Method for improving signal value of photo-induced chemiluminescence detection and application

Abstract

This application relates to a method and its application for improving the detection signal value of photo-induced chemiluminescence (PET). The method includes: mixing a companion reagent with antibody-coated luminescent microparticles, antibody-coated biotin, avidin-coated photosensitive microparticles, and a sample to be tested, reacting the mixture to generate a test mixture; exciting the test mixture at least once with excitation light, and measuring the amount of emitted photons to obtain a light signal value; wherein the companion reagent includes an oxygen-generating agent and a catalyst, the oxygen-generating agent undergoes a catalytic reaction under the action of the catalyst to generate oxygen, and the oxygen provides singlet oxygen for PET detection. The solution provided in this application can improve the detection signal value of PET, thereby enhancing the stability, sensitivity, and precision of the detection.

Description

Technical Field

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

[0002] Photoinduced chemiluminescence (PRC) is a laser-excited chemiluminescence process involving photosensitive and luminescent microparticles. In this process, the photosensitive microparticles contain photosensitive substances such as phthalocyanines, while the luminescent microparticles contain dimethylthiophene derivatives and europium chelates. When the target antigen is present, a sandwich immune complex can be formed, tightly binding the photosensitive and luminescent microparticles. Under 680 nm laser excitation, the photosensitive substances in the photosensitive microparticles activate and catalyze the formation of high-energy singlet oxygen molecules from surrounding oxygen molecules. This high-energy singlet oxygen diffuses into the luminescent microparticles, triggering a series of chemical reactions that ultimately transfer energy to europium, causing the luminescent microparticles to emit a fluorescence signal in the 520-620 nm range. The concentration of the target antigen can then be obtained by converting the number of photons using a single-photon counter and mathematical fitting. However, when the sample does not contain the target antigen, an immune complex cannot form between the two types of microparticles. The distance between the two microparticles exceeds the propagation range of the singlet oxygen, which is rapidly quenched in the liquid phase, resulting in no high-energy red light signal during detection.

[0003] According to the principle of photo-induced chemiluminescence analysis, when the concentration level of the target to be measured is low, the light signal value generated by photo-induced chemiluminescence is also low. In addition, the noise interference of the instrument makes the detection results unstable and the precision poor, which further leads to a decrease in detection capability. Summary of the Invention

[0004] To address or partially address the problems existing in related technologies, this application provides a method and application for improving the detection signal value of photo-induced chemiluminescence, which can increase the light signal value when the concentration level of the analyte is low, thereby improving the detection capability of the analyte.

[0005] The first aspect of this application provides a method for improving the detection signal value in photo-induced chemiluminescence immunoassay, comprising:

[0006] After adding a companion reagent to the test mixture, which includes the sample to be tested and the photo-induced chemiluminescence detection reagent, the test mixture is irradiated with a laser, and the amount of emitted photons is measured to obtain the light signal value.

[0007] The companion reagent includes an oxygen-generating agent and a catalyst, wherein the oxygen-generating agent reacts under the action of the catalyst to generate oxygen.

[0008] The method 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] In the method described above, 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 method described above, wherein the concentration of the oxygen-generating agent is 10 mg / mL to 100 mg / mL, and / or

[0013] The concentration of the catalyst is 0.5 mg / mL to 100 mg / mL.

[0014] In the method described above, the concentration ratio of the oxygen-generating agent to the catalyst is (1-10):(1-20);

[0015] Preferably, the concentration ratio of the oxygen-generating agent to the catalyst is 10:1.

[0016] The method described above includes the following specific steps:

[0017] S1. Mix the sample to be tested with the luminescent reagent and the labeling reagent, and incubate for the first time to obtain the first reaction solution;

[0018] S2. Add the donor reagent to the first reaction solution, and after a second incubation, obtain the second reaction solution;

[0019] S3. Add the companion reagent to the second reaction solution, and after a third incubation, obtain the third reaction solution;

[0020] S4. The light signal is detected by irradiating the third reaction liquid with excitation light;

[0021] The luminescent reagent includes luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles can react with reactive oxygen species to generate a detectable light signal; the labeling reagent includes a second biomolecule labeled with a tag; the donor reagent can generate reactive oxygen species under laser irradiation of a certain wavelength; both the first biomolecule and the second biomolecule can specifically bind to the target molecule in the sample to be tested.

[0022] In the method described above, the three incubation temperatures are the same; preferably, the incubation temperature is 37–42°C; more preferably, it is 37°C.

[0023] In the method described above, the companion reagent is premixed and then added to the second reaction solution; preferably, the premixing time is 1 to 10 minutes; more preferably, the premixing time is 3 minutes.

[0024] In the method described above, the sample to be tested is cerebrospinal fluid, whole blood, serum, or plasma.

[0025] A second aspect of this application provides the application of the method described above in the detection of neurodegenerative markers; preferably in the detection of neurofilament light chains, glial fibrillary acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

[0026] The technical solution provided in this application may include the following beneficial effects:

[0027] This application incorporates a companion reagent into the photo-induced chemiluminescence (PET) detection process. The companion reagent includes an oxygen-generating agent and a catalyst. The oxygen-generating agent reacts under the action of the catalyst to release oxygen. This oxygen is activated into reactive oxygen species, increasing the reactive oxygen content in the detection system and thus enhancing the light signal intensity of PET. This improves the stability, sensitivity, and precision of PET detection, effectively addressing issues such as low luminescence signal values, significant interference from instrument noise, and poor precision in detecting low-level analytes.

[0028] 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

[0029] 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.

[0030] 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.

[0031] 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.

[0032] Terminology Explanation

[0033] The term "sample to be tested" as used in this application refers to a mixture containing or suspected of containing the target molecule. Samples to be tested that can be used in this application include bodily fluids, such as blood (which may be anticoagulated blood commonly seen in collected blood samples), plasma, serum, cerebrospinal fluid, etc. Other types of samples to be tested include solvents, seawater, industrial water samples, food samples, environmental samples such as soil or water, plant material, eukaryotic cells, bacteria, plasmids, viruses, fungi, and cells derived from prokaryotes. Samples to be tested can be diluted with a diluent as needed before use. For example, to avoid the hook effect, the sample can be diluted with a diluent before being tested on the instrument.

[0034] 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.

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] The first aspect of this application provides a method for improving the detection signal value of photo-induced chemiluminescence, comprising: adding a companion reagent to a mixture of a sample to be tested and a photo-induced chemiluminescence detection reagent, irradiating the mixture of the sample to be tested with a laser, and measuring the amount of emitted photons to obtain a light signal value; wherein the companion reagent comprises an oxygen-generating agent and a catalyst, and the oxygen-generating agent reacts under the action of the catalyst to generate oxygen.

[0043] Specifically, the sample to be tested is mixed with a photo-induced chemiluminescence detection reagent to obtain a test mixture. Then, a companion reagent is added to the test mixture and mixed. The test mixture is then irradiated with a laser, and finally, the amount of emitted photons is measured to obtain the light signal value.

[0044] The chaperone reagent includes an oxygen-generating agent and a catalyst. The oxygen-generating agent reacts under the action of the catalyst to release oxygen. This oxygen can be activated into reactive oxygen species, thereby increasing the amount of reactive oxygen species in photochemiluminescence, thus improving the luminescence efficiency of photochemiluminescence and enhancing the light signal intensity of photochemiluminescence.

[0045] This application does not limit the specific selection of the analyte in the sample to be tested; the selection can be made according to actual needs. It is understood that the photochemiluminescence detection reagent of this application corresponds to the analyte in the sample to be tested. For example, if the analyte is an antigen, then the photochemiluminescence detection reagent includes the antibody corresponding to the antigen.

[0046] This application does not limit the specific selection of photo-induced chemiluminescence detection reagents. They can be selected according to actual needs. Commonly used reagents, including antibody-coated luminescent microspheres, biotin-labeled antibodies, and avidin-coated photosensitive microspheres, can be selected as photo-induced chemiluminescence detection reagents.

[0047] This application adds a companion reagent to the photo-induced chemiluminescence detection process, which can increase the content of reactive oxygen species in the detection system, thereby enhancing the light signal intensity of photo-induced chemiluminescence. This improves the stability, sensitivity, and precision of the detection when applied to photo-induced chemiluminescence detection, and helps to solve the problems of low luminescence signal value, large interference of instrument noise, and poor precision when detecting low-level analytes.

[0048] 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. The above-mentioned oxygen-generating agents have high oxygen production, high stability, good solubility, and high environmental friendliness. When applied in photochemiluminescence detection systems, they can efficiently generate ground-state oxygen while avoiding side reactions and environmental pollution caused by the oxygen-generating agent and photochemiluminescence detection reagents, thereby improving the stability, sensitivity, and precision of the detection.

[0049] 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. The above-mentioned catalysts have advantages such as high catalytic activity, high stability, high environmental friendliness, and low cost. They can efficiently and stably catalyze oxygen production from oxygen-generating agents and do not interfere with photo-induced chemiluminescence detection, thus improving the stability, sensitivity, and precision of the detection.

[0050] 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.

[0051] 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.

[0052] It should be noted that the concentrations of the oxygen-generating agent and the catalyst mentioned above in this application are the concentrations of the oxygen-generating agent and the catalyst when they are prepared and stored separately, that is, their respective concentrations before they are 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, increase the active oxygen content in the photo-induced chemiluminescence system, reduce the waste of raw materials caused by excessive oxygen-generating agent or catalyst, improve the stability, sensitivity and precision of detection, and save costs.

[0053] 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 concentration ratio of the oxygen-generating agent to the catalyst is within the above range, the matching between the oxygen-generating agent and the catalyst is higher, the oxygen-generating agent can better generate oxygen under the action of the catalyst, increase the oxygen content in the photo-induced chemiluminescence detection system, and make the detection stability, sensitivity and precision better, while reducing the excess of oxygen-generating agent or catalyst and reducing costs.

[0054] In some embodiments, the specific steps of the method for improving the detection signal value of photo-induced chemiluminescence in this application include:

[0055] S1. Mix the sample to be tested with the luminescent reagent and the labeling reagent, and incubate for the first time to obtain the first reaction solution.

[0056] The luminescent reagent includes luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles can react with reactive oxygen species to generate a detectable light signal; the labeling reagent includes a second biomolecule labeled with a tag; both the first biomolecule and the second biomolecule can specifically bind to the target molecule in the sample to be tested.

[0057] Taking the target molecule as an antigen as an example, both the first and second biomolecules can be antibodies that specifically bind to the antigen. The first and second biomolecules can be the same antibody or different antibodies.

[0058] In some implementations, the label of the labeling reagent may be, for example, biotin.

[0059] It is understandable that the first reaction solution after the reaction is completed contains a complex of "luminescent microparticles-first biomolecule-target molecule-second biomolecule-biotin".

[0060] In some implementations, the incubation time for the first incubation is 15 minutes.

[0061] S2. Add the donor reagent to the first reaction solution, and after a second incubation, obtain the second reaction solution.

[0062] The donor reagent can generate reactive oxygen species under laser irradiation of a certain wavelength. In some embodiments, the donor reagent is also called a photosensitive reagent, which includes photosensitive microparticles filled with photosensitive material. 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 is conducive to binding with biotin, preferably streptavidin.

[0063] It is understandable that the second reaction solution after the reaction is completed contains a sandwich immune complex consisting of "luminescent microparticles - first biomolecule - target molecule - second biomolecule - biotin - avidin - photosensitive microparticles".

[0064] In some implementations, the second incubation period is 10 minutes.

[0065] S3. Add the companion reagent to the second reaction solution, and after a third incubation, obtain the third reaction solution.

[0066] The companion reagent comprises a premixed oxygen-generating agent and a catalyst. In some embodiments, the companion reagent is premixed and then added to the second reaction solution. Premixing the oxygen-generating agent and catalyst in the companion reagent prevents the oxygen-generating agent from decomposing upon contact with the catalyst after mixing and leaving the mixture, thus avoiding premature oxygen release and ensuring no improvement in detection stability, sensitivity, or precision. Preferably, the premixing time is 1–10 min, for example, 1 min, 2 min, 3 min, 4 min, 5 min, 6 min, 7 min, 8 min, 9 min, or 10 min; more preferably, the premixing time is 3 min. Within this premixing time, the oxygen-generating agent and catalyst are in sufficient contact, allowing the oxygen-generating agent to begin producing oxygen under the action of the catalyst, thereby increasing the intensity of the detected optical signal.

[0067] It is understandable that the third reaction solution includes a sandwich immune complex, a catalyst, and an oxygen-generating agent after the reaction. The oxygen-generating agent decomposes under the action of the catalyst to produce ground-state oxygen, thereby increasing the oxygen content in the third reaction solution.

[0068] In some implementations, the third incubation period is 3 minutes.

[0069] S4. The light signal is detected by irradiating the third reaction liquid with excitation light.

[0070] The third reaction solution is irradiated with excitation light. Under 680nm laser irradiation, the ground-state oxygen in the third reaction solution is converted into reactive oxygen. The luminescent particles react with the reactive oxygen to generate a light signal, which is then detected to obtain the light signal value. In the specific steps described above, to obtain more accurate detection results, in some embodiments, the concentration of the luminescent reagent can be 10μg / mL to 100μg / mL, for example, the concentration of the luminescent reagent can be 10μg / mL, 20μg / mL, 30μg / mL, 40μg / mL, 50μg / mL, 60μg / mL, 70μg / mL, 80μg / mL, 90μg / mL, or 100μg / mL. In some embodiments, the concentration of the labeling reagent can be 0.5μg / mL to 5μg / mL, for example, the concentration of the labeling reagent can be 0.5μg / mL, 1μg / mL, 2μg / mL, 3μg / mL, 4μg / mL, or 5μg / 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.

[0071] In some implementations, the incubation temperatures are the same for the three incubation steps S1 to S3, which helps the binding of the first biomolecule, the second biomolecule and the target molecule to be tested, as well as the binding of the donor reagent and the tag in the labeling reagent, reduces the influence of temperature on detection, improves the stability and accuracy of detection results, and simplifies the detection process and reduces detection costs.

[0072] In some preferred embodiments, the incubation temperature for each step is 37–42°C, for example, 37°C, 38°C, 39°C, 40°C, 41°C, or 42°C; more preferably, it is 37°C. When the incubation temperature is within the above range, the activity of each substance in the photo-induced chemiluminescence detection reagent and the oxygen production efficiency of the companion reagent can be guaranteed, avoiding excessively high temperatures that lead to substance inactivation or excessively low temperatures that lead to low oxygen production and binding efficiency, thereby improving the stability, sensitivity, and precision of the detection.

[0073] In some implementations, the sample to be tested is cerebrospinal fluid, whole blood, serum, or plasma. The selection of cerebrospinal fluid, whole blood, serum, or plasma as the sample ensures the stable presence of the target molecules and facilitates sample collection, which is beneficial for the widespread application of photochemiluminescence detection methods in clinical medicine.

[0074] In summary, the method for improving photochemiluminescence detection signal values ​​provided in this application involves adding a chaperone reagent after forming a sandwich immune complex to increase the oxygen content in the system. Then, the donor reagent is irradiated with excitation light to convert the oxygen into reactive oxygen species, thereby increasing the reactive oxygen species content in the system and ultimately enhancing the light signal intensity. This method significantly improves the stability, sensitivity, and precision of the detection, and is simple to operate with fewer raw material requirements, which facilitates its widespread application.

[0075] A second aspect of this application provides the application of the above-described method in the detection of neurodegenerative markers; preferably, its application in the detection of neurofilament light chains, glial fibrillary acidic proteins, ubiquitin C-terminal hydrolases, or laminin. Neurodegenerative markers are present at low concentrations in the sample. The photo-induced chemiluminescence detection method provided in this application can improve the intensity of the detected light signal, enhance the stability, sensitivity, and accuracy of the detection, thereby improving the detection capability for low concentrations of neurodegenerative markers.

[0076] 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.

[0077] The following example uses urea peroxide as the oxygen-generating agent and catalase as the catalyst to prepare a companion reagent. Then, the companion reagent is used to mix and react with the luminescent reagent, labeling reagent, donor reagent and the sample to be tested. Finally, the light signal value is detected on a photo-induced chemiluminescence platform.

[0078] Example 1: Preparation of companion reagent

[0079] 1.1 The main experimental materials are shown in Table 1

[0080] Table 1

[0081] peroxyurea Purchased from Sigma catalase Purchased from Sigma

[0082] 1.2 Catalase was diluted with 20 mM HEPES buffer to obtain different concentrations of catalase reagent as catalysts. The concentrations of catalase reagent were 0.5 mg / mL, 1 mg / mL, 5 mg / mL, and 10 mg / mL. 、 100mg / mL.

[0083] 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.

[0084] 1.4 Store the catalase reagent and urea peroxide reagent separately in the companion reagent, and then mix them at a volume ratio of 1:1 during the test. The mixing time is 3 minutes.

[0085] Example 2: Verification of the effect of the companion reagent on the detection signal of glial fibrillary acidic protein (GFAP)

[0086] 2.1 The main experimental materials and equipment are shown in Table 2.

[0087] Table 2

[0088]

[0089]

[0090] 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., the luminescent reagent; 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., the labeling reagent; 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.

[0091] 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%.

[0092] 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.

[0093] Table 3 (The concentration of urea peroxide reagent in the companion reagent of each experimental group is 10 mg / mL)

[0094]

[0095]

[0096] Table 4 (The concentration of urea peroxide reagent in the companion reagent of each experimental group is 10 mg / mL)

[0097]

[0098] 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.

[0099] 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.

[0100] 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).

[0101] Table 5

[0102]

[0103] Table 6

[0104]

[0105] 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.

[0106] 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.

[0107] Example 3: Verification of the effect of the companion reagent on the detection signal of neurofilament light chain (NfL)

[0108] 3.1 The main experimental materials are shown in Table 7.

[0109] Table 7

[0110] 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

[0111] 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., the luminescent reagent; 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., the labeling reagent; 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.

[0112] 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%.

[0113] 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.

[0114] Table 8

[0115]

[0116] Table 9

[0117]

[0118] 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.

[0119] Example 4: Verification of the effect of the companion reagent on the detection signal of ubiquitin carboxyl-terminal hydrolase (UCH-L1)

[0120] 4.1 The main experimental materials are shown in Table 10.

[0121] Table 10

[0122]

[0123]

[0124] 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., a luminescent reagent; 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., a labeling reagent; the analyte UCH-L1 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.

[0125] 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 amplitudes (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; 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 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 to obtain the corresponding signal enhancement amplitude. The calculation formula is: Signal enhancement amplitude (%) = (Light signal value of experimental group - Light signal value without mate reagent) / Light signal value without mate reagent * 100%, which gives the signal enhancement amplitude.

[0126] 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.

[0127] Table 11

[0128]

[0129]

[0130] Table 12

[0131]

[0132] 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 as high as 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.

[0133] Example 5: Verification of the effect of the companion reagent on the detection signal of the laminin (LN) assay kit (photochemical photoimmunoassay).

[0134] 5.1 The main experimental materials are shown in Table 13.

[0135] Table 13

[0136]

[0137] 5.2 Follow the instructions for the laminin (LN) assay kit (photochemical immunoassay).

[0138] 5.3 The concentrations of urea peroxide reagent and catalase reagent in the companion reagent were controlled at 50 mg / mL and 5 mg / mL, respectively, and then mixed at a volume ratio of 1:1 during detection. 50 μL of the mixed companion 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.

[0139] Table 14

[0140]

[0141] 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.

[0142] 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 method for improving the detection signal value in photo-induced chemiluminescence immunoassay, characterized in that, include: After adding a companion reagent to the test mixture, which includes the sample to be tested and the photo-induced chemiluminescence detection reagent, the test mixture is irradiated with a laser, and the amount of emitted photons is measured to obtain the light signal value. The companion reagent includes an oxygen-generating agent and a catalyst, wherein the oxygen-generating agent reacts under the action of the catalyst to generate oxygen.

2. The method 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 method 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 method 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 method 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 method according to claim 1, characterized in that, The specific steps include: S1. Mix the sample to be tested with the luminescent reagent and the labeling reagent, and incubate for the first time to obtain the first reaction solution; S2. Add the donor reagent to the first reaction solution, and after a second incubation, obtain the second reaction solution; S3. Add the companion reagent to the second reaction solution, and after a third incubation, obtain the third reaction solution; S4. The light signal is detected by irradiating the third reaction liquid with excitation light; The luminescent reagent includes luminescent microparticles and a first biomolecule bound thereto, wherein the luminescent microparticles can react with reactive oxygen species to generate a detectable light signal; the labeling reagent includes a second biomolecule labeled with a tag; the donor reagent can generate reactive oxygen species under laser irradiation of a certain wavelength; both the first biomolecule and the second biomolecule can specifically bind to the target molecule in the sample to be tested.

7. The method according to claim 6, characterized in that, The three incubations are at the same temperature; preferably, the incubation temperature is 37-42°C; more preferably, it is 37°C.

8. The method according to claim 6, characterized in that, The companion reagent is premixed and then added to the second reaction solution; preferably, the premixing time is 1 to 10 minutes; more preferably, the premixing time is 3 minutes.

9. The method according to any one of claims 1 to 8, characterized in that, The sample to be tested is cerebrospinal fluid, whole blood, serum, or plasma.

10. The application of the method according to any one of claims 1 to 9 in the detection of neurodegenerative markers; preferably in the detection of neurofilament light chains, glial fibrillary acidic proteins, ubiquitin carboxyl-terminal hydrolases, or laminin.

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