In some embodiments of this application, the tag protein contains lysine residues. By using a tag protein, the lysine content on the catcher carrier protein can be increased, thereby increasing the number of binding sites on the catcher, improving the efficiency of biomolecule coating of luminescent microparticles, enhancing the stability of the biomolecule coating, improving the stability of the detection reagent, and ultimately increasing the sensitivity of the detection reagent.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] In some embodiments of this application, the tag protein can bind 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] When Catcher binds to luminescent microparticles, the microparticles can bind to either the C-terminus or N-terminus of the Catcher protein. Alternatively, the Catcher protein can be linked to the luminescent microparticle via a tag protein at one end, achieving Catcher coating on the microparticle to facilitate antigen / antibody binding. To further improve the stability of the Catcher-luminescent microparticle relationship, the tag protein can be linked to the Catcher via a (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5) connection at one end, and then linked to the luminescent microparticle.

[0075] The Catcher, which completes the directional binding of luminescent microparticles, can couple with any protein containing a tag, enabling directional coating of proteins from different projects.

[0076] 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.

[0077] This application also relates to a photo-induced chemiluminescence detection kit, which includes the above-mentioned detection reagents, specifically including luminescent reagents, wherein the luminescent reagents achieve directional coating of biomolecules on luminescent microparticles through a Catcher-Tag system.

[0078] In addition, the kit includes a biotin reagent and a photosensitive reagent. The luminescent reagent comprises luminescent microparticles and a first biomolecule coated on the surface of the luminescent microparticles via a Catcher-Tag system; the biotin reagent comprises a second biomolecule and biotin labeled to the second biomolecule; the photosensitive reagent comprises photosensitive microparticles and avidin coated on the surface of the photosensitive microparticles; wherein the first and second biomolecules are capable of specifically binding to the target substance to be tested, respectively.

[0079] The preparation method of the photo-induced chemiluminescence detection reagent described in this application includes coating a Cater onto luminescent microparticles, mixing them with fused-expressed Tag-biomolecules, and forming heteropeptide bonds through the interaction between the Cater and the Tag to obtain luminescent microparticles oriented with biomolecules.

[0080] Specifically, this may include: (1) Inserting the Tag sequence into the biomolecule sequence and co-expressing it in cells to obtain a fusion biomolecule; (2) Mix the luminescent particles with the Catcher to couple the Catcher to the surface of the luminescent particles; (3) The luminescent microparticles coupled with Catcher are mixed with the fused biomolecules and purified to obtain luminescent microparticles oriented with biomolecules.

[0081] In the reagents and kits of this application, the Cater is covalently coupled to specific functional groups on the surface of luminescent microparticles via chemical coupling. Subsequently, it reacts with Tag fusion antigen / antibody to form heteropeptide bonds, achieving directional coating of antigen / antibody on luminescent microparticles. This increases the content of biomolecules with specific binding and recognition capabilities on the surface of luminescent microparticles, and ensures that the specific binding and recognition ends of the coated biomolecules are fully exposed. This ensures that the detection reagent can fully react with the target substance, enabling rapid and effective immune and chemiluminescent reactions between antigen and antibody. It also enhances the detection system's ability to resist the HOOK effect, improves detection sensitivity, and increases the accuracy of detection results.

[0082] 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.

[0083] The following example uses antibodies as biomolecules. Luminescent reagents are prepared by directionally coating antibody-coated luminescent microparticles using the Catcher-Tag system. The luminescent reagents prepared by directionally coating antibody-coated luminescent microparticles are then used to detect the test samples on a photo-induced chemiluminescence platform. Qualitative comparisons are made using the light signal values ​​to determine the improvement effect of the photo-induced chemiluminescence detection performance of directionally coated antibodies.

[0084] Example 1: Preparation of Detection Reagent 1. Preparation of reagent R1 (luminescent reagent) 1.1 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). (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.

[0085] (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.

[0086] (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.

[0087] Prepare S Catcher Pro containing the tagged proteins in Table 1 following the steps described above: Table 1

[0088] 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.

[0089] 1.3 Luminescent microparticles coated with antibodies 1.3.1 Preparation of directionally coated luminescent microparticles (1) ① Dialyze the purified S Catcher 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 S Catcher 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, centrifuge and wash with luminescent buffer. ⑦ Adjust the volume to 20 mg / mL with 10 mM PBS, 50 mM Tris-HCl, 50 mM HEPES buffer (pH 6-8).

[0090] (2) The FG microparticles coated with S Catcher and the antibody fused with S tag (hereinafter collectively referred to as Ab1-tag) were mixed at a mass ratio of FG:Ab1-tag=10:(0.25~5) with a particle concentration of 10mg / mL. The mixture was stirred and reacted at room temperature for 1h. ② The microparticles were washed with luminescent buffer and any free antibody that might be present was removed by centrifugation. The centrifugation and washing were repeated 3 times. Finally, the microparticles were resuspended with luminescent buffer and the volume was adjusted to 10mg / mL to obtain the luminescent microparticles of the directionally conjugated antibody (hereinafter collectively referred to as the directionally coated microparticles as FG-Ab1-tag).

[0091] 1.3.2 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.

[0092] 1.3.3 The antibody-directed 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.

[0093] 2. Preparation of reagent R2 (biotin reagent) Biotin and antibody were mixed in a molar ratio of 1:(1~4) to obtain Bio-Ab2; then, it was prepared with biotin buffer to obtain R2 reagent containing 5 μg / mL Bio-Ab2.

[0094] Example 2: Performance testing of luminescent reagents for S Catcher-Tag targeted antibody coating 1. Experimental Procedure (1) Both the FG-Ab1-tag of the S Catcher-Tag-coated antibody and the FG-Ab1 of the directly coated antibody were reacted with the biotinylated antibody Bio-Ab2 on a photoluminescence platform. 228 serum samples were tested simultaneously to compare the reactivity of the FG-Ab1-tag and the directly coated antibody with Bio-Ab2. Both antibodies Ab1 and Ab2 were CA19-9 antibodies.

[0095] (2) Reaction system: 25 μL R1 reagent (containing 50 μg / mL FG-Ab1 or FG-Ab-tag1) + 25 μL R2 reagent (containing 5 μg / mL Bio-Ab2) + 25 μL 10 mM PBS. After incubation for 17 min, add 175 μL R3 universal photosensitive solution (containing photosensitive microparticles coated with streptavidin, purchased from PerkinElmer) and incubate for 10 min. Read the values. Compare the test data with the results from Roche reagent detection and calculate the correlation. The test data are shown in the table below.

[0096] 2. Experimental Results 2.1 Sample test results.

[0097] Table 2

[0098] 2.2 The statistical analysis method of constructing BA and PB diagrams was used to perform statistical analysis on the detection results of the above samples, and the following performance comparison results were obtained.

[0099] Table 3

[0100] 3. Analysis of Experimental Results Reagents that directionally coat antibodies with luminescent microparticles outperform reagents that directly coat antibodies in all aspects: ① The consistency range reflects the detection stability of the reagent: reagents with targeted antibody coating have better stability during detection than reagents with direct antibody coating; ②Spearman correlation coefficient is the correlation coefficient between the test results and those of Roche reagents: reagents with directed antibody coating have a better correlation than reagents with direct antibody coating, reaching 0.99; ③ The intercept reflects the magnitude of the reagent's deviation at the low end of the test: the deviation of reagents with directionally coated antibodies is smaller than that of reagents with directly coated antibodies.

[0101] Therefore, based on experimental data, it is shown that when the antibody at the coated end of the photo-induced chemiluminescence detection reagent is directionally coated, it is superior to direct coating in terms of correlation and test accuracy.

[0102] Example 3: Anti-HOOK ability of luminescent reagents for S Catcher-Tag targeted antibody coating 1. Experimental Procedure As shown in Example 2, reagent R1, prepared by directionally coating FG-Ab1-tag with antibody and directly coating FG-Ab1 with antibody, was used in combination with biotinylate reagent (R2 reagent) and universal solution to detect serum samples, and the results were compared with those obtained using Roche reagent. The test data are shown in the table below.

[0103] 2. Experimental Results Table 4

[0104] 3. Analysis of Experimental Results When the sample concentration reaches about 10,000 U / mL, the detection signal value of the reagent directly coated with antibody reaches its highest value. As the sample concentration increases thereafter, the signal value drops instead. That is, the detection reagent directly coated with antibody has an anti-HOOK ability of about 10,000 U / mL.

[0105] The detection reagents that use isopeptide-coupled antibody technology to directionally coat antibodies have an anti-HOOK ability of about 60,000 U / mL, and the signal value discrimination and abundance are higher.

[0106] Example 4: Anti-HOOK ability of receptor reagents for S Catcher Pro-Tag targeted antibody coating The following uses the CEA (carcinoembryonic antigen, purchased from Fitzgerald, USA) photochemiluminescence detection reagent as an example to prepare a detection reagent labeled with anti-CEA (carcinoembryonic antigen antibody) according to the method in Example 1, and conducts a comparative experiment.

[0107] 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).

[0108] The specific CEA reagents are shown in Table 5.

[0109] Table 5

[0110] Note: Spy Catcher Pro is Spy Catcher containing a tagged protein, the molecular weight of which is approximately 16.5 kDa.

[0111] (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.

[0112] (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.

[0113] 2. Experimental Results Table 6

[0114] 3. Experimental Data Analysis Compared to R1 reagents that directly coat antibodies, Spy Catcher (Pro)-Tag-mediated targeted antibody coating can enhance anti-HOOK ability, especially Spy Catcher Pro-Tag, which shows a more significant enhancement effect.

[0115] Example 5: Sensitivity Comparison of Spy Catcher (Pro)-Tag-Mediated Directional Coating of Luminescent Reagents Taking the glial fibrillary acidic protein (GFAP) detection reagent as an example, the detection performance of the directional coated antibody FG-Ab1-tag and the directly coated antibody FG-Ab1 was compared using GFAP calibrators.

[0116] 1. Experimental Procedure Dilute the coated ends FG-Ab1-tag and FG-Ab1 to 20 μg / mL with luminescent diluent, and the labeled end Bio-Ab2 to 5 μg / mL with diluent. Place them in the corresponding reagent racks of the photochemiluminescence instrument. Place the GFAP calibrators C1, C2, C3, C4, C5, and C6 standards in the corresponding reagent racks of the photochemiluminescence instrument.

[0117] 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.

[0118] 2. Experimental Results Table 7

[0119] Note: In FG-I-Ab1-tag, the Catcher is S Catcher Pro fused with tag protein I; in FG-II-Ab1-tag, the Catcher is S Catcher Pro fused with tag protein II; in FG-III-Ab1-tag, the Catcher is S Catcher Pro fused with tag protein III; and in FG-IV-Ab1-tag, the Catcher is S Catcher Pro fused with tag protein IV.

[0120] 3. Analysis of Experimental Results Combinations ①-⑤ showed good linear correlation with the calibrators, exceeding 0.99. Different proteins fused to the Catcher resulted in varying reactivity. Combinations ①-④ all employed isopeptide-coupled antibody technology at the coating end, 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, higher correlation, and a steeper slope. Therefore, the optimal molecular weight of the tag protein is 15kDa–30kDa, with an isoelectric point (PI) range of 6.5–7.5.

[0121] 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.

[0122] Example 6: Comparison of the stability of luminescent reagents with different coating methods 1. Experimental Procedure (1) Accelerated test at 37℃ using reagent R1. The R1 reagent described in Example 5 (containing 50 μg / mL FG-II-Ab1-tag and 50 μg / mL FG-Ab1) was subjected to an accelerated experiment at 37°C: the R1 reagent was placed in an incubator at 37°C for a total of 7 days; and on days 0 (D0, before the accelerated experiment), 1 (D1), 3 (D3), 5 (D5), and 7 (D7), the R1 reagent was reacted with the directly labeled antibody R2 reagent in a photoluminescence platform using a universal photosensitive solution for detection, comparing the stability of the luminescent microparticle reagent with the directionally coated antibody and the directly coated antibody. The antibody Ab1 coated by the luminescent microparticles in the R1 reagent was a mouse IgG antibody, and the biotin-labeled antibody Ab2 in the R2 reagent was a secondary antibody against mouse IgG (purchased from Invitrogen).

[0123] (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.

[0124] 2. Experimental Results (1) Stability test results of luminescent microparticle reagents for directional coating of antibodies.

[0125] Table 8

[0126] (2) Stability test results of luminescent microparticle reagents directly coated with antibodies.

[0127] Table 9

[0128] 3. Analysis of Experimental Results 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%.

[0129] The stability of luminescent microparticles is significantly improved by using directional coupling technology compared to reagents that are directly coated with antibodies.

[0130] Therefore, in photochemiluminescence platforms, antibody coating using isopeptide-coupled antibody technology can significantly improve the signal abundance, sensitivity, stability, detection range, correlation, and anti-hooking ability of reagents.

[0131] 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 luminescent microparticles and biomolecules directionally coated on the luminescent microparticles; the luminescent microparticles can react with singlet oxygen to generate a detectable signal, and the biomolecules can specifically bind to the target analyte; the biomolecules are directionally coated on the luminescent microparticles using a Catcher-Tag system.

2. The reagent according to claim 1, characterized in that, In the Catcher-Tag system, the Catcher is coated on the luminescent microparticles, and the Tag is co-expressed and linked with the biomolecule to form a fused biomolecule; the directional coating of the biomolecule on the luminescent microparticles is achieved through the isopeptide bonds between the Catcher and the Tag on the luminescent microparticles.

3. The reagent according to claim 2, characterized in that, The mass ratio of the luminescent particles to the catcher is 10:(0.05~1); and / or, The mass ratio of the luminescent microparticles coated with Catcher to the fused biomolecules is 10:(0.25~5).

4. The reagent according to claim 2, characterized in that, In the fused biomolecule, the Tag is bound to the N-terminus and / or C-terminus of the biomolecule; Preferably, the tag is connected to the biomolecule via (Gly-Gly-Gly-Gly-Ser)n at one end; more preferably, n is selected from natural numbers 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 comprises a tag protein; Preferably, the molecular weight of the tag protein is 15-50 kDa; Preferably, the tag protein contains lysine residues; more preferably, the proportion of lysine residues in the tag protein is 0.05~5wt%; even more preferably, the number of lysine residues in the tag protein is 3~50. Preferably, the isoelectric point of the tag protein is between 4 and 7.

5.

6. The reagent according to claim 5, characterized in that, The tag protein and the Catcher protein are co-expressed and linked; Preferably, the tag protein is attached to the C-terminus and / or N-terminus of the Catcher protein; Preferably, the tag protein is connected to the Catcher via (Gly-Gly-Gly-Gly-Ser)n at one end; more preferably, n is selected from natural numbers from 1 to 5.

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 luminescent microparticles were coated with a Catcher and then mixed with the fused-expressed Tag-biomolecules. Through the interaction between the Catcher and the Tag, heteropeptide bonds were formed, resulting in luminescent microparticles with directional coating of biomolecules.

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