Partner reagent for optical chemiluminescence detection and optical chemiluminescence detection kit

Abstract

This application relates to a companion reagent and a photo-induced chemiluminescence detection kit for photo-induced chemiluminescence detection. The companion reagent includes an oxygen-generating agent and a catalyst. The oxygen-generating agent reacts with the catalyst to produce oxygen. During photo-induced chemiluminescence detection, the oxygen generated by the companion reagent increases the oxygen content in the reaction system. The solution provided in this application can improve the optical signal value even at low analyte concentrations, which is beneficial for improving the stability, precision, and sensitivity of the detection.

Description

Technical Field

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

[0002] Photo-induced chemiluminescence assay combines photo-induced chemiluminescence measurement with highly specific immunoreaction. It is used for the detection and analysis of various antigens, haptens, antibodies, hormones, enzymes, fatty acids, vitamins, and drugs. It is a new immunoassay technique developed after radioimmunoassay, enzyme immunoassay, fluorescence immunoassay, and time-resolved fluorescence immunoassay.

[0003] According to the principle of photo-induced chemiluminescence analysis, when the concentration level of the analyte is low, the signal value generated by photo-induced chemiluminescence is also low, which leads to the detection results being greatly affected by instrument noise, resulting in unstable detection results, poor precision, and low sensitivity.

[0004] Therefore, improving the stability, precision, and sensitivity of photo-induced chemiluminescence immunoassay is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] To address or partially address the problems existing in related technologies, this application provides a companion reagent and a photo-induced chemiluminescence detection kit for photo-induced chemiluminescence detection, which can improve the light signal value when the concentration level of the analyte is low, thereby improving the stability, precision and sensitivity of the detection.

[0006] The first aspect of this application provides a companion reagent for photo-induced chemiluminescence detection, comprising an oxygen-generating agent and a catalyst, wherein the oxygen-generating agent reacts under the action of the catalyst to generate oxygen.

[0007] During photo-induced chemiluminescence detection, the oxygen generated by the companion reagent is used to increase the oxygen content in the reaction system.

[0008] The companion reagent for photo-induced chemiluminescence detection as described above, wherein the oxygen-generating agent comprises a metal-peroxide salt and / or a hydrogen peroxide complex;

[0009] Preferably, the oxygen-generating agent includes at least one of urea peroxide, calcium peroxide, calcium hydroxide, magnesium peroxide, sodium percarbonate, and internal peroxide.

[0010] The companion reagent for photo-induced chemiluminescence detection as described above, wherein the catalyst comprises at least one of an enzyme compound, a halide, and a metal oxide;

[0011] Preferably, the catalyst comprises at least one selected from catalase, iodide, manganese dioxide, iron(III), silver and dichromate.

[0012] The companion reagent for photo-induced chemiluminescence detection as described above, wherein,

[0013] The concentration of the oxygen-generating agent is 10 mg / mL to 100 mg / mL, and / or

[0014] The concentration of the catalyst is 0.5 mg / mL to 100 mg / mL.

[0015] The companion reagent for photo-induced chemiluminescence detection as described above, wherein,

[0016] The concentration ratio of the oxygen-generating agent to the catalyst is (1-10):(1-20);

[0017] Preferably, the concentration ratio of the oxygen-generating agent to the catalyst is 10:1. 。

[0018] The companion reagent for photo-induced chemiluminescence detection as described above, wherein,

[0019] The oxygen-generating agent and the catalyst are stored separately and premixed before use.

[0020] Preferably, the volume ratio of the oxygen-generating agent to the premixed catalyst is 1:1.

[0021] The companion reagent for photo-induced chemiluminescence detection as described above is added to the reaction system before the photo-induced chemiluminescence reaction excitation reading.

[0022] The companion reagent for photo-induced chemiluminescence detection as described above, wherein the operating temperature of the companion reagent is consistent with the photo-induced chemiluminescence reaction temperature.

[0023] A second aspect of this application provides a photo-induced chemiluminescence detection kit, comprising:

[0024] Reagent 1, wherein reagent 1 comprises the companion reagent as described above;

[0025] Reagent 2, comprising luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles are capable of reacting with reactive oxygen species to generate a detectable light signal;

[0026] Reagent 3, wherein reagent 3 comprises a label-tagged second biomolecule;

[0027] Both the first biomolecule and the second biomolecule can specifically bind to the target molecule in the sample to be tested.

[0028] Preferably, it further includes a donor reagent, which can convert ground-state oxygen into reactive oxygen under laser irradiation of a certain wavelength; more preferably, the donor reagent includes photosensitive microparticles filled with photosensitive material.

[0029] A third aspect of this application provides the application of the companion reagent as described above or the photo-induced chemiluminescence detection kit as described above in the detection of target molecules;

[0030] Preferably, the target molecule to be tested is selected from neurodegenerative markers;

[0031] More preferably, the target molecule to be tested is selected from neurofilament light chains, glial fibrous acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

[0032] The technical solution provided in this application may include the following beneficial effects:

[0033] The oxygen-generating agent of this application can react under the action of a catalyst to release oxygen. This oxygen generation increases the oxygen content in the photochemiluminescence detection system, and the oxygen can be activated into singlet oxygen, thereby increasing the singlet oxygen content in the system. Subsequently, the singlet oxygen is captured by luminescent particles, thus enhancing the light signal intensity of the photochemiluminescence. Therefore, when applied to photochemiluminescence detection, the companion reagent can improve the stability, sensitivity, and precision of the detection results, effectively addressing the problems of low luminescence signal values, significant interference from instrument noise, and poor precision in the detection of low-level analytes.

[0034] 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

[0035] To facilitate understanding of this application, it will be described in detail below. However, before describing this application in detail, it should be understood that this application is not limited to the specific embodiments described. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be restrictive.

[0036] Where a numerical range is 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 within this application. The upper and lower limits of these smaller ranges may be independently included in the smaller range and are also covered within this application, subject to any explicitly excluded limits within the specified range. Where the specified range includes one or two limits, the range excluding any or both of those included limits is also included within this application.

[0037] 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 application pertains. While the methods and materials described herein, or any equivalent methods and materials, may also be used in the implementation or testing of this application, preferred methods and materials are now described.

[0038] Terminology Explanation

[0039] The term "oxygen-generating agent" as used in this application refers to a chemical substance capable of producing oxygen, which undergoes a decomposition reaction to release oxygen. In some specific embodiments of this application, the oxygen-generating agent may be, for example, compounds such as hydrogen peroxide, sodium peroxide, and urea peroxide. Other oxygen-generating agents known to those skilled in the art may also be used in this application.

[0040] The term "catalyst" as used in this application refers to a substance capable of altering the chemical reaction rate of an oxygen-generating agent, while maintaining essentially unchanged mass and chemical properties before and after the reaction. In some specific embodiments of this application, the catalyst may be, for example, platinum, palladium, manganese dioxide, ferric oxide, enzymes, etc. Other catalysts known to those skilled in the art may also be used in this application.

[0041] The term "luminescent microparticle" as used in this application refers to particles containing compounds capable of reacting with reactive oxygen species to generate a detectable signal. In some specific embodiments of this application, the luminescent microparticles comprise a luminescent composition and a carrier, wherein the luminescent composition is filled in the carrier and / or coated on the surface of the carrier.

[0042] The term "luminescent microparticle and first biomolecule bound thereto" as used in this application refers to binding (coating) a first biomolecule onto the surface of the luminescent microparticle. The first biomolecule is an organic molecule that can specifically bind to the target molecule in the sample to be tested. For example, the first biomolecule can be an antibody, enzyme, receptor, nucleic acid aptamer, etc.

[0043] The term "tagged second biomolecule" as used in this application refers to a biomolecule labeled with a marker, such as biotin, fluorescent, or radioactive markers. A second biomolecule is an organic molecule capable of specifically binding to a target molecule in a test sample; for example, a second biomolecule can be an antibody, enzyme, receptor, or nucleic acid aptamer.

[0044] The term "bonding" as used in this application refers to the direct union between two molecules caused by interactions such as covalent, electrostatic, hydrophobic, ionic, and / or hydrogen bonding, including but not limited to interactions such as salt bridges and water bridges.

[0045] The term "specific binding" as used in this application refers to a mutually distinguishable and selective binding reaction between two substances, which, from a stereostructural perspective, refers to the conformational correspondence between the corresponding reactants. Under the technical concept disclosed in this application, detection methods for specific binding reactions include, but are not limited to: double-antibody sandwich assays, competitive assays, neutralization competitive assays, indirect assays, or capture assays.

[0046] The term "donor reagent" as used in this application refers to a reagent containing a sensitizer that, upon activation by energy, can generate an active intermediate, such as reactive oxygen species, that reacts with the luminescent microparticles. Donor reagents include photosensitive microparticles filled with a photosensitive substance. In some specific embodiments of this application, the photosensitive microparticles are polymeric microparticles filled with a photosensitizer, and the photosensitive substance is a photosensitizer. The photosensitizer can be a photosensitizer known in the art, preferably a relatively photostable compound that does not react effectively with singlet oxygen, such as compounds like methylene blue, rose red, porphyrin, phthalocyanine, and chlorophyll, as well as derivatives of these compounds having 1-50 substituents, the substituents being used to make these compounds more lipophilic or more hydrophilic, and / or as linking groups to specifically binding pairing members. Other examples of photosensitizers known to those skilled in the art may also be used in this application.

[0047] The term "ground state oxygen" as used in this application refers to the lowest energy state of the oxygen molecule O2. Ground state oxygen is a stable state of gaseous oxygen and the most common state of oxygen, occupying the lowest energy level.

[0048] The term "reactive oxygen species" as used in this application refers to a general term for substances composed of oxygen in the body or natural environment that contain oxygen and are reactive in nature. It mainly refers to an excited-state oxygen molecule, 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 singlet oxygen (1O2), etc.

[0049] The photosensitive microparticles of this application are induced and activated by energy or active compounds to release high-energy reactive oxygen species. These high-energy reactive oxygen species are captured by nearby luminescent microparticles, thereby transferring energy to activate the luminescent microparticles and emitting light signals.

[0050] The term "antigen" as used in this application refers to a substance that can stimulate the body to produce an immune response and can bind to immune response products, antibodies and sensitized lymphocytes, in vivo and in vitro to produce an immune effect.

[0051] The first aspect of this application provides a companion reagent for photo-induced chemiluminescence detection, the companion reagent comprising an oxygen-generating agent and a catalyst, wherein the oxygen-generating agent reacts under the action of the catalyst to generate oxygen; during photo-induced chemiluminescence detection, the oxygen generated by the companion reagent is used to increase the oxygen content in the reaction system.

[0052] The oxygen-generating agent of this application can undergo a decomposition reaction under the action of a catalyst to release oxygen. This oxygen generation increases the oxygen content in the photochemiluminescence detection system, and the oxygen can be activated into singlet oxygen, thereby increasing the singlet oxygen content in the system. Subsequently, the singlet oxygen is captured by luminescent particles, thus enhancing the light signal intensity of the photochemiluminescence. Therefore, when used in detection, the companion reagent can improve the stability, sensitivity, and precision of the detection results, effectively addressing the problems of low luminescence signal values, significant interference from instrument noise, and poor precision in the detection of low-level analytes.

[0053] In some embodiments, the oxygen-generating agent includes a metal-peroxide salt and / or a hydrogen peroxide complex. Preferably, the oxygen-generating agent includes at least one or more combinations of urea peroxide, calcium peroxide, calcium hydroxide, magnesium peroxide, sodium percarbonate, and internal peroxides. Of course, the oxygen-generating agent can also be other metal-peroxide salts or hydrogen peroxide complexes; this is merely illustrative and not intended to be limiting. When the above-mentioned compounds are selected as the oxygen-generating agent, the oxygen-generating agent has high oxygen production, high stability, good solubility, and high environmental friendliness. When applied to photo-induced chemiluminescence detection systems, it can efficiently generate ground-state oxygen while avoiding side reactions between the oxygen-generating agent and other components, as well as environmental pollution, thereby improving the stability, sensitivity, and precision of the detection.

[0054] In some embodiments, the catalyst includes at least one selected from enzyme compounds, halides, and metal oxides. Preferably, the catalyst includes at least one or more combinations of catalase, iodides, manganese dioxide, iron(III), silver, and dichromates. Of course, the catalyst can also be other enzyme compounds, halides, or metal oxides; this is merely illustrative and not limiting. When the above-mentioned compounds are selected as the catalyst, the catalyst has advantages such as high catalytic activity, high stability, high environmental friendliness, and low cost. It can efficiently and stably catalyze oxygen production from oxygen-generating agents and will not interfere with the detection of photo-induced chemiluminescence, which is beneficial to improving the stability, sensitivity, and precision of the detection.

[0055] In some embodiments, the concentration of the oxygen-generating agent is 10 mg / mL to 100 mg / mL. For example, the concentration of the oxygen-generating agent can be 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, or 100 mg / mL.

[0056] In some embodiments, the concentration of the catalyst is 0.5 mg / mL to 100 mg / mL, for example, the concentration of the catalyst can be 0.5 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, 9 mg / mL, 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL or 100 mg / mL, etc.

[0057] It should be noted that the concentrations of the oxygen-generating agent and the catalyst mentioned above in this application are the concentrations when the oxygen-generating agent and the catalyst are prepared and stored separately, that is, their respective concentrations before being mixed and used. When the concentrations of the oxygen-generating agent and the catalyst are within the above-mentioned ranges, the oxygen-generating agent can fully and efficiently release oxygen under the action of the catalyst, increasing the oxygen content in the photo-induced chemiluminescence system. At the same time, it can avoid the waste of oxygen-generating agent and catalyst due to excessive amounts, thereby improving the stability, sensitivity, and precision of the detection and saving costs.

[0058] In some embodiments, the concentration ratio of the oxygen-generating agent to the catalyst is (1-10):(1-20); for example, the concentration ratio of the oxygen-generating agent to the catalyst can be 1:1, 1:5, 1:10, 1:20, 5:1, 1:2, 5:20, 10:1, etc. Preferably, the concentration ratio of the oxygen-generating agent to the catalyst is 10:1. For example, when the concentration of the oxygen-generating agent is 50 mg / mL, the concentration of the catalyst is 5 mg / mL, and so on. The concentration ratio of the oxygen-generating agent to the catalyst in this application refers to the ratio of the concentration of the oxygen-generating agent to the concentration of the catalyst after mixing in the companion reagent. When the concentrations of the oxygen-generating agent and the catalyst are within the above range, the reaction ratio of the oxygen-generating agent and the catalyst is more reasonable, and the oxygen-generating agent can generate oxygen more fully and efficiently under the action of the catalyst, thereby increasing the oxygen content in the photo-induced chemiluminescence detection system and making the detection stability, sensitivity, and precision better.

[0059] In some embodiments, the oxygen-generating agent and catalyst in the companion reagent are stored separately and premixed before use. In this application, the oxygen-generating agent and catalyst are prepared to appropriate concentrations and stored independently. They are then mixed before application in the photo-induced chemiluminescence detection system. This avoids the oxygen-generating agent decomposing upon contact with the catalyst during mixed storage, preventing premature oxygen release and ensuring no improvement in detection stability, sensitivity, or precision. Preferably, the volume ratio of the oxygen-generating agent to the catalyst in the premix is ​​1:1. Preferably, the oxygen-generating agent and catalyst are mixed according to the above-mentioned concentration ratio and premix volume ratio.

[0060] In some implementations, the chaperone reagent is added to the reaction system before the photo-induced chemiluminescence reaction excitation reading. That is, after the components have mixed to form a sandwich immune complex during the photo-induced chemiluminescence reaction, the chaperone reagent is added, followed by laser excitation reading. Adding the chaperone reagent after the formation of the sandwich immune complex serves only to increase the oxygen content of the system, preventing the oxygen-generating agent and catalyst in the chaperone reagent from reacting with other components in the system, thereby improving the stability, sensitivity, and precision of the detection.

[0061] In some implementations, the companion reagent is used at the same temperature as the photochemiluminescence reaction temperature, which simplifies the application process of the companion reagent in photochemiluminescence detection. At the same time, it avoids the adverse effects of different operating temperatures on the sandwich immune complex, reduces the probability of decomposition or mutation of the sandwich immune complex, and thus ensures the enhancement effect of the companion reagent on the light signal intensity of photochemiluminescence detection.

[0062] A second aspect of this application provides a photochemiluminescence detection kit, comprising reagent 1, reagent 2, and reagent 3. Reagent 1 includes the aforementioned companion reagent; reagent 2 includes luminescent microparticles and a first biomolecule bound thereto, the luminescent microparticles being capable of reacting with reactive oxygen species to generate a detectable light signal; reagent 3 includes a tag-labeled second biomolecule; both the first and second biomolecules are capable of specifically binding to the target molecule in the sample to be tested.

[0063] Specifically, the photo-induced chemiluminescence detection kit of this application includes reagent 1, which includes a companion reagent. The companion reagent includes an oxygen-generating agent and a catalyst. The oxygen-generating agent reacts under the action of the catalyst to generate oxygen. During photo-induced chemiluminescence detection, the oxygen generated by the companion reagent is used to increase the oxygen content in the reaction system.

[0064] Specifically, the photoluminescence detection kit of this application includes reagent 2, which comprises luminescent microparticles and a first biomolecule bound thereto. The luminescent microparticles can react with reactive oxygen species to generate a detectable light signal, and the first biomolecule can specifically bind to the target molecule in the sample to be tested. This application does not limit the specific selection of the luminescent microparticles, which can be selected according to actual needs. This application does not limit the specific selection of the first biomolecule, as long as the first biomolecule can specifically bind to the target molecule in the sample to be tested. For example, when the target molecule in the sample to be tested is an antigen, the first biomolecule can be an antibody that can specifically bind to the antigen. For example, if the target molecule is neurofilament light chain, the first biomolecule in reagent 2 should be a neurofilament light chain antibody; for example, if the target molecule is glial fibrillary acidic protein, the first biomolecule in reagent 2 should be a glial fibrillary acidic protein antibody.

[0065] Specifically, the photochemiluminescence detection kit of this application includes reagent 3, which includes a tag-labeled second biomolecule capable of specifically binding to the target molecule in the test sample. This application does not limit the specific choice of the tag; it can be selected according to actual needs, such as biotin. This application also does not limit the specific choice of the second biomolecule, as long as it can specifically bind to the target molecule in the test sample. For example, when the target molecule in the test sample is an antigen, the second biomolecule can be an antibody capable of specifically binding to the antigen. For example, if the target molecule is neurofilament light chain, the second biomolecule in reagent 3 should be a neurofilament light chain antibody; for example, if the target molecule is glial fibrillary acidic protein, the second biomolecule in reagent 3 should be a glial fibrillary acidic protein antibody. For the same target molecule, the aforementioned first and second biomolecules can be the same antibody or different antibodies.

[0066] In some embodiments, the photoluminescence detection kit of this application may further include a donor reagent, which can convert ground-state oxygen into reactive oxygen species under laser irradiation of a certain wavelength. Preferably, the donor reagent is also called a photosensitive reagent, which includes photosensitive microparticles filled with a photosensitive substance. The surface of the photosensitive microparticles is bonded with a substance that can specifically bind to the label of reagent 3, such as avidin, which facilitates binding with biotin, preferably streptavidin. This application does not limit the specific selection of photosensitive microparticles, which can be selected according to actual needs, as long as they can convert the ground-state oxygen generated by reagent 1 into reactive oxygen species under laser irradiation of a certain wavelength. This application does not limit the specific selection of the photosensitive substance, as long as it can receive a laser signal of a certain wavelength.

[0067] When using a photochemiluminescence platform for detection, following the sandwich detection method, reagents 2 and 3 from the photochemiluminescence detection kit of this application are mixed and incubated with the sample to be tested, and then mixed and incubated with the donor reagent to form a sandwich immune complex. Reagent 1 is then added, and the reaction solution is irradiated with excitation light to detect the light signal. The companion reagent in reagent 1 increases the oxygen content in the photochemiluminescence detection kit system, thereby increasing the content of singlet oxygen in the system, thus increasing the intensity of the photochemiluminescence light signal, and consequently improving the stability, sensitivity, and precision of the detection.

[0068] To obtain more accurate detection results, in some embodiments, the concentration of reagent 2 can be from 10 μg / mL to 100 μg / mL, for example, the concentration of reagent 2 can be 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL. In some embodiments, the concentration of reagent 3 can be from 0.5 μg / mL to 5 μg / mL, for example, the concentration of reagent 3 can be 0.5 μg / mL, 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, or 5 μg / mL. In some embodiments, the concentration of the donor reagent is 10 μg / mL to 100 μg / mL, for example, the concentration of the donor reagent can be 10 μg / mL, 20 μg / mL, 30 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL, 80 μg / mL, 90 μg / mL, or 100 μg / mL. The donor reagent can be used as a universal reagent, suitable for photochemiluminescence detection kits that detect different target molecules.

[0069] A third aspect of this application provides the application of the above-described companion reagent or the above-described photochemiluminescence detection kit in the detection of target molecules.

[0070] Preferably, the target molecule to be tested is selected from neurodegenerative markers.

[0071] More preferably, the target molecule to be tested is selected from neurofilament light chains, glial fibrous acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

[0072] The companion reagent provided in this application can improve the stability, sensitivity, and precision of photochemiluminescence detection. When the target molecule is a neurodegenerative marker, the companion reagent or a photochemiluminescence kit including the companion reagent can improve the detection capability of low concentrations of neurodegenerative markers in the test sample.

[0073] To make this application easier to understand, the following detailed description will be provided with reference to embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of application of this application. Unless otherwise specified, the raw materials or components used in this application can be obtained commercially or by conventional methods.

[0074] The following example uses urea peroxide as the oxygen-generating agent and catalase as the catalyst. Reagent 1, i.e., the companion reagent, is prepared. Then, the companion reagent is used to mix and react with reagent 2, reagent 3, donor reagent and the sample to be tested. Finally, the light signal value is detected on a photo-induced chemiluminescence platform.

[0075] Example 1: Preparation of companion reagent

[0076] 1.1 The main experimental materials are shown in Table 1

[0077] Table 1

[0078] peroxyurea Purchased from Sigma catalase Purchased from Sigma

[0079] 1.2 Catalase was diluted with 20 mM HEPES buffer to obtain catalase reagents of different concentrations as catalysts. The concentrations of catalase reagents were 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL and 100 mg / mL.

[0080] 1.3 Urea peroxide was prepared using 20 mM HEPES buffer to obtain urea peroxide reagents of different concentrations as oxygen generating agents, namely 10 mg / mL, 50 mg / mL and 100 mg / mL.

[0081] 1.4 The catalase reagent obtained in step 1.2 and the urea peroxide reagent obtained in step 1.3 are stored separately, and then mixed at a volume ratio of 1:1 during detection. The mixing time is 1 min to 10 min, preferably 3 min.

[0082] Example 2: Verification of the effect of the companion reagent on the detection signal of glial fibrillary acidic protein (GFAP)

[0083] 2.1 The main experimental materials and equipment are shown in Table 2.

[0084] Table 2

[0085] Glial fibrillary acidic protein (GFAP) Purchased from Oukai Bio GFAP antibody-coated luminescent microspheres (FG-GFAP antibody) Purchased from Huakui Gold Biotin-labeled GFAP antibody (Biotin-GFAP antibody) Purchased from Huakui Gold Donor reagents (including avidin-coated photosensitive microparticles) Purchased from PerkinElmer

[0086] 2.2 The FG-GFAP antibody was diluted with HEPES diluent to obtain an FG-GFAP antibody reagent with a concentration of 25 μg / mL, i.e., reagent 2; the Biotin-GFAP antibody was diluted with HEPES diluent to obtain a Biotin-GFAP antibody reagent with a concentration of 2 μg / mL, i.e., reagent 3; the analyte GFAP with a concentration of 1 mg / mL was diluted with HEPES diluent to obtain calibrators 1 to 6 with different concentrations, i.e., CAL1 to CAL6; the concentrations of calibrators 1 to 6 were 0, 50 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 10000 pg / mL, respectively.

[0087] 2.3 The six calibrators prepared in step 2.2 were subjected to photo-induced chemiluminescence detection according to this procedure to obtain the corresponding light signal values ​​(see Table 3) and signal enhancement amplitudes (see Table 4). Specifically, 25 μL of calibrator, 25 μL of FG-GFAP antibody reagent, and 25 μL of Biotin-GFAP antibody reagent were mixed sequentially; then, a first incubation was performed at 37°C for 15 min to obtain the first reaction solution; then, 175 μL of donor reagent was added, and a second incubation was performed at 37°C for 10 min to obtain the second reaction solution; then, 50 μL of companion reagent (in which the volume ratio of catalyst to oxygen-generating agent is 1:1) was added, the mixture was shaken evenly, and a third incubation was performed at 37°C for 3 min to obtain the third reaction solution; after photoexcitation reaction in the photo-induced chemiluminescence detector, the light signal value was read. The corresponding signal enhancement amplitude was calculated based on the light signal value to obtain the corresponding signal enhancement amplitude. The calculation formula is: Signal enhancement amplitude (%) = (Light signal value of experimental group - Light signal value of un-partnered reagent) / Light signal value of un-partnered reagent * 100%.

[0088] It should be noted that in the control groups of Tables 3 and 4, "without companion reagent" means that the test is performed according to step 2.3, and the difference from the experimental group is that no companion reagent is added during the reaction process; "HEPES buffer" means that the test is performed according to step 2.3, and the difference from the experimental group is that an equal volume of 20mM HEPES buffer is used instead of companion reagent during the reaction process.

[0089] Table 3 (The concentration of urea peroxide reagent in the companion reagent of each experimental group is 10 mg / mL)

[0090]

[0091]

[0092] Table 4 (The concentration of urea peroxide reagent in the companion reagent of each experimental group is 10 mg / mL)

[0093]

[0094] Table 4 shows that comparing the detection results of the control group without the chaperone reagent and the control group with HEPES buffer, it is clear that adding HEPES buffer has no effect on improving the light signal value. Comparing the experimental group with the chaperone reagent and the control group without the chaperone reagent, the experimental group showed a significant increase in signal strength for all concentrations of calibrators after adding the chaperone reagent, with the highest increase reaching 110%, indicating that adding the chaperone reagent can improve the detected light signal. Comparing the light signal improvement of the chaperone reagents containing different concentrations of catalase in the experimental group, it was found that when the catalase concentration reached 1 mg / mL, the light signal improvement plateaued, and further increasing the catalase concentration had a limited effect on improving the light signal.

[0095] The following steps will involve increasing the concentrations of catalase and urea peroxide compared to the previous year to further investigate the effects of the concentrations of the catalyst and oxygen-generating agent on oxygen production and the detection signal.

[0096] The ratio of urea peroxide reagent to catalase reagent concentration in the mixing process was set to 10:1. Various concentrations of urea peroxide reagent were mixed with corresponding concentrations of catalase reagent to obtain the corresponding companion reagents. Detection was performed according to the method in step 2.3, and the corresponding optical signal values ​​were obtained (see Table 5). Subsequently, the signal enhancement amplitude was calculated based on the optical signal values, and the signal enhancement amplitude was obtained (see Table 6).

[0097] Table 5

[0098]

[0099] Table 6

[0100]

[0101] Table 5 shows that when the concentration ratio of the catalyst and oxygen-generating agent is fixed, the light signal value increases to a certain extent and then plateaus, meaning that the light signal value does not increase significantly with further increases in the concentration of the catalyst and oxygen-generating agent. Table 6, combined with comparisons of calibrators with different concentrations of catalase and urea peroxide, reveals that when the catalase concentration reaches 5 mg / mL and the urea peroxide concentration reaches 50 mg / mL, the increase in light signal reaches a plateau; further increases in the concentrations of catalase and urea peroxide have a relatively small effect on increasing the light signal.

[0102] In summary, the addition of the companion reagent effectively enhances the optical signal value, which is beneficial for improving the detection capability of GFAP. The preferred concentrations of the companion reagents are: 50 mg / mL for urea peroxide reagent and 5 mg / mL for catalase reagent; the volume ratio of urea peroxide reagent to catalase reagent is 1:1. This design maximizes the enhancement of the optical signal value while controlling the raw material cost of the companion reagent and avoiding waste.

[0103] Example 3: Verification of the effect of the companion reagent on the detection signal of neurofilament light chain (NfL)

[0104] 3.1 The main experimental materials are shown in Table 7.

[0105] Table 7

[0106] Neurofilament light chain (NfL) Purchased from Wuxi Aoyue Dongyuan NfL antibody-coated luminescent microspheres (FG-NfL antibody) Purchased from Wuxi Aoyue Dongyuan Biotin-labeled NfL antibody (Biotin-NfL antibody) Purchased from Wuxi Aoyue Dongyuan Donor reagents (including avidin-coated photosensitive microparticles) Purchased from PerkinElmer

[0107] 3.2 The FG-NfL antibody was diluted with HEPES diluent to obtain an FG-NfL antibody reagent with a concentration of 25 μg / mL, i.e., reagent 2; the Biotin-NfL antibody was diluted with HEPES diluent to obtain a Biotin-NfL antibody reagent with a concentration of 2 μg / mL, i.e., reagent 3; the analyte NfL with a concentration of 1 mg / mL was diluted with HEPES diluent to obtain calibrators 1 to 6 with different concentrations, i.e., CAL1 to CAL6; the concentrations of calibrators 1 to 6 were 0, 50 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 10000 pg / mL, respectively.

[0108] 3.3 The six calibrators prepared in step 3.2 were subjected to photo-induced chemiluminescence detection according to this procedure to obtain the corresponding optical signal values ​​(see Table 8) and signal enhancement amplitude (see Table 9). Specifically, 25 μL of calibrator, 25 μL of FG-NfL antibody reagent, and 25 μL of Biotin-NfL antibody reagent were mixed sequentially; then, a first incubation was performed at 37°C for 15 min to obtain the first reaction solution; then, 175 μL of donor reagent was added, followed by a second incubation at 37°C for 10 min to obtain the second reaction solution; then, 50 μL of companion reagent was added, shaken thoroughly, and a third incubation was performed at 37°C for 3 min to obtain the third reaction solution; after photoexcitation, the optical signal value was read. The corresponding signal enhancement amplitude was calculated based on the optical signal value. The calculation formula is: Signal enhancement amplitude (%) = (Optical signal value of experimental group - Optical signal value without companion reagent) / Optical signal value without companion reagent * 100%.

[0109] It should be noted that in the control groups of Tables 8 and 9, "without companion reagent" means that the test is performed according to step 3.3, and the difference from the experimental group is that no companion reagent is added during the reaction process.

[0110] Table 8

[0111]

[0112]

[0113] Table 9

[0114]

[0115] As shown in Tables 8 and 9, when comparing the detection light signal values ​​of the experimental group with the added chaperone reagent and the control group without the chaperone reagent, the signal enhancement of each concentration of calibrator in the experimental group with the added chaperone reagent was greater, with the highest reaching 103%. This indicates that adding the chaperone reagent can improve the detection light signal, which means that adding the chaperone reagent can improve the detection capability of NfL.

[0116] Example 4: Verification of the effect of the companion reagent on the detection signal of ubiquitin carboxyl-terminal hydrolase (UCH-L1)

[0117] 4.1 The main experimental materials are shown in Table 10.

[0118] Table 10

[0119] Ubiquitin C-terminal hydrolase (UCH-L1) Purchased from Huamei Bio UCH-L1 antibody-coated luminescent microspheres (FG-UCH-L1 antibody) Purchased from Baixinyi Biotin-labeled UCH-L1 antibody (Biotin-UCH-L1 antibody) Purchased from Wuxi Aoyue Dongyuan Donor reagents (including avidin-coated photosensitive microparticles) Purchased from PerkinElmer

[0120] 4.2 The FG-UCH-L1 antibody was diluted with HEPES diluent to obtain an FG-UCH-L1 antibody reagent with a concentration of 25 μg / mL, i.e., reagent 2. The Biotin-UCH-L1 antibody was diluted with HEPES diluent to obtain a Biotin-UCH-L1 antibody reagent with a concentration of 2 μg / mL, i.e., reagent 3. The analyte UCH-L1 with a concentration of 1 mg / mL was diluted with HEPES diluent to obtain calibrators 1–6 with different concentrations, i.e., CAL1–CAL6; the concentrations of calibrators 1–6 were 0, 50 pg / mL, 200 pg / mL, 500 pg / mL, 1000 pg / mL, and 10000 pg / mL, respectively.

[0121] 4.3 The six calibrators prepared in step 4.2 were subjected to photo-induced chemiluminescence detection according to this procedure to obtain the corresponding optical signal values ​​(see Table 11) and signal enhancement amplitude (see Table 12). Specifically, 25 μL of calibrator, 25 μL of FG-UCH-L1 antibody reagent, and 25 μL of Biotin-UCH-L1 antibody reagent were mixed sequentially; followed by a first-stage incubation treatment at 37°C for 15 min; then 175 μL of donor reagent was added, followed by a second-stage incubation treatment at 37°C for 10 min; then 50 μL of companion reagent was added and the mixture was shaken thoroughly; after photoexcitation in the photo-induced chemiluminescence detector, the optical signal value was read. The corresponding signal enhancement amplitude was calculated based on the optical signal value. The calculation formula is: Signal enhancement amplitude (%) = (Optical signal value of experimental group - Optical signal value without companion reagent) / Optical signal value without companion reagent * 100%.

[0122] It should be noted that in the control groups of Tables 11 and 12, "without companion reagent" means that the test is performed according to step 4.3, and the difference from the experimental group is that no companion reagent is added during the reaction process.

[0123] Table 11

[0124]

[0125] Table 12

[0126]

[0127]

[0128] As shown in Tables 11 and 12, the experimental group with added chaperone reagent and the control group without chaperone reagent showed a greater signal enhancement for each concentration of calibrator in the experimental group with added chaperone reagent. In particular, the signal enhancement was the highest at 101% for the detection of calibrators with concentrations as low as 50 pg / mL. This indicates that adding chaperone reagent can improve the detection light signal, which also means that adding chaperone reagent can improve the detection capability for low concentrations of UCH-L1.

[0129] Example 5: Verification of the effect of the companion reagent on the detection signal of the laminin (LN) assay kit (photochemical photoimmunoassay).

[0130] 5.1 The main experimental materials are shown in Table 13.

[0131] Table 13

[0132] Laminin (LN) Assay Kit (Photochemical Immunoassay) Purchased from Komei Diagnostics (batch 2301)

[0133] 5.2 Follow the instructions for the laminin (LN) assay kit (photochemical immunoassay).

[0134] 5.3 The concentration of urea peroxide reagent was controlled at 50 mg / mL, and the concentration of catalase reagent was controlled at 5 mg / mL. These reagents were then mixed at a volume ratio of 1:1 during detection. 50 μL of the mixed chaperone reagent was added to the laminin (LN) assay kit, shaken to mix, and the optical signal value was obtained. The corresponding signal enhancement amplitude was then calculated based on the optical signal value, as shown in Table 14.

[0135] Table 14

[0136]

[0137]

[0138] As shown in Table 14, based on the comparison between the experimental group with the chaperone reagent and the control group without the chaperone reagent, the signal enhancement of the calibrators of each concentration in the experimental group was relatively large after the addition of the chaperone reagent, with the highest reaching 104%, and the signal enhancement of low concentration CAL2 reached 97%. This indicates that the addition of the chaperone reagent can effectively enhance the light signal value of low concentration target molecules during detection.

[0139] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A companion reagent for photo-induced chemiluminescence detection, characterized in that, It includes an oxygen-generating agent and a catalyst, wherein the oxygen-generating agent reacts under the action of the catalyst to generate oxygen; During photo-induced chemiluminescence detection, the oxygen generated by the companion reagent is used to increase the oxygen content in the reaction system.

2. The companion reagent according to claim 1, characterized in that, The oxygen-generating agent includes metal-peroxide salts and / or hydrogen peroxide complexes; Preferably, the oxygen-generating agent includes at least one of urea peroxide, calcium peroxide, calcium hydroxide, magnesium peroxide, sodium percarbonate, and internal peroxide.

3. The companion reagent for photo-induced chemiluminescence detection according to claim 1, characterized in that, The catalyst includes at least one of enzyme compounds, halides, and metal oxides; Preferably, the catalyst comprises at least one selected from catalase, iodide, manganese dioxide, iron(III), silver and dichromate.

4. The companion reagent for photo-induced chemiluminescence detection according to claim 1, characterized in that: The concentration of the oxygen-generating agent is 10 mg / mL to 100 mg / mL, and / or The concentration of the catalyst is 0.5 mg / mL to 100 mg / mL.

5. The companion reagent for photo-induced chemiluminescence detection according to claim 4, characterized in that: The concentration ratio of the oxygen-generating agent to the catalyst is (1-10):(1-20); Preferably, the concentration ratio of the oxygen-generating agent to the catalyst is 10:

1. 。 6. The companion reagent for photo-induced chemiluminescence detection according to claim 4, characterized in that: The oxygen-generating agent and the catalyst are stored separately and premixed before use. Preferably, the volume ratio of the oxygen-generating agent to the premixed catalyst is 1:

1.

7. The companion reagent for photo-induced chemiluminescence detection according to claim 1, characterized in that, The companion reagent is added to the reaction system before the excitation reading of the photo-induced chemiluminescence reaction.

8. The companion reagent according to claim 1, characterized in that, The companion reagent is used at the same temperature as the photo-induced chemiluminescence reaction.

9. A photo-induced chemiluminescence detection kit, characterized in that, include: Reagent 1, wherein reagent 1 comprises the companion reagent according to any one of claims 1 to 5; Reagent 2, comprising luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles are capable of reacting with reactive oxygen species to generate a detectable light signal; Reagent 3, wherein reagent 3 comprises a label-tagged second biomolecule; Both the first biomolecule and the second biomolecule can specifically bind to the target molecule in the sample to be tested. Preferably, it further includes a donor reagent, which can convert ground-state oxygen into reactive oxygen under laser irradiation of a certain wavelength; more preferably, the donor reagent includes photosensitive microparticles filled with photosensitive material.

10. The application of any one of the companion reagents of claims 1 to 8 or the photo-induced chemiluminescence detection kit of claim 9 in the detection of target molecules; Preferably, the target molecule to be tested is selected from neurodegenerative markers; More preferably, the target molecule to be tested is selected from neurofilament light chains, glial fibrous acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

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