A photo-induced chemiluminescence detection kit and its application

Abstract

This application relates to a photocatalytic chemiluminescence detection kit and its application. The kit includes: reagent R1, comprising luminescent microspheres and a first biomolecule bound thereto, the luminescent microspheres being capable of reacting with singlet oxygen to generate a detectable signal; and reagent R2, comprising a second biomolecule and one of its specific binding pairs; wherein the biomolecule is capable of specifically binding to the analyte; the first biomolecule binds to the luminescent microspheres via a Catcher-Tag system and / or the second biomolecule binds to one of its specific binding pairs via a Catcher-Tag system. The technical solution of this application can improve the stability of the detection reagent, retain the activity of the biomolecules in the detection reagent, thereby enhancing the ability of the detection reagent to specifically bind to the analyte in the sample, and improving detection sensitivity and accuracy.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to a photo-induced chemiluminescence assay kit and its application. Background Technology

[0002] Photocatalytic chemiluminescence analysis is a new generation of immunoassay technology based on nanoscale polymer particles. Its core principle is the generation and transfer of reactive oxygen species (ROS): when an antibody / antigen labeled on a photosensitive microsphere (GG) binds to an antigen / antibody in the sample, and another antibody / antigen labeled on a luminescent microsphere (FG) binds to the antigen / antibody in the sample, the distance between the photosensitive microsphere and the luminescent microsphere is shortened, realizing the energy transfer of ROS. When the energy level transitions, a high-energy red light is generated. By using a single-photon counter and mathematical fitting, the number of photons is converted into a relative light signal, thus determining the concentration of the target analyte in the sample.

[0003] However, in photochemiluminescence detection, if the key components in the detection reagent, such as the antibodies / antigens labeled on the photosensitive microspheres / luminescent microspheres, are unstable, it may lead to a decrease in the intensity and stability of the relative light signal, thereby affecting the accuracy and reliability of the detection results. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, this application provides a photo-induced chemiluminescence detection kit and its application, which can improve the stability of the detection reagent, retain the activity of biomolecules in the detection reagent, thereby enhancing the ability of the detection reagent to specifically bind to the analyte in the sample, and improving the detection sensitivity and the accuracy of the detection results.

[0005] The first aspect of this application provides a photo-induced chemiluminescence detection kit, comprising: R1 reagent comprises luminescent microspheres and a first biomolecule, wherein the luminescent microspheres are capable of reacting with singlet oxygen to generate a detectable signal; R2 reagent, which includes a second biomolecule; The biomolecules are capable of specifically binding to the analyte; the first biomolecule binds to the luminescent microspheres via a Catcher-Tag system and / or the second biomolecule binds to one of the specific binding pairs via a Catcher-Tag system.

[0006] In some implementations, the Catcher in the Catcher-Tag system contains a tag protein.

[0007] In some embodiments, the molecular weight of the tag protein is 15kDa to 50kDa.

[0008] In some embodiments, the tag protein contains lysine residues.

[0009] In some embodiments, the lysine residues in the tagged protein account for 0.05 to 5 wt%.

[0010] In some embodiments, the tag protein contains 3 to 50 lysine residues.

[0011] In some embodiments, the isoelectric point of the tag protein is between 4 and 7.5.

[0012] In some implementations, the tag protein binds to the N-terminus or C-terminus of the catcher.

[0013] In some embodiments, the tag protein binds to the catcher via a linker peptide at one end.

[0014] In some embodiments, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n; preferably, n is a natural number from 1 to 5.

[0015] 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.

[0016] In some embodiments, the tag protein is selected from at least one of 6xHIS, Flag, GST, MBP, Nus A, Myc, eGFP, eCFP, eYFP, mCherrye GFP, HA, and SUMO.

[0017] 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.

[0018] In some embodiments, the first biomolecule is directionally bound to the luminescent microsphere via a first Cater-Tag system, and the second biomolecule is directionally bound to one of the specific binding pair members via a second Cater-Tag system; the first Cater-Tag system and the second Cater-Tag system are different.

[0019] In some implementations, in the first Catcher-Tag system, the Catcher is coated onto luminescent microspheres, and the Tag is co-expressed and linked with the first biomolecule to form a first fusion protein.

[0020] In some embodiments, the mass ratio of the luminescent microspheres to the Catcher is 10:(0.05~1).

[0021] In some embodiments, the mass ratio of the luminescent microspheres coated with Catcher to the first fusion protein is 10:(0.25~5).

[0022] In some implementations, in the second Catcher-Tag system, the Catcher binds to one of the specific binding pair members, and the Tag is co-expressed with the second biomolecule to form a second fusion protein.

[0023] In some implementations, the molar ratio of the Catcher to one of the specifically binding pair members is 1:(10~45).

[0024] In some embodiments, the molar ratio of the Catcher, which binds to one of the specific binding pairing members, to the second fusion protein is 1:(1~4).

[0025] In some embodiments, the kit further includes: an R3 reagent comprising photosensitive microspheres and a substance bound to the photosensitive microspheres capable of interacting with one of the specific binding pair members.

[0026] In some embodiments, the specific binding pairing member is selected from biotin or avidin.

[0027] The second aspect of this application provides the application of the above-mentioned photochemiluminescence detection kit in the detection of blood biomarkers.

[0028] The technical solution provided in this application may include the following beneficial effects: by introducing a new antigen-antibody conjugation technology into the photo-induced chemiluminescence detection reagent, that is, by using the Catcher-Tag system to achieve directional coating of biomolecules on the luminescent microspheres and / or directional labeling of one of the specific binding pairs on the biomolecules, the influence of coating and / or labeling methods on the activity of biomolecules is reduced, the stability of the detection reagent is improved, and the accuracy and sensitivity of the detection results are improved.

[0029] 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

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 containing the antigen-binding portion of the antibody and non-antibody proteins. Where necessary, antibodies may be further conjugated to other parts, such as specifically binding pairing members, for example, biotin or avidin. The location capable of specific binding to the antigen in an immune response, known as the complementarity determining region (CDR), is located at the Fab end. Therefore, in this application, the Fab end of the antibody is referred to as the specific binding region, and the opposite end is referred to as the non-specific binding region.

[0035] 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.

[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 Catcher-Tag system described in this article is a protein covalent linking technology, which consists of two parts: a Tag composed of multiple amino acid residues and a Catcher. Covalent linking of proteins can be achieved through the specific reaction between the Tag and the Catcher.

[0039] The S Catcher described in this article is a Spy Catcher, Snoop Catcher, or other Cater protein that can form isopeptide bonds with the corresponding tag without a tag protein. The Cater Pro described in this article is a SCatcher protein that contains a tag protein, which is linked to the C-terminus or N-terminus of the S Catcher via (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5).

[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 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, 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.

[0043] 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.

[0044] 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.

[0045] In this application, "specifically binding pair" refers to a pair of molecules that can specifically bind to each other, such as enzyme-substrate, antigen-antibody, or ligand-receptor pairs. A specific example of a specific binding pair is the biotin-streptavidin system.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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+ .

[0050] 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.

[0051] 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.

[0052] II. Specific Implementation Plan This application will now be described in more detail.

[0053] 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 the stability of the detection reagent, and consequently affecting the detection sensitivity and the accuracy of the detection results.

[0054] The photo-induced chemiluminescence detection kit disclosed in this application includes reagent R1 and reagent R2. Reagent R1 comprises luminescent microspheres and a first biomolecule, wherein the luminescent microspheres react with singlet oxygen to generate a detectable signal; reagent R2 comprises a second biomolecule; both the first and second biomolecules are capable of specifically binding to the analyte. In some embodiments, the first biomolecule binds to the luminescent microspheres via a Catcher-Tag system; in some embodiments, the second biomolecule binds to one of its specific binding pairs via a Catcher-Tag system; in some embodiments, the first biomolecule binds to the luminescent microspheres via a Catcher-Tag system and the second biomolecule binds to one of its specific binding pairs via a Catcher-Tag system.

[0055] In the Catcher-Tag system of this application, the spontaneously formed isopeptide bonds between the Catcher and the Tag enable biomolecules to bind stably and irreversibly to the luminescent microspheres and / or specifically bind to one of the paired members. This avoids the presence of a large number of free specific antigens / antibodies in the detection reagent, which could affect the stability of the detection reagent. Moreover, these specific antigens / antibodies have good activity, which can ensure the immune reaction between them and the analyte, improve the anti-hooking ability of the detection system, and improve the overall detection sensitivity and accuracy.

[0056] The luminescent microspheres comprise a carrier and a luminescent composition coated on or filled within the carrier, enabling them to react with reactive oxygen species to generate a detectable light signal. The carrier can be a polymer microparticle; suitable polymer microparticles for this application are polystyrene microspheres. Of course, other detectable microspheres made of other materials are also possible and are not limited thereto. Examples of luminescent compositions include 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+ .

[0057] 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; further, it may be selected from the S Catcher-Tag system with or without a tag protein; preferably, it is an S Catcher-Tag system that includes a tag protein.

[0058] The tag protein can bind to the Catcher protein. Furthermore, it can bind to the Catcher protein through co-expression or chemical coupling; co-expression is preferred.

[0059] 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.

[0060] In some embodiments of this application, the tag protein contains lysine residues. Using a tag protein containing lysine residues can increase the lysine content on the Catcher carrier protein, which on the one hand improves the efficiency of biomolecule coating or labeling, and on the other hand increases the number of binding sites on the Catcher that can bind to one of the luminescent microspheres or specifically bind to one of the paired members, thereby improving the stability of the biomolecule after coating or labeling, enhancing the stability of the detection reagent, and ultimately improving the sensitivity of the detection reagent.

[0061] 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%; preferably 0.2~3 wt%. In some specific embodiments of this application, the proportion of lysine residues in the tag protein can be 0.05 wt%, 0.1 wt%, 0.2 wt%, 0.5 wt%, 0.8 wt%, 1 wt%, 1.2 wt%, 1.5 wt%, 2 wt%, 3 wt%, 4 wt%, 5 wt%, etc.

[0062] In some embodiments of this application, the number of lysine residues in the tagged protein is 3 to 50.

[0063] In some embodiments of this application, the isoelectric point of the tag protein is between 4 and 7.5; preferably between 6.5 and 7.5.

[0064] The tag protein can be attached to either the N-terminus or C-terminus of the catcher protein to facilitate binding between the catcher and biomolecules. When a tag protein is introduced to both the N-terminus and C-terminus of the catcher protein, the introduced tag proteins can be the same or different. Preferably, the tag protein is introduced to only one end of the catcher protein, i.e., the N-terminus or the C-terminus.

[0065] To further improve the binding stability of the tag protein to the catcher, 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 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 6xHIS, Flag, GST, MBP, NusA, Myc, eGFP, eCFP, eYFP, mCherryeGFP, HA, and SUMO.

[0067] 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 (Gly-Gly-Gly-Gly-Ser)n (n=1 to 5).

[0068] In some embodiments of this application, a first biomolecule is directionally bound to a luminescent microsphere via a first Cater-Tag system, and a second biomolecule is directionally bound to one of the specific binding pair members via a second Cater-Tag system.

[0069] Using a Catcher-Tag system to directionally couple the target substance to both the coated end of the first biomolecule and the labeled end of the second biomolecule can further improve detection sensitivity. The first and second Catcher-Tag systems can be the same or different; preferably, they are different. Using different types of Catcher-Tag systems for the coated end of the first biomolecule and the labeled end of the second biomolecule 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.

[0070] In some embodiments of this application, in the first Cater-Tag system, the Cater is coated onto luminescent microspheres, and the Tag is co-expressed and linked with a first biomolecule to form a first fusion protein. In some specific embodiments of this application, when the Cater binds to the luminescent microspheres, the mass ratio of the luminescent microspheres to the Cater is 10:(0.05~1). In some specific embodiments of this application, the mass ratio of the luminescent microparticles coated with the Cater to the first fusion protein is 1:(0.25~5).

[0071] In the second Catcher-Tag system, the Catcher binds to one of the specific binding pair members, and the Tag is co-expressed and linked with a second biomolecule to form a second fusion protein. In some specific embodiments of this application, the molar ratio of the Catcher to one of the specific binding pair members is 1:(10~45). In some specific embodiments of this application, the molar ratio of the Catcher bound to one of the specific binding pair members to the second fusion protein is 1:(1~4).

[0072] Catchers that have completed the directional coating of luminescent microspheres or specifically bound to one of the paired members can be coupled to any protein containing a tag, enabling directional coating or directional labeling of proteins from different projects.

[0073] In some embodiments, the kit may further include a photosensitive agent, also known as a photosensitive solution, universal solution, or universal photosensitive liquid. The photosensitive agent includes photosensitive microparticles and a substance coated on the surface of the photosensitive microparticles that can interact with one of the specifically binding pair members. For example, when the second biomolecule is labeled with biotin, the photosensitive microparticles are coated with avidin. The photosensitive microparticles include a carrier and a photosensitive substance carried by the carrier. The carrier may be a polymeric microparticle, and the photosensitive substance may be coated on the surface of the carrier and / or filled inside the carrier. The photosensitive substance may be able to generate reactive oxygen species (e.g., singlet oxygen) upon photoexcitation. The polymeric microparticles may be polystyrene microspheres, but other microspheres of other detectable materials are also possible and are not limited thereto. The photosensitive substance may be, for example, a photosensitizer or a photosensitive dye, and may be a photosensitive substance known in the art, such as methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, but is not limited thereto. Photosensitive microspheres can also be filled with other sensitizers, non-limiting examples of which are certain compounds that catalyze the conversion of hydrogen peroxide into 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 singlet oxygen.

[0074] The kit described in this application can be used in the detection of blood biomarkers. The specific detection method includes: contacting the sample to be tested with reagents R1, R2, and R3 to obtain a reaction product formed by a first biomolecule, the target antigen / antibody, and a second biomolecule; contacting the reaction product with singlet oxygen; and detecting the light signal intensity of the reaction product. Based on the light signal intensity, it is determined whether the sample contains the target antigen / antibody and the content of the target antigen / antibody in the sample, thus achieving qualitative or quantitative analysis of the target antigen / antibody in the blood sample.

[0075] 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.

[0076] The following example uses Spy Catcher-Tag. A luminescent reagent (R1 reagent) prepared by directly coating an antibody and using Spy Catcher-directedly coated antibodies is compared with a biotin reagent (R2 reagent) prepared by directly labeling biotin and using Spy Catcher-directedly labeled biotin. The samples are then tested using a sandwich immunoassay principle in a photochemiluminescence detection platform. The light signal values ​​are measured and compared to determine the detection performance of the directed coating and / or directed labeling antibodies.

[0077] Example 1: Preparation of Spy Catcher, Spy Catcher Pro and Tag fusion antibodies 1. Preparation and purification of S Catcher (Pro) (1) Construction of Catcher plasmid expression vector: The expression vector of S Catcher (E. coli vector-pet series expression plasmid) was constructed based on the sequence of Spy Catcher (GenBank accession number: JQ478411.1).

[0078] (2) Catcher expression induced by E. coli: The E. coli expression vector was used to induce the expression of S Catcher. The culture conditions were as follows: ① Self-induction medium (10g peptone, 5g yeast extract, 1x NPS, 1mM MgCl2, 1x 5052, pH=7.4), with 100nM ampicillin added; ② Induction conditions: overnight culture at 30℃ and 200rpm in a shaker.

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

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

[0081] Prepare S Catcher Pro containing the tagged proteins in Table 1 following the steps described above: Table 1

[0082] 2. Preparation and purification of S Tag-antibodies The Spy Tag (sequence AHIVMVDAYKPTK) was linked to the antibody using pClick technology to obtain the Spy Tag-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.

[0083] Example 2: Preparation of antibody coated with luminescent microparticles 1. Luminescent microparticles directionally coated with antibodies (1) S Catcher (Pro) coating luminescent microparticles ① Dialyze the purified S Catcher / S Catcher Pro to 0.05M CB, pH 9.6 buffer and determine the protein concentration. ② Dialyze 10 mg of luminescent microparticles (purchased from PerkinElmer) 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 (0.8 mg of Tween-20 per 10 mg of luminescent microparticles). ④ After homogenization by sonication, add 0.05–1 mg of S Catcher / SCatcher 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, centrifuge and wash with luminescent buffer (HEPES, pH=8.0). 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).

[0084] (2) Screening of luminescent microparticles FG microparticles coated with S Catcher / S 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.

[0085] Screening for FG-catchers with strong reaction signals indicates that the luminescent microparticles were successfully coated with SCatcher / S Catcher Pro, which was then used for subsequent coupling reactions.

[0086] (3) Conjugation of luminescent microparticles with S Tag-antibody FG microparticles coated with S Catcher / S Catcher Pro were mixed with antibodies fused with the S tag (hereinafter collectively referred to as Ab1-tag; both Ab1-tag and Catcher FG microparticles were stored in CB buffer) at a mass ratio of FG:Ab1-tag = 10:(0.25~5), with a microparticle concentration of 10 mg / mL. The mixture was stirred and reacted at room temperature for 1 h. The microparticles were then washed with luminescent buffer and centrifuged to remove any remaining free antibody. This centrifugation and washing process was repeated three times. Finally, the microparticles were resuspended in luminescent buffer and brought to a final volume of 10 mg / mL to obtain the luminescent microparticles with the directionally conjugated antibody. The directionally coated microparticles are referred to as FG-Ab1-tag.

[0087] 2. Luminescent microparticles are directly coated with antibodies. 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.

[0088] Example 3: Preparation of Biotin-Labeled Antibodies 1. Biotin-labeled antibody (1) S Catcher (Pro) labeled biotin Purified S Catcher and S Catcher Pro were dialyzed into 0.1 M NaHCO3, pH 8.3 buffer. NHS-biotin was added at a molar ratio of S Catcher / S Catcher Pro to biotin of 1:(10–45), and the mixture was incubated overnight at 4°C. Free biotin was removed by dialysis, desalting, or other similar methods. The final product was stored in 0.01 M NaHCO3, pH 8.3 buffer, and protein concentration was determined.

[0089] (2) The reaction between the Catcher and the Tag-antibody The labeled S Catcher or S Catcher Pro was mixed with the S Tag-antibody 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) Catcher, yielding the directionally conjugated biotinylated antibody (Ab2-tag biotin). The protein concentration of the collected fraction was determined using the BCA method, and the fraction containing protein was then subjected to electrophoresis. Collection tubes matching the molecular weight after conjugation were selected and dialyzed into biotinylate storage buffer. Hereinafter, the directionally labeled antibody is referred to as Ab2-tag biotin.

[0090] 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.

[0091] Example 4: 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 FG-Ab1-tag with directional antibody coating and FG-Ab1 with direct antibody coating is qualitatively compared using the secondary antibody.

[0092] 1. Experimental Procedure (1) As in Example 2, the directed antibody-coated microparticles FG-Ab1-tag and the directly antibody-coated microparticles FG-Ab1 contain mouse IgG antibody (purchased from Invitrogen). The R1 reagent, containing 50 μg / mL FG-Ab1-tag or FG-Ab1, was prepared using luminescent buffer. As in Example 3, the biotinylated antibody Ab2 biotin contains mouse IgG secondary antibody (purchased from Invitrogen). The R2 reagent, containing 2 μg / mL Bio-mouse IgG secondary antibody, was prepared using biotinylated buffer. These were then reacted in a photoluminescence platform with a universal solution of photosensitive microparticles (purchased from PerkinElmer), i.e., the R3 reagent. The reaction signals of the directed antibody-coated luminescent microparticles and the directly antibody-coated luminescent microparticles with the biotinylated antibody were compared. A higher signal indicates better antibody activity after coating.

[0093] (2) Reaction system: 25 μL R1 reagent + 25 μL R2 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. The readings were then taken. The detection results are shown in the table below.

[0094] 2. Experimental Results Table 2

[0095] 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.

[0096] 3. Experimental Data Analysis 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.

[0097] 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.

[0098] Example 5: Comparison of the stability of coated end reagents Comparison of the stability of FG-Ab1-tag with directed antibody coating and FG-Ab1 with direct antibody coating.

[0099] 1. Experimental Procedure (1) The R1 reagent described in Example 4 (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 day 0 (D0, before the accelerated experiment), day 1 (D1), day 3 (D3), day 5 (D5) and day 7 (D7), the R1 reagent was reacted with the R2 reagent containing 2 μg / mL Bio-mouse IgG secondary antibody in a photoluminescence platform with a universal photosensitive solution to compare the stability of the luminescent microparticle reagent with the antibody-directly coated luminescent microparticle reagent.

[0100] (2) Reaction system: 25 μL R1 reagent + 25 μL R2 reagent + 25 μL 10 mM PBS, incubated for 17 min, then 175 μL R3 reagent (general photosensitive solution) was added and incubated for 10 min. The readings were then taken. The detection results are shown in the table below.

[0101] 2. Experimental Results (1) Stability test results of luminescent microparticle reagents for directional coating of antibodies.

[0102] Table 3

[0103] (2) Stability test results of luminescent microparticle reagents directly coated with antibodies.

[0104] Table 4

[0105] 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%.

[0106] The stability of the coated end is significantly improved compared to reagents that are directly coated with antibodies after using directional coupling technology.

[0107] Example 6: 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.

[0108] 1. Experimental Procedure (1) As in Example 2, Ab1 in the directly coated antibody microparticles FG-Ab1 is a mouse IgG secondary antibody. R1 reagent containing 50 μg / mL FG-Ab1 was prepared using luminescent buffer. As in Example 3, Ab2 in the biotin-directed labeled antibody Ab2-tag biotin and the biotin-directed labeled antibody Ab2 biotin is a mouse IgG antibody. R2 reagent containing 2 μg / mL Bio-mouse IgG antibody was prepared using biotin buffer. These were reacted on a photoluminescence platform with photosensitive microparticles (purchased from PerkinElmer) universal solution, and the signal was detected. A higher signal indicates better antibody activity after labeling.

[0109] (2) Reaction system: 25 μL R1 reagent + 25 μL R1 reagent + 25 μL 10 mM PBS, incubate for 17 min, then add 175 μL R3 reagent (general photosensitive solution) and incubate for 10 min, then read the value. The detection results are shown in the table below.

[0110] 2. Experimental Results Table 5

[0111] 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.

[0112] 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.

[0113] Example 7: Comparison of the stability of labeled reagents A comparison of the stability of Ab2-tag biotin with biotin-directed labeling and Ab2 biotin with direct labeling.

[0114] 1. Experimental Procedure Both the directionally labeled Ab2-tag biotin and the directly labeled Ab2 biotin were prepared into R2 reagent (containing 2 μg / mL Bio-mouse IgG antibody) with biotin-buffered buffer at a concentration of 2 μg / mL for accelerated testing at 37°C. The R2 reagent was incubated at 37°C for a total of 7 days. On days 0 (D0, before the accelerated test), 1 (D1), 3 (D3), 5 (D5), and 7 (D7), the R2 reagent was reacted with R1 reagent (containing 50 μg / mL FG-mouse IgG secondary antibody) in a photochemiluminescence platform using a universal photosensitive solution for detection. The stability of the biotin-directly labeled antibody reagent and the biotin-directly labeled antibody reagent was compared. The results are shown in the table below.

[0115] 2. Experimental Results (1) Stability test results of biotin-directed labeled antibody reagent.

[0116] Table 6

[0117] (2) Stability test results of biotin-labeled antibody reagent.

[0118] Table 7

[0119] 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.

[0120] The reagent stability was good after the labeling end adopted the directional coupling technology, which was significantly improved compared with direct labeling.

[0121] Example 8: Preparation of CEA reagent The following uses 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) through the methods of Examples 1 to 3.

[0122] CEA detection kit assembly: R1 reagent: luminescent microspheres and anti-CEA bound to the luminescent microspheres; R2 reagent: Biotin-labeled anti-CEA; The specific differences between CEA reagents are shown in Table 8.

[0123] Table 8

[0124] Pair with R3 reagent: photosensitive reagent (streptavidin-photosensitive microsphere solution).

[0125] Example 9: 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

[0126] 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.

[0127] (2) 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.

[0128] 2. Experimental Results Table 10

[0129] 3. Experimental Data Analysis Spy Catcher (Pro)-Tag-mediated coating of R1 reagent can enhance anti-HOOK ability compared to R1 reagent that directly coats antibodies, especially Spy Catcher Pro-Tag, which has a more significant enhancement effect.

[0130] Example 10: 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

[0131] Following the method of Example 9, 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.

[0132] 2. Experimental Results Table 12

[0133] 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.

[0134] Example 11: 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

[0135] Following the method of Example 9, 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.

[0136] 2. Experimental Results Table 14

[0137] 3. Experimental Data Analysis The use of Spy Catcher (Pro)-Tag-mediated coating of R1 reagent and Spy Catcher (Pro)-Tag-mediated labeling of R2 reagent can further enhance the anti-hooking ability of the reagents, especially the Spy Catcher Pro-Tag, which has a more significant enhancement effect.

[0138] 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.

[0139] 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 photo-induced chemiluminescence detection kit, characterized in that, include: R1 reagent comprises luminescent microspheres and a first biomolecule, wherein the luminescent microspheres are capable of reacting with singlet oxygen to generate a detectable signal; R2 reagent, which includes a second biomolecule; The biomolecules are capable of specifically binding to the analyte; the first biomolecule binds to the luminescent microspheres via a Catcher-Tag system and / or the second biomolecule binds to one of the specific binding pairs via a Catcher-Tag system.

2. The reagent kit according to claim 1, characterized in that, In the Catcher-Tag system, the Catcher contains 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.

3. The reagent kit according to claim 2, characterized in that, The tag protein binds to the N-terminus or C-terminus of the Catcher; Preferably, the tag protein binds to the Catcher via a linker peptide at one end; more preferably, the linker peptide is selected from (Gly-Gly-Gly-Gly-Ser)n; even more preferably, n is a natural number from 1 to 5.

4. The kit according to claim 2 or 3, 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.

5. The kit according to any one of claims 1 to 4, characterized in that, The first biomolecule is directionally bound to the luminescent microsphere via a first Cater-Tag system, and the second biomolecule is directionally bound to one of the specific binding pair members via a second Cater-Tag system; the first Cater-Tag system and the second Cater-Tag system are different.

6. The reagent kit according to claim 5, characterized in that, In the first Catcher-Tag system, the Catcher is coated with luminescent microspheres, and the Tag is co-expressed and linked with the first biomolecule to form the 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 reagent kit according to claim 5, characterized in that, In the second Catcher-Tag system, the Catcher binds to one of the specific binding pair members, and the Tag is co-expressed and linked with the second biomolecule to form the second fusion protein. Preferably, the molar ratio of the Catcher to one of the specifically binding pair members is 1:(10~45); Preferably, the molar ratio of the Catcher, which binds to one of the specific binding pairing members, to the second fusion protein is 1:(1~4).

8. The reagent kit according to claim 1, characterized in that, The kit also includes: The R3 reagent comprises photosensitive microspheres and a substance bound to the photosensitive microspheres that can interact with one of the specific binding pair members.

9. The kit according to any one of claims 1 to 8, characterized in that, The specific binding pair member is selected from biotin or avidin.

10. The application of the photochemiluminescence detection kit as described in any one of claims 1 to 9 in the detection of blood biomarkers.

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