Methods, reagents, and instrument systems for homogeneous immunoassay of target antibody analytes

Abstract

This invention provides a method for homogeneous immunoassay of target antibody analytes, along with its reagents and instrument system. This method eliminates the conventionally used luminescent bulbs and employs reactive oxygen species (ROS) that react with oxygen-sensitive substances to generate electrical signals detectable by electrodes. These ROS are generated by the excitation of photosensitive compounds, eliminating the need for complex instrument modules. This method is more conducive to the reaction between the antibody analyte and the antigen, shortens the reaction time, and improves antibody utilization, resulting in higher sensitivity and accuracy in the detection instrument of this invention.

Description

Technical Field

[0001] This invention belongs to the field of medical diagnostics, specifically relating to a method, reagents, and instrument system for homogeneous immunoassay of target antibody analytes. Background Technology

[0002] Immunoassay technology has wide applications in medical diagnostics. Commonly used immunoassay methods include enzyme-linked immunosorbent assay (ELISA), electrochemical immunoassay, and homogeneous immunochemiluminescence. Antibody detection technology has seen various improvements in recent years, further enhancing detection performance through rapid and point-of-care testing (POCT).

[0003] Homogeneous chemiluminescence, due to its lack of cleaning involved, suffers from limitations in instrument miniaturization, relatively high instrument costs, and background signal issues. Furthermore, its detection method requires whole blood separation before testing to ensure accuracy, making it complex and unsuitable for home-based point-of-care testing (POCT) scenarios. Additionally, patients with mild symptoms or those requiring long-term monitoring increase the risk of cross-infection when visiting hospitals; the methods mentioned above are only suitable for hospital settings and difficult to promote in home use. Therefore, to address the issues of home-based compatibility, whole blood testing capability, and reduced background signal, the development of a simple, compact instrument and accompanying diagnostic reagents for mobile monitoring of disease levels is urgently needed.

[0004] Electrochemical immunoassay (EIA) targets antibody analytes based on immune recognition. It achieves specific detection of specific substances through the immune recognition reaction between antigens and antibodies, with the output being an electrical signal. Traditional immunoelectrochemical assays typically require multiple washing steps to remove excess antibodies before accurate testing. Compared to traditional immunoelectrochemical assays, EIA eliminates these intermediate washing steps, enabling accurate testing. It combines the high specificity of immunoassay with the advantages of electrochemical detection, such as low cost, high sensitivity, cost-effectiveness, and small instrument size. EIA instruments do not require complex structures or optical components, allowing for miniaturization and portability. Furthermore, because it detects electrical signals, it can use whole blood samples, making it more suitable for point-of-care testing (POCT) scenarios. Summary of the Invention

[0005] The purpose of this invention is to provide a method, reagents, and instrument system for detecting target antibody analytes using homogeneous immunoassay technology. This method eliminates the conventionally used luminescent bulbs and employs reactive oxygen species (ROS) that react with oxygen-sensitive substances to generate electrical signals detectable by electrodes. These ROS are generated by the excitation of photosensitive compounds, eliminating the need for complex instrument modules. This method is more conducive to the reaction between the antibody target analyte and the antigen, shortens the reaction time, and improves antibody utilization, resulting in a detection instrument with higher sensitivity and accuracy.

[0006] To achieve the above objectives, the main technical solution of the present invention is as follows:

[0007] In a first aspect, the present invention provides a kit of reagents for detecting a target antibody analyte, characterized in that the target antibody analyte is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment; the kit of reagents comprises a first structural substance and a second structural substance capable of binding the target antibody analyte, the first structural substance being coupled with a photosensitive compound that can be activated under preset conditions, the activated photosensitive compound being capable of generating reactive oxygen species; the second structural substance being coupled with an oxygen-receiving composition capable of receiving reactive oxygen species and generating a preset electrical signal; the first structural substance and the second structural substance are not simultaneously one of an antigen, a hapten, a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment; the target antibody analyte specifically binds to at least one of the first structural substance and the second structural substance; wherein, when the first structural substance and the second structural substance contact the target antibody analyte, the activated photosensitive compound transfers reactive oxygen species to the oxygen-receiving composition, the oxygen-receiving composition generates an electrical signal, and the information of the electrical signal is used to characterize the presence and / or quantity of the target antibody analyte.

[0008] Furthermore, the first and second structural substances are antigens or haptens, which can specifically bind to the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment; when the first and second structural substances come into contact with the target antibody analyte, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound to generate an electrical signal.

[0009] Furthermore, the reagent kit also includes a third structural substance coupled to the photosensitive compound. When the third structural substance comes into contact with the first structural substance, the third structural substance undergoes acceptor-ligand affinity adsorption with the first structural substance. The photosensitive compound is excited by light of a certain wavelength and reacts with the oxygen-receiving composition to generate an electrical signal.

[0010] Furthermore, the intensity of the electrical signal is positively correlated with the quantity of the target antibody analyte.

[0011] Further, the second structural substance is an antigen or hapten, and the first structural substance is a polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment, wherein the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen or hapten, wherein:

[0012] When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the target antibody analyte competitively binds to the second structural substance with the first structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated composition of the second structural substance to generate an electrical signal.

[0013] Further, the first structural substance is an antigen or hapten, and the second structural substance is a polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment, wherein the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen or hapten, wherein:

[0014] When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the target antibody analyte and the second structural substance competitively bind to the first structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated composition of the second structural substance to generate an electrical signal.

[0015] Furthermore, the second structural substance is an antigen or hapten, and the first structural substance is not directly coupled to the photosensitive compound;

[0016] The kit of reagents also includes a third structural substance conjugated to the photosensitive compound. The third structural substance is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment. The third structural substance is capable of receptor-ligand affinity adsorption with the first structural substance. The polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen or hapten.

[0017] When the target antibody analyte comes into contact with the second and third structural substances, the target antibody analyte competitively binds to the second structural substance with the third structural substance. The photosensitive compound on the third structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated composition of the second structural substance to generate an electrical signal.

[0018] Furthermore, the intensity of the electrical signal is negatively correlated with the quantity of the target antibody analyte.

[0019] Secondly, the present invention provides a method for detecting a target antibody analyte, comprising incubating a sample to be tested with the kit of reagents described in the present invention; detecting the background electrical signal of the incubated sample using an electrode; exciting a photosensitive compound of the kit of reagents with light of a specific wavelength and detecting the excited electrical signal; and comparing the background electrical signal and the excited electrical signal to obtain sample electrical signal information and determine the presence and / or quantity of the target antibody analyte.

[0020] Thirdly, the present invention provides an instrument system for detecting a target antibody analyte, comprising a liquid reaction module configured to incubate a sample to be tested and the reagent kit described in the present invention; an electrode module configured to contact the liquid in the liquid reaction module and detect an electrical signal; an optical module configured to emit light of a certain wavelength into the liquid reaction module; and a processor configured to receive and process the signal from the electrode module to determine the presence and / or quantity of the target antibody analyte.

[0021] Optionally, the liquid reaction module is a container capable of containing liquid, preferably a cuvette, a cuvette array, or a well plate;

[0022] Optionally, the liquid reaction module is a test strip coated with the complete set of reagents described in this invention.

[0023] Fourthly, the present invention provides a target antibody analyte detection system, comprising an electrode that adsorbs or couples an immune molecule; photosensitive microspheres and an oxygen-receiving compound reagent, wherein the photosensitive microspheres contain a photosensitive compound, which generates reactive oxygen species capable of reacting with the oxygen-receiving compound reagent under light excitation at a certain wavelength, thereby generating an electrical signal detectable by the electrode; wherein the photosensitive microspheres further contain a binding ligand.

[0024] Furthermore, the binding ligand is an antigen or hapten, the immune molecule adsorbed or coupled by the electrode is an antibody, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten.

[0025] When the target antibody analyte comes into contact with the photosensitive microsphere and the electrode, the target antibody analyte and the antibody on the electrode competitively bind to the photosensitive microsphere, and the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

[0026] Furthermore, the binding ligand is an antigen or hapten, the immune molecule adsorbed or coupled by the electrode is an antigen, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen or hapten.

[0027] When the target antibody analyte comes into contact with the electrode and the photosensitive microsphere, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

[0028] Furthermore, the immune molecules adsorbed or coupled by the electrode are polyclonal antibodies, monoclonal antibodies, ScFv, or antibody fragments. The detection system also includes antigens labeled with receptor-ligand affinity adsorption. The antigens labeled with receptor-ligand affinity adsorption can undergo affinity adsorption with the photosensitive ball, and the polyclonal antibodies, monoclonal antibodies, ScFv, or antibody fragments immunize the antigens.

[0029] When the target antibody analyte comes into contact with the photosensitive microspheres and the electrode, the target antibody analyte and the antibody on the electrode competitively bind to the receptor-ligand affinity-adsorbed antigen, and the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

[0030] Furthermore, the detection system further includes receptor-ligand affinity-labeled antigens adsorbed or coupled to the electrode, and the receptor-ligand affinity-labeled antibody can undergo affinity adsorption with the photosensitive ball. The polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen.

[0031] When the target antibody analyte comes into contact with the electrode and the receptor-ligand affinity-adsorbed antigen, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

[0032] Fifthly, the present invention provides a method for detecting a target antibody analyte, comprising the detection system described in the present invention, incubating with a sample to be tested; using electrodes to detect the background electrical signal of the incubated sample; exciting the photosensitive compound with light of a specific wavelength and detecting the excited electrical signal; and comparing the background electrical signal and the excited electrical signal to obtain sample electrical signal information and determine the presence and / or quantity of the target antibody analyte.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] 1) The homogeneous immunoreaction of the present invention can solve the problem of poor detection specificity caused by the need for intermediate washing process in traditional electrochemical methods.

[0035] 2) The back-end signal output and detection of the present invention use electrical signals, which, compared with the optical signals of the prior art, reduce the size of the instrument detection part, making the instrument portable and reducing the cost of signal acquisition.

[0036] 3) The detection method of the present invention adopts electrochemical detection, which requires relatively simple instruments and can realize product integration and miniaturization.

[0037] 4) The instrument system for detecting target analytes in this invention employs screen-printed electrodes. Electrode materials include, but are not limited to, carbon, silver, gold, copper, and combinations thereof. This solves the problems of high operational difficulty and poor reproducibility associated with traditional three-electrode systems, which hinder commercialization. Furthermore, the instrument system of this invention is low-cost, disposable, and free from cross-interference, making it suitable for home and other POCT applications. Users can import test results to their mobile phones via Bluetooth, USB, TPC, etc., to monitor the significance and fluctuations of the test results in real time.

[0038] 5) The test samples of the present invention are not limited to fingertip blood; urine, saliva, etc. can also be included in the method of the present invention for testing. Attached Figure Description

[0039] Figure 1 This is a schematic diagram of the reaction process and testing of the present invention;

[0040] Figure 2 This is a peak-shaped graph showing the relationship between current and antibody concentration, specifically the relationship between current and hepatitis B surface antibody (HbsAb) concentration.

[0041] Figure 3 Photoexcitation signal gradient diagrams for different concentrations of hepatitis B surface antibody (HbsAb);

[0042] Figure 4 The graph shows the relationship between current and the concentration of hepatitis B surface antibody (HBsAb).

[0043] Figure 5 This is a diagram illustrating the consistency evaluation between the method of this invention and the testing methodology of the Roche biochemical analyzer. Detailed Implementation

[0044] This invention discloses a novel homogeneous immunoassay detection method, along with its reagents and instruments. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments; those skilled in the art will clearly be able to modify or appropriately alter and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0045] Terminology Explanation

[0046] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0047] The term "photosensitive compound" refers to a photosensitive substance that can be excited by excitation light of a certain wavelength, such as rose bengal, methylene blue, phthalocyanine complexes, naphtholine complexes, and combinations thereof.

[0048] The term "oxygenated compound" refers to a product that can undergo a redox reaction with reactive oxygen species to produce an electrochemical signal. Oxygenated compounds themselves do not produce electrochemical signals; they can be any one or a combination of hydroquinone, resorcinol, dopamine, acetaminophen, para-aminophenol, ferrocene, and their derivatives. Oxygenated compounds can be categorized into chemical substances, silica microspheres containing oxygenated chemical substances, and polystyrene microspheres containing oxygenated chemical substances.

[0049] The term "antibody" refers to immunoglobulins and immunoglobulin fragments, whether naturally occurring or partially or wholly synthetic (e.g., recombinant), including any fragment that retains the binding specificity of the full-length immunoglobulin molecule, containing at least a portion of the variable region of the immunoglobulin molecule. The term antibody includes polyclonal antibodies, monoclonal antibodies, ScFv, or antibody fragments.

[0050] The term "monoclonal antibody" refers to a group of identical antibodies, meaning that each individual antibody molecule in the monoclonal antibody group is identical to the other antibody molecules. "Polyclonal antibody" refers to a group of antibodies containing multiple different sequences.

[0051] The term "scFv fragment" refers to an antibody composed of variable regions of the antibody heavy chain and light chain linked by a short peptide linker of 5–20 amino acids. The linker length allows the two variable domains to be bridged with minimal interference.

[0052] The term "antibody fragment" refers to any portion of a full-length antibody that is less than the full length but contains at least a portion of the antibody's variable region (e.g., one or more CDRs and / or one or more antibody binding sites) that binds to the antigen, and thus retains binding specificity as well as at least a portion of the full-length antibody's specific binding ability.

[0053] The term "antigen" refers to either an isolated antigen or an antigen present in a biological sample.

[0054] The term "hapten" is a compound that can specifically bind to a corresponding antibody but does not itself act as an immunogen (or antigen) for antibody production. Haptens are typically linked to an antigen carrier used to generate antibodies.

[0055] The terms "antigen carrier" or "immunogenic carrier" are used interchangeably to refer to a group or portion that, when conjugated to a hapten and injected into a mammal or otherwise used as an immunogen, induces an immune response and triggers the production of antibodies that bind to the hapten. Poly(amino acid) antigen carriers have molecular weights (in Daltons) ranging, for example, from about 5,000 to about 10,000,000, or from about 20,000 to about 600,000, or from about 25,000 to about 250,000. Poly(amino acid) antigen carriers include proteins such as, for example, albumins, serum proteins such as globulins, lens proteins, and lipoproteins. Illustrative proteins include, but are not limited to, bovine serum albumin (BSA), keyhole cyanin (KLH), ovalbumin, and bovine gamma globulin (BGG). Non-poly(amino acid) antigen carriers include polysaccharides, nucleic acids, and particles (biological and synthetic materials).

[0056] The term "specific binding" for an antibody or its antigen-binding fragment refers to the ability of an antibody or antigen-binding fragment to form one or more non-covalent bonds with the same antigen through a non-covalent interaction between the antibody and the antigen's antibody-binding site. "Competitive binding" refers to the competitive binding of two or more antigens or haptens to an antibody, antigen carrier, or antigen-binding fragment in a limited number of samples.

[0057] The term "biotin" refers to a substance that can be coupled with an antigen / antibody; it is essentially a vitamin.

[0058] The term "avidin" refers to a glycoprotein that can be extracted from egg white. Avidin includes avidin, neutralizing avidin, streptavidin, etc.

[0059] The term "receptor-ligand affinity adsorption label" refers to a specific molecule or particle that has a certain tag (such as fluorescein, enzyme, radioisotope, etc.).

[0060] Figure 1 The diagram illustrates the reaction process and testing schematic of this invention. The target antibody analyte is in contact with a photosensitive sphere. The photosensitive sphere contains a photosensitive substance that can be excited by excitation light of a certain wavelength. The activated photosensitive compound can generate reactive oxygen species. The reactive oxygen species undergoes a redox reaction with the oxygen-receiving compound to produce products that can induce electrochemical signals, which can generate electrical signals under a specific potential.

[0061] Figure 2The typical test result graph (reduction peak of BQ) is shown, that is, the peak shape graph of the relationship between current and antibody concentration, that is, the reduction peak signal of the target antibody as the product (BQ) after oxidation by oxygenated compound (HQ); specifically, the linear graph of current and hepatitis B surface antibody (HbsAb) concentration verified in the embodiments of the present invention, that is, the electrical signal of the target antibody analyte antibody as the product (BQ) after oxidation by oxygenated compound (HQ) is plotted against the standard concentration to obtain the standard curve of hepatitis B surface antibody (HbsAb) concentration.

[0062] Figure 3 The photoexcitation signal gradient diagram shows different concentrations of hepatitis B surface antibody (HbsAb).

[0063] Figure 4 The graph shows the relationship between current and the concentration of hepatitis B surface antibody (HBsAb).

[0064] Figure 5 The diagram shows the consistency evaluation between the method of the present invention and the testing methodology of the Roche biochemical analyzer.

[0065] Unless otherwise specified, the experimental methods used in the embodiments are conventional methods, and the materials and reagents used can be purchased commercially unless otherwise specified.

[0066] In the following examples, hepatitis B surface antibody (HBsAb) was used as the target antibody analyte; silica (SiO2) was synthesized in step 1.1 of Example 1; tetraethyl orthosilicate (TEOS) was purchased from Sigma-Aldrich, catalog number 8006580250; 3-aminopropyltrimethoxysilane (APTMS) was purchased from Sigma-Aldrich, catalog number 281778-5mL; hydroquinone oxygenated compound (HQ) was purchased from Sigma-Aldrich, catalog number H9003-100G; p-benzoquinone (B... Q) is the oxidation product of HQ; hepatitis B surface antibody (Ab) and hepatitis B surface antibody antigen (Ag) were purchased from Hangzhou Boyue Biotechnology Co., Ltd.; SiO2-HQ-Ab1 is SiO2 microspheres coated with HQ conjugated with hepatitis B surface antibody (HbsAb)-specific antigen Ag1; photosensitive microspheres (GG) were purchased from Xi'an Qiyue Biotechnology Co., Ltd.; streptavidin (SA) was purchased from Sigma-Aldrich, catalog number S6402-1ML; biotin conjugated with hepatitis B surface antibody (HbsAb)-antigen (Bio-Ag2).

[0067] The present invention will be further illustrated below with reference to the embodiments:

[0068] Example 1: Synthesis of SiO2 microspheres

[0069] (1) Take 60 mL of cyclohexane, 5 mL of n-hexanol, 20 mL of Triton X-100, and 5 mL of purified water and add them to a 250 mL clean conical flask. Place a 2 cm diameter magnetic stir bar in the flask and stir at 100 RPM for 20 min. Add 0.5 mL of ammonia and 1 mL of TEOS, and continue stirring for 24 h. After stirring is complete, aspirate the solution from the conical flask and transfer it to two 50 mL centrifuge tubes for centrifugation. Collect the supernatant.

[0070] (2) Add 10 mL of anhydrous ethanol and 10 mL of purified water respectively, resuspend by sonication, centrifuge at 10000 RPM for 25 minutes, and take out the supernatant.

[0071] Repeat step (2).

[0072] (3) Add 6 mL of anhydrous ethanol and 6 mL of purified water again, resuspend by sonication, centrifuge at 10000 RPM for 25 minutes, and take out the supernatant.

[0073] Repeat step (3).

[0074] (4) Add 3 mL of anhydrous ethanol to each and resuspend to prepare SiO2 microspheres for later use.

[0075] Example 2: Synthesis of SiO2-HQ microspheres

[0076] (1) Take out a 10ml round bottom flask, clean it, and rinse it once with pure water and once with ethanol.

[0077] (2) Take 2 ml of the SiO2 microspheres synthesized in Example 1 and add them to a round-bottom flask. Add 0.5 μl of ATPMS and stir magnetically for 2 hours in the dark.

[0078] (3) Bathe in a 65℃ water bath for 30 minutes.

[0079] (4) Clean with anhydrous ethanol 3 times, 12800RPM, 5 minutes.

[0080] (5) Add hydroquinone (4 mg / ml) and stir magnetically in the dark for 2 hours at 400 μL.

[0081] (6) Wash with anhydrous ethanol, centrifuge 3 times at 12800RPM for 5 min, and resuspend in 2 mL of anhydrous ethanol.

[0082] (7) Add 4uL of TEOS and react overnight for 15 hours. Wash with anhydrous ethanol and centrifuge once at 12800RPM for 5 min.

[0083] Repeat steps (2)-(7) 4 times.

[0084] (8) Take 2 mL of silica microspheres and add them to a round-bottom flask. Add 6 μL of ATPMS and stir magnetically for 2 h in the dark.

[0085] (9) Bath in 65℃ water for 5 minutes, wash 3 times with anhydrous ethanol, 12800RPM, 5 minutes, and resuspend in 2mL of anhydrous ethanol.

[0086] Example 3: Synthesis of SiO2-HQ-Ag1 microspheres, namely, SiO2 microspheres coated with HQ containing hepatitis B surface antibody (HBsAb)-specific antigen Ag1.

[0087] Take 208.3 μL of SiO2-HQ microspheres (12 mg / mL) into a centrifuge tube. Add 100 μL of 2 mg / mL glutaraldehyde to the microsphere solution and react at 1300 RPM for 2 h at room temperature. After the reaction, wash five times with 10 mM PBS (pH 7.4) and dialyze overnight; centrifuge, add 76.39 μL of 10 mM PBS (pH 7.2) buffer solution, and sonicate.

[0088] Add 22.4 μL of hepatitis B surface antibody (HbsAb)-specific antigen Ag1 (5.0 mg / mL) to the microspheres, mix well, then add 4 μL of sodium cyanoborohydride (50 mg / mL), and react at 1300 rpm and 37℃ for 1 h in a constant temperature mixer. Then add 2 μL of TW-20 and continue the reaction for 23 h.

[0089] Add 10 μL of 150 mg / mL Gly and shake for 2 h, then add 20 μL of PBS at pH 7.2 and react for 2 h.

[0090] Add 100 μL of 10 mM PBS pH 7.4, take 80 μL and add it to the bottom A of the centrifuge tube, then freeze-dry. After freezing, store at -20℃ to prepare SiO2-HQ-Ag1 microspheres for later use.

[0091] Example 4: Synthesis of GG-SA microspheres, i.e., photosensitive microspheres coupled with streptavidin.

[0092] Add 1 mL of carboxyl-containing photosensitive microspheres to a centrifuge tube, followed by 100 μL of 50 mM MES buffer (pH 5.5) and 3.2 μL of EDC. Vortex rapidly to mix. Add 14 μL of LTW-20 (0.5%, v / v), vortex rapidly to mix, and discard the supernatant. Add 100 μL of (pH 650 mM MES) buffer, resuspend by sonication, and add 500 μg of hepatitis B surface antibody (HBsAb) antigen.

[0093] The above reaction solution was placed at 37℃ for 2 hours. 10 μL of Gly was added and rapidly vortexed to mix. 10 μL of 100 mg / mL BSA was added, mixed, and centrifuged, discarding the supernatant. 100 μL of (pH 650 mM MES) buffer solution was added, and the microspheres were sonicated to suspend them. The mixture was then vortexed to mix, and the entire mixture was added to the bottom (B) of the centrifuge tube. The tube was then lyophilized and stored at -20℃ to prepare GG-SA microspheres for later use.

[0094] Example 5: Synthesis of Bio-Ag2, i.e., biotinylated hepatitis B surface antibody (HBsAb) antigen.

[0095] Add 36.3 μL of 10 mM PBS pH 7.2 buffer and 11.8 μL of hepatitis B surface antibody (HbsAb) antigen to a centrifuge tube and mix well.

[0096] Add 1.8 μL of biotin, mix well, and incubate with shaking for 2 hours. Add 1 μL of Gly and react at room temperature for 5 hours. Then add all of it to the bottom C of the centrifuge tube, freeze dry, and store at 2-8℃ to prepare Bio-Ag2 for later use.

[0097] Example 6: Establishment of the Standard Curve

[0098] (1) Incubation

[0099] Add 150 μL of purified water to a centrifuge tube containing the lyophilized material prepared in Example 5 to obtain HbsAb antibody samples with different concentration gradients. Determine the concentration using a Roche instrument. Add the samples of different concentrations to centrifuge tubes (sample concentrations of 0, 5.5, 11.71, 19.19, 38.33, and 53.55 mIU / mL, respectively), vortex to mix, and incubate at 37°C for 10 min. The target antibody analyte, hepatitis B surface antibody (HbsAb, Ab), GG-SA, Bio-Ag2, and SiO2-HQ-Ag1 form a sandwich (GG-SA-Bio-Ag2-Ab-Ag1-HQ-SiO2).

[0100] (2) Sample loading

[0101] Following the method in CN114216949B, insert the screen-printed electrode into an electrochemical analyzer such as CHI660 or a portable electrochemical detector. Use a dropper to draw an appropriate amount of the reaction solution and add it to the working surface of the screen-printed electrode. The solution needs to cover all three electrodes to ensure circuit continuity.

[0102] (3) Background signal test

[0103] After sample loading, the electrochemical analyzer was run using the differential pulse voltammetry program. The program parameters were: (start potential: 0.05V, end potential: -0.3V, potential increment: 0.005V, amplitude: 0.06V, pulse width: 0.07s, sampling width: 0.02s, pulse period: 0.15s). The target analyte was the reduction peak signal of the product (BQ) after oxidation by an oxygen compound (HQ). The test results were automatically recorded by the instrument. Figure 2 The figure shown is a typical test result graph, which is the reduction peak of BQ at -0.15V. The reaction that occurs at this time is BQ + e- → HQ.

[0104] (4) Excitation signal test

[0105] The electrode surface is excited by light with a wavelength of 680 nm for 2 minutes. The photosensitive sphere is excited and energy transfer produces singlet oxygen. The singlet oxygen reacts with oxygenated compounds (HQ) in the diffusion range, continuously oxidizing HQ to BQ.

[0106] (5) Test excitation signal

[0107] Subtracting the background signal from the excitation signal yields the sample electrical signal. Plotting the sample electrical signal against the standard concentration yields the standard curve for hepatitis B surface antibody (HbsAb).

[0108] Example 7, Sample Testing

[0109] (1) Synthesis: The synthesis of reagents in the establishment of the reference standard curve.

[0110] (2) Incubation: Add 150 μL of purified water to the centrifuge tube containing the above-mentioned lyophilized material, add 10 μL of antibody sample to the reaction vessel, vortex to mix, incubate at 37°C for 10 min, and then test. The target antibody analytes, hepatitis B surface antibody (HBsAb), GG-SA, Bio-Ag2, and SiO2-HQ-Ag1, form a sandwich (GG-SA-Bio-Ag2-Ab-Ag1-HQ-SiO2).

[0111] (3) Sample loading: Insert the screen-printed electrode into an electrochemical analyzer such as CHI660 or a portable electrochemical detector. Use a dropper to draw an appropriate amount of the reaction solution and add it to the working surface of the screen-printed electrode. The solution needs to cover all three electrodes to ensure the circuit is conductive.

[0112] (4) Background signal test: After sample loading, the electrochemical analyzer was run using the differential pulse voltammetry program with the following parameters: (starting potential: 0.05V, ending potential: -0.3V, potential increment: 0.005V, amplitude: 0.06V, pulse width: 0.07s, sampling width: 0.02s, pulse period: 0.15s). The target analyte was the reduction peak signal of the product (BQ) after oxidation by an oxygen compound (HQ). The test results were automatically recorded by the instrument.

[0113] (5) Excitation signal test: The electrode surface is excited using light with a wavelength of 680 nm for 2 minutes. The photosphere is excited and energy transfer produces singlet oxygen. The singlet oxygen reacts with the oxygenated compound (HQ) within the diffusion range, continuously oxidizing HQ to BQ. At this time, the differential pulse voltammetry program with the parameters described in step (4) is run to test the excitation signal. Figure 3 The image shows the gradient of light-excited signals at different concentrations.

[0114] (6) Subtracting the background signal from the excitation signal yields the sample's electrical signal. The concentration of hepatitis B surface antibody (HBsAb) in the sample can then be obtained from the standard curve. The relationship between its concentration and the current gradient is as follows: Figure 4 As shown, the correlation coefficient R 2 =0.9976.

[0115] Example 8, Performance Evaluation

[0116] The test protocol described in Example 7 was used to test 20 clinical samples. Simultaneously, a Roche instrument (Roche E11) was used to test these samples. The Roche instrument test values ​​were plotted on the X-axis, and the test results of this method were plotted on the Y-axis to calculate the correlation. The results are as follows: Figure 5 As shown, the test results indicate that the method of this invention has a good correlation with the test results of Roche instruments, with a correlation coefficient R. 2 =0.9841.

[0117] Taking the hepatitis B surface antibody (HBsAb) project as an example, the precision of mild and moderate inflammation samples at two medical decision levels was tested. The two samples were tested 10 times consecutively using this method, and the precision CV was calculated.

[0118] Table 1 Precision Evaluation

[0119]

[0120]

[0121] As shown in Table 1, the precision CV of the kit of the present invention for testing three concentrations of hepatitis B surface antibody (HbsAb) was less than 10%, indicating that the kit of the present invention has good precision.

[0122] This embodiment uses the it current curve (constant initial voltage, measuring the change of current over time) as the testing and detection method, but it is not limited to this method. Similar methods such as differential pulse voltammetry, square wave pulse method (SMV), or other commonly used electrochemical detection methods can also be used in the electrochemical detection method of this invention.

[0123] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A complete set of reagents for detecting a target analyte, characterized in that, The target analyte is a target antibody analyte, which is a polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment; The kit of reagents contains a first structural substance and a second structural substance capable of binding to the target antibody analyte; The first structural material is coupled with a photosensitive compound that can be activated under preset conditions, and the activated photosensitive compound can generate reactive oxygen species. The second structural material is coupled with an oxygen-receiving compound that can accept reactive oxygen species to generate a preset electrical signal; The first and second structural substances are not simultaneously one of the following: antigen, hapten, polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment; The target antibody analyte specifically binds to at least one of the first structural substance and the second structural substance; When the first and second structural substances come into contact with the target antibody analyte, the activated photosensitive compound transfers reactive oxygen species to the oxygen-receiving compound, which generates an electrical signal. The information in the electrical signal is used to characterize the presence and / or quantity of the target antibody analyte.

2. The complete set of reagents according to claim 1, wherein, The first and second structural substances are antigens, and the antigens are capable of specifically binding to polyclonal antibodies, monoclonal antibodies, ScFv, or antibody fragments. When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound to generate an electrical signal.

3. The kit of reagents according to claim 1, wherein the first structural substance and the second structural substance are haptens, and the haptens are capable of specifically binding to polyclonal antibodies, monoclonal antibodies, ScFv or antibody fragments; When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound to generate an electrical signal.

4. The reagent kit according to claim 2 or 3, wherein the first structural substance is not directly coupled to the photosensitive compound, and the reagent kit further comprises a third structural substance coupled to the photosensitive compound; when the third structural substance comes into contact with the first structural substance, the third structural substance undergoes acceptor-ligand affinity adsorption with the first structural substance; the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygen-receiving compound to generate an electrical signal.

5. The reagent kit according to claim 3 or 4, wherein the intensity of the electrical signal is positively correlated with the quantity of the target antibody analyte.

6. The kit of reagents according to claim 1, wherein the second structural substance is an antigen, the first structural substance is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment, and the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen, wherein: When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the target antibody analyte competitively binds to the second structural substance with the first structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated compound of the second structural substance to generate an electrical signal.

7. The kit of reagents according to claim 1, wherein the second structural substance is a hapten, the first structural substance is a polyclonal antibody, a monoclonal antibody, ScFv, or an antibody fragment, and the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the hapten, wherein: When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the target antibody analyte competitively binds to the second structural substance with the first structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated compound of the second structural substance to generate an electrical signal.

8. The complete set of reagents according to claim 1, wherein, The first structural substance is an antigen, and the second structural substance is a polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment. The polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen, wherein: When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the target antibody analyte and the second structural substance competitively bind to the first structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated compound of the second structural substance to generate an electrical signal.

9. The reagent kit according to claim 1, wherein, The first structural substance is a hapten, and the second structural substance is a polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment. The polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the hapten, wherein: When the target antibody analyte comes into contact with the first structural substance and the second structural substance, the target antibody analyte and the second structural substance competitively bind to the first structural substance. The photosensitive compound on the first structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated compound of the second structural substance to generate an electrical signal.

10. The complete set of reagents according to claim 1, wherein, The second structural substance is an antigen, and the first structural substance is not directly coupled to the photosensitive compound; The kit of reagents also includes a third structural substance conjugated to the photosensitive compound. The third structural substance is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment. The third structural substance is capable of receptor-ligand affinity adsorption with the first structural substance. The polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen. When the target antibody analyte comes into contact with the second and third structural substances, the target antibody analyte competitively binds to the second structural substance with the third structural substance. The photosensitive compound on the third structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated compound of the second structural substance to generate an electrical signal.

11. The reagent kit according to claim 1, wherein, The second structural substance is a hapten, and the first structural substance is not directly coupled to the photosensitive compound; The kit of reagents also includes a third structural substance conjugated to the photosensitive compound. The third structural substance is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment. The third structural substance is capable of receptor-ligand affinity adsorption with the first structural substance. The polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the hapten. When the target antibody analyte comes into contact with the second and third structural substances, the target antibody analyte competitively binds to the second structural substance with the third structural substance. The photosensitive compound on the third structural substance is excited by light of a certain wavelength to generate an oxygenated compound, which reacts with the oxygenated compound of the second structural substance to generate an electrical signal.

12. The kit of reagents according to any one of claims 6 to 9, wherein the intensity of the electrical signal is negatively correlated with the quantity of the target antibody analyte.

13. A method for detecting a target antibody analyte, comprising: The sample to be tested is incubated with the reagent kit according to any one of claims 1 to 12. The background electrical signal of the incubated sample was detected using electrodes; The photosensitive compound of the kit of reagents is excited by light of a specific wavelength, and the electrical signal after excitation is detected. as well as By comparing the background electrical signal and the excited electrical signal, sample electrical signal information is obtained, and the presence and / or quantity of the target antibody analyte is determined.

14. An instrument system for detecting a target antibody analyte, comprising: A liquid reaction module is configured to incubate the sample to be tested and the kit of reagents according to any one of claims 1 to 12; The electrode module is configured to contact the liquid in the liquid reaction module and detect electrical signals; An optical module is configured to emit light of a certain wavelength into the liquid reaction module; as well as The processor is configured to receive and process signals from the electrode module to determine the presence and / or quantity of the target antibody analyte. The liquid reaction module is a container capable of holding liquid, and the container is a cuvette, a cuvette array, or a perforated plate; The liquid reaction module is a test strip coated with the complete set of reagents according to any one of claims 1 to 12.

15. A target antibody analyte detection system, comprising: Electrodes that adsorb or couple immune molecules; Using the kit of reagents according to any one of claims 1 to 12, the kit of reagents includes photosensitive microspheres and oxygen-receiving compound reagents, wherein the photosensitive microspheres contain photosensitive compounds, and the photosensitive compounds generate active oxygen substances that can react with oxygen-receiving compound reagents under light excitation of a certain wavelength, thereby generating an electrical signal that can be detected by the electrode; The photosensitive microspheres also contain binding ligands.

16. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, an ScFv or an antibody fragment, the binding ligand is an antigen, the immune molecule adsorbed or coupled to the electrode is an antibody, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target antibody analyte comes into contact with the photosensitive microsphere and the electrode, the target antibody analyte and the antibody on the electrode competitively bind to the photosensitive microsphere, and the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

17. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, an ScFv or an antibody fragment, the binding ligand is a hapten, the immune molecule adsorbed or coupled by the electrode is an antibody, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the hapten; When the target antibody analyte comes into contact with the photosensitive microsphere and the electrode, the target antibody analyte and the antibody on the electrode competitively bind to the photosensitive microsphere, and the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

18. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, an ScFv or an antibody fragment, the binding ligand is an antigen, the immune molecule adsorbed or coupled by the electrode is an antigen, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the antigen; When the target antibody analyte comes into contact with the electrode and the photosensitive microsphere, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

19. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, an ScFv or an antibody fragment, the binding ligand is a hapten, the immune molecule adsorbed or coupled by the electrode is an antigen, and the polyclonal antibody, monoclonal antibody, ScFv or antibody fragment immunizes the hapten or antigen. When the target antibody analyte comes into contact with the electrode and the photosensitive microsphere, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

20. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment, the immune molecule adsorbed or coupled by the electrode is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment, the detection system further includes a receptor-ligand affinity-labeled antigen, wherein the receptor-ligand affinity-labeled antigen can undergo affinity adsorption with the photosensitive microspheres, and the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen; When the target antibody analyte comes into contact with the photosensitive microspheres and the electrode, the target antibody analyte and the antibody on the electrode competitively bind to the receptor-ligand affinity-adsorbed antigen, and the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

21. The detection system according to claim 15, wherein the target antibody analyte is a polyclonal antibody, a monoclonal antibody, an ScFv, or an antibody fragment, the electrode adsorbs or conjugates an immunomolecular antigen, the detection system further includes a receptor-ligand affinity-labeled antigen, the receptor-ligand affinity-labeled antibody being able to undergo affinity adsorption with the photosensitive microspheres, and the polyclonal antibody, monoclonal antibody, ScFv, or antibody fragment immunizes the antigen; When the target antibody analyte comes into contact with the electrode and the receptor-ligand affinity-adsorbed antigen, the photosensitive compound is excited by light of a certain wavelength and reacts with the oxygenated compound reagent.

22. A method for detecting a target antibody analyte, comprising: Using the detection system according to any one of claims 15 to 21, incubate with the sample to be tested; The background electrical signal of the incubated sample was detected using electrodes; The photosensitive compound is excited using light of a specific wavelength, and the electrical signal after excitation is detected. as well as By comparing the background electrical signal and the excited electrical signal, sample electrical signal information is obtained, and the presence and / or quantity of the target antibody analyte is determined.

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