Full-automatic immunodetection method for amplifying signal, detection reagent and sample analyzer

Abstract

The invention provides a full-automatic immunodetection method for amplifying a signal, which comprises the following steps: controlling a pipetting device to transfer a sample to be detected, a reagent 1 containing a solid phase carrier coated with a first ligand, a reagent 2 containing a second ligand labeled with biotin and a reagent 3 containing streptavidin labeled with a signal substance into a reaction container; a second compound is formed, and the second compound is a solid phase carrier-first ligand-object to be detected-second ligand-biotin-streptavidin-signal substance compound; the first ligand and the second ligand are specifically combined with different structural domains of an object to be detected; in the second compound, the molar ratio of the biotin to the second ligand is greater than or equal to 2: 1, the molar ratio of the signal substance to the streptavidin is greater than or equal to 2: 1, and the molar ratio of the streptavidin to the second ligand is greater than or equal to 1: 1. According to the detection method, cascade amplification of detection signals is achieved, the detection sensitivity is greatly improved, and accurate detection of the to-be-detected substance with extremely low concentration is achieved.

Description

Technical Field

[0001] This invention relates to the field of immunoassay, and more specifically, to a fully automated immunoassay method that amplifies signals. Background Technology

[0002] Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive cognitive decline, including memory impairment, disorientation, language impairment, and behavioral abnormalities. It primarily occurs in the elderly or pre-elderly and is a leading cause of dementia in people aged 65 and older. With the increasing aging of the population, the number of patients is constantly rising, further increasing the socioeconomic costs.

[0003] The core pathological features of Alzheimer's disease (AD) are neurofibrillary tangles, neuroinflammation, and senile plaques formed by the deposition of amyloid β-protein (Aβ) due to abnormally phosphorylated Tau protein. The formation of these plaques affects signal transmission between nerve cells, leading to nerve cell death and ultimately brain atrophy. Currently, there is no effective treatment for AD, and treatment is usually initiated in the late stages of the disease. However, changes in related proteins at the molecular level occur far earlier than the appearance of neurodegenerative symptoms; therefore, early diagnosis of AD is crucial for delaying disease progression.

[0004] According to the consensus of Alzheimer's disease experts, proteins such as β-amyloid and p-tau protein are currently key biomarkers for AD diagnosis. They can be accurately detected in cerebrospinal fluid samples from AD patients. However, collecting cerebrospinal fluid samples is an invasive technique, carrying certain risks and incurring high costs. Blood samples are safer, but due to the presence of the blood-brain barrier, the concentration of these AD biomarkers in blood is extremely low, typically at the pg / mL level. This results in insufficient sensitivity and low detection signals in traditional chemiluminescent immunoassay methods, limiting their application in AD diagnosis.

[0005] Therefore, there is an urgent need to develop a method to improve detection sensitivity and achieve accurate detection of extremely low concentrations of analytes in samples, thereby supporting clinical diagnostic needs. Summary of the Invention

[0006] The main objective of this invention is to provide a fully automated immunoassay method that amplifies signals, thereby solving the problem that existing technologies cannot accurately detect extremely low concentrations of analytes in blood.

[0007] To achieve the above objectives, according to a first aspect of the present invention, a fully automated immunoassay method for amplifying signals is provided, comprising controlling a pipette to transfer a sample to be tested, a reagent 1 containing a solid-phase carrier coated with a first ligand, a reagent 2 containing a biotin-labeled second ligand, and a reagent 3 containing streptavidin labeled a signal substance into a reaction vessel to form a second complex, wherein the second complex is a solid-phase carrier-first ligand-analyte-second ligand-biotin-streptavidin-signal substance complex; the first ligand and the second ligand specifically bind to different structural domains of the analyte; in the second complex, the molar ratio of biotin to the second ligand is ≥2:1, the molar ratio of the signal substance to streptavidin is ≥2:1, and the molar ratio of streptavidin to the second ligand is ≥1:1.

[0008] According to another aspect of the present invention, an immunoassay reagent for amplifying a signal is provided, comprising reagent 1, reagent 2, and reagent 3. Reagent 1 comprises a solid-phase carrier coated with a first ligand that specifically binds to an analyte; reagent 2 comprises a second ligand labeled with biotin that specifically binds to an analyte; reagent 3 comprises streptavidin labeled with a signal substance, wherein the molar ratio of biotin to the second ligand is ≥2:1, the molar ratio of the signal substance to the streptavidin is ≥2:1, and the molecular weight of the streptavidin is 50-200 kD; optionally, the molecular weight of the streptavidin is 110-200 kD; optionally, the molecular weight of the signal substance is less than 1000 kD.

[0009] According to another aspect of the present invention, a sample analyzer is provided, characterized in that it comprises: a pipetting device for transferring a sample or reagent into a reaction vessel; a transfer device for transferring a reaction vessel; a detection device for detecting the sample to be tested and obtaining detection results; a controller for controlling the pipetting device to transfer the sample to be tested, reagent 1 containing a solid-phase carrier coated with a first ligand, reagent 2 containing a biotin-labeled second ligand, and reagent 3 containing a streptavidin-labeled signal substance into the reaction vessel to form a second complex, the second complex being a solid-phase carrier-first ligand-analyte-second ligand-biotin-streptavidin-signal substance complex; and controlling the transfer device to transfer the reaction vessel into the detection device, determining the content of the analyte in the sample by detecting the amount of the second complex; the first ligand and the second ligand specifically bind to different structural domains of the analyte; in the second complex, the molar ratio of biotin to the second ligand is ≥2:1, the molar ratio of the signal substance to streptavidin is ≥2:1, and the molar ratio of streptavidin to the second ligand is ≥1:1. Further, the method for controlling a pipette to transfer the test sample, reagent 1 containing a solid support coated with a first ligand, reagent 2 containing a biotin-labeled second ligand, and reagent 3 containing streptavidin containing a labeled signal substance into a reaction vessel to form a second complex includes: Step 1: Controlling a pipette to transfer the test sample, reagent 1 containing a solid support coated with a first ligand, and reagent 2 containing a biotin-labeled second ligand into a reaction vessel to form a first reaction solution containing the first complex; Step 2: Controlling a pipette to transfer reagent 3 containing streptavidin containing the labeled signal substance into a reaction vessel containing the first complex, forming a second reaction solution containing the second complex; Step 3: Controlling a transfer device to transfer the reaction vessel containing the second complex into a detection device, and determining the content of the analyte in the sample by detecting the amount of the second complex. Further, the molecular weight of streptavidin is 50-200 kDa, further, the molecular weight of streptavidin is 110-200 kDa, and further, the molecular weight of the signal substance is less than 1000 kDa.

[0010] Applying the technical solution of this invention, in the aforementioned fully automated immunoassay method for amplified signals, a solid-phase carrier is coated with a first ligand that specifically binds to the analyte, biotin is labeled on a second ligand that specifically binds to the analyte, and streptavidin is labeled with a signal substance, ultimately forming a complex of solid-phase carrier-first ligand-analyte-second ligand-biotin-streptavidin-signal substance. Specifically, the molar ratio of biotin to the second ligand is set to ≥2:1; the molar ratio of the signal substance to streptavidin is ≥2:1; and the molar ratio of streptavidin to the second ligand is ≥1:1. The detection method of this invention achieves cascaded amplification of the detection signal, greatly improving detection sensitivity and enabling accurate detection of extremely low concentrations of the analyte in the sample. Detailed Implementation

[0011] It should be noted that, unless otherwise specified, the embodiments and features described in the embodiments of this invention can be combined with each other. The invention will now be described in detail with reference to the embodiments.

[0012] Terminology Explanation:

[0013] Biotin: A water-soluble vitamin (vitamin B7) with a small molecular weight (244 Da). It can be covalently bound to antibodies, enzymes or other proteins through chemical modification (such as amino activation) without affecting their biological activity.

[0014] Streptavidin (SA): A tetrameric glycoprotein derived from *Streptomyces avermitilis*, with a molecular weight of approximately 60 kDa. It exhibits extremely high affinity for the small-molecule vitamin biotin, with a binding constant reaching 10. - 15 M-level interactions are among the strongest non-covalent interactions in nature, and their binding is almost irreversible. Each streptavidin molecule can specifically bind four biotin molecules.

[0015] Feed ratio and labeling ratio: These are two closely related data parameters. Taking the labeling reaction of a signal substance with streptavidin as an example: the feed ratio refers to the molar ratio of signal substance to streptavidin used in the preparation of the signal substance-streptavidin conjugate; it is a preset process control parameter. The labeling ratio, on the other hand, refers to the molar ratio of signal substance to streptavidin in the final signal substance-streptavidin conjugate; it is a result characterization parameter. In a defined reaction system, by optimizing and controlling the feed ratio, conjugates with specific labeling ratios can be stably and reproducibly prepared.

[0016] As mentioned in the background section, in the field of chemiluminescence detection, some analytes have low concentrations in the blood, especially some Alzheimer's disease biomarkers. Due to the presence of the blood-brain barrier, the concentration of such biomarkers in the blood is usually at the pg / mL or even fg / mL level. Therefore, existing technologies usually collect cerebrospinal fluid samples for the detection of Alzheimer's disease biomarkers. However, the method of collecting cerebrospinal fluid samples is an invasive technique, which is not only costly but also carries certain risks. Therefore, there is an urgent need to develop a fully automated immunoassay method that amplifies the signal. Based on this, the present invention proposes a series of methods for the accurate detection of analytes at extremely low concentration levels in samples.

[0017] In a first typical embodiment of the present invention, a fully automated immunoassay method for amplifying signals is provided. The assay method is executed by a controller and includes controlling a pipette to transfer the sample to be tested, reagent 1 containing a solid-phase carrier coated with a first ligand, reagent 2 containing a biotin-labeled second ligand, and reagent 3 containing streptavidin labeled a signal substance into a reaction vessel to form a second complex. The second complex is a solid-phase carrier-first ligand-analyte-second ligand-biotin-streptavidin-signal substance complex. The first ligand and the second ligand specifically bind to different structural domains of the analyte. In the second complex, the molar ratio of biotin to the second ligand is ≥2:1, the molar ratio of the signal substance to streptavidin is ≥2:1, and the molar ratio of streptavidin to the second ligand is ≥1:1.

[0018] The first and second ligands in this article are proteins that specifically bind to different domains of the analyte. In a preferred embodiment, the first and second ligands are antibodies. In one embodiment of the present invention, the first ligand is antibody 1 and the second ligand is antibody 2.

[0019] In the above immunological detection method, the pipette is controlled to transfer reagent 1, reagent 2 and reagent 3 to the reaction vessel for reaction and to form a second complex. In the second complex, since the molar ratio of biotin to the second ligand is ≥2:1, the molar ratio of the signal substance and streptavidin is ≥2:1. At the same time, after the reagents react, the molar ratio of streptavidin to the second ligand is ≥1:1.

[0020] The detection method of this invention enables cascaded amplification of the detection signal. Specifically, a single second ligand of the analyte binds to two or more molecules of biotin; furthermore, each biotin binds to streptavidin carrying two or more signal molecules. This dual amplification mechanism ultimately transforms a single analyte molecule into a complex of multiple signal molecules. On the one hand, the detection method of this invention significantly multiplies the signal intensity through cascade reaction amplification, greatly improving detection sensitivity and enabling accurate detection of extremely low concentrations of analytes. On the other hand, because this process is based on specific coupling relationships and a clear molecular quantity relationship, the amplification factor triggered by each analyte molecule is more uniform, thus ensuring good repeatability of the detection results.

[0021] In a preferred embodiment, a pipetting device is controlled to transfer the sample to be tested, reagent 1 containing a solid support coated with a first ligand, reagent 2 containing a biotin-labeled second ligand, and reagent 3 containing streptavidin containing a labeled signal substance into a reaction vessel to form a second complex, comprising: Step 1: Control the pipette to transfer the sample to be tested, reagent 1 containing a solid support coated with the first ligand, and reagent 2 containing a biotin-labeled second ligand into the reaction vessel to form a first reaction solution containing the first complex; Step 2: Control the pipette to transfer reagent 3 containing streptavidin labeled with a signaling substance to a reaction vessel containing the first complex; forming a second reaction solution containing the second complex; Step 3: Control the transfer mechanism to transfer the reaction vessel containing the second complex to the detection device, and determine the content of the analyte in the sample by detecting the amount of the second complex.

[0022] In a preferred embodiment, the molecular weight of streptavidin is 50-200 kDa.

[0023] In a more preferred embodiment, the molecular weight of streptavidin is 110-200 kD. Based on the above detection method, by modifying streptavidin into polystreptavidin, which has a larger molecular weight, each polystreptavidin molecule can carry significantly more signal substances than the monomer. Therefore, on the basis of existing cascade amplification, the luminescence intensity can be multiplied, and the detection sensitivity can be greatly improved to meet the detection requirements of extremely low concentration analytes.

[0024] Polystreptavidin can be a polymer formed by combining two or more streptavidin monomers through chemical cross-linking, or it can be prepared by fusion expression using genetic engineering techniques to obtain streptavidin with higher molecular weight.

[0025] In a more preferred embodiment, streptavidin is an octamer or a dodecamer.

[0026] In a preferred embodiment, the molecular weight of the signaling substance is less than 1000D.

[0027] Based on the use of streptavidin or polystreptavidin, further selection is made to use signal substances with a molecular weight of less than 1000D for streptavidin labeling, so as to achieve the effect of labeling more signal substances, thereby further amplifying the detection signal and improving the detection sensitivity. In a more preferred embodiment, the signal substance is selected from chemiluminescent labels, and further selected from the following compounds: isoluminol, ABEI, AHEI, ABEN, ABAH, HEMA, glossin, acridine ester, ruthenium terpyridine, and derivatives containing the basic skeleton of the above compounds.

[0028] In a preferred embodiment, the labeling molar ratio of the signal substance and streptavidin in reagent 3 is 5:1-12:1, and the feed ratio is 10:1-20:1. This labeling ratio allows streptavidin to carry more signal substance, while the nonspecificity of detection is relatively low, resulting in optimal detection light intensity and sensitivity.

[0029] In a more preferred embodiment, the labeling molar ratio of the signaling substance and streptavidin is 9:1, and the feed ratio is 15:1.

[0030] In a preferred embodiment, the second ligand in reagent 2 is a full-length antibody.

[0031] Full-length antibodies have more primary amino groups than Fab or F(ab')2, enabling them to carry more biotin. This gives the antibodies a higher probability of binding to streptavidin labeled with the signaling substance, thereby improving detection light intensity and sensitivity.

[0032] Meanwhile, when the second ligand is a full-length antibody and streptavidin is polystreptavidin, based on the reaction principle and experimental data, the inventors believe that during the reaction, streptavidin has difficulty binding to multiple biotins labeled on different antibodies, resulting in one molecule of streptavidin binding to multiple antibodies, thus affecting the detection results. This is because the molecular weight of streptavidin and the full-length antibody is relatively large during the reaction, and there is a certain steric hindrance after binding. Once streptavidin binds to a biotin labeled on one antibody, it is difficult for other biotin sites on other antibodies to be bound. Even if streptavidin binds to biotin labeled on other antibodies, the formation of a solid-carrier-first ligand-analyte-second ligand-biotin-streptavidin (-biotin-second ligand-analyte-first ligand-solid-carrier)-signal complex is difficult due to the large steric hindrance of the solid-phase carrier. The excellent reproducibility in the experimental data of this invention also indicates that if streptavidin could bind multiple biotin, the reaction might become uncontrollable, leading to poor reproducibility. Therefore, in the second complex, streptavidin binds only one molecule of biotin.

[0033] In a preferred embodiment, the molar ratio of biotin to the second ligand in reagent 2 is 14:1-28:1, and the feeding ratio is 20:1-40:1. Within this molar ratio range, the detection light intensity and sensitivity can be effectively improved.

[0034] In a more preferred embodiment, the biotin to second ligand labeling ratio in reagent 2 is 21:1, and the feed ratio is 30:1. Under this molar ratio condition, with minimal impact on antibody activity, more streptavidin labeled with the signal substance can bind to the first complex, thereby improving the detection light intensity and sensitivity.

[0035] In the second complex, the specific coupling and quantitative relationships between the components are crucial for achieving efficient signal amplification. Specifically, a molar ratio of biotin to the second ligand ≥ 2:1 ensures that each second ligand molecule can bind multiple biotin molecules, laying the foundation for subsequent cascade amplification; a molar ratio of the signal substance to streptavidin ≥ 2:1 ensures that each streptavidin molecule can carry a sufficient number of signal units, thereby significantly enhancing the output signal. In this application, when using octamer streptavidin, based on the reaction principle, the inventors hypothesize that, due to its similar molecular weight and spatial size to the second ligand, their binding is limited by steric hindrance, making it difficult for a single second ligand to simultaneously bind multiple streptavidin molecules. The actual binding ratio of streptavidin to the second ligand is typically 1:1 or 2:1.

[0036] In the above detection method, reagent 3 is added in saturated or excessive amounts. Those skilled in the art can add sufficient or excessive amounts of reagent 3 according to the upper limit of the concentration or the linear range of the analyte to ensure that the second complex in the embodiments of this application can be formed for each molecule of the analyte.

[0037] In a preferred embodiment, controlling the pipetting device to transfer reagent 3 containing streptavidin with a labeled signal substance to a reaction vessel containing the first complex includes: controlling the pipetting device to directly transfer reagent 3 containing streptavidin with a labeled signal substance to a reaction vessel containing the first reaction solution.

[0038] After the first reaction solution has completed incubation, reagent 3 is added directly to the first reaction solution without washing or separation steps to initiate subsequent reactions, ensuring maximum utilization of the reaction components. Since no washing is required during the process, all incompletely bound components from the first reaction stage are completely retained within the system and can continue to participate in subsequent cascade reactions initiated by reagent 3. This avoids the loss of reaction components caused by washing steps, and is particularly effective for low-abundance analytes, preventing further attenuation of the detection signal, significantly reducing the loss of effective signal, and further ensuring signal amplification and detection sensitivity.

[0039] In a preferred embodiment, at least one of reagents 1 and 2 further includes polyvinylpyrrolidone (PVP). Since the detection method of this application employs a signal cascade approach, the complex reaction components involved can easily lead to non-specific adsorption. Furthermore, when streptavidin is a polymer, its large molecular weight further increases the risk of non-specific binding. Therefore, the inventors creatively added PPVP to the reagents, effectively eliminating non-specific binding in the reaction. In a more preferred embodiment, PPVP is added to reagent 2, allowing it to participate in the reaction system at the initial stage, forming a steric hindrance layer. This effectively inhibits the non-specific adsorption between the antibody-biotin conjugate and the solid-phase support in reagent 2. Simultaneously, this method avoids the potential impact on the solid-phase interface properties and the activity of the first ligand that might occur during storage when PPVP is added to reagent 1. Thus, while fully utilizing its function of inhibiting non-specific binding, the long-term stability of reagent 1 is ensured.

[0040] In a more preferred embodiment, the concentration of polyvinylpyrrolidone added is 0.2%-0.6% (w / v), and the molecular weight is 8000D-58000D.

[0041] In a preferred embodiment, at least one of reagents 1 and 2 further includes sodium polyacrylate. Since the addition of polyvinylpyrrolidone leads to a decrease in detection light intensity, the inventors unexpectedly discovered that the detection light intensity was improved to a certain extent after further adding sodium polyacrylate to reagent 1 or reagent 2. The inventors speculate that polyvinylpyrrolidone and sodium polyacrylate have complementary effects, and their synergistic effect can ensure that the detection light intensity is not affected while reducing nonspecificity. This not only improves the signal-to-noise ratio of the detection, but also ensures a high detection light intensity and detection sensitivity. In a preferred embodiment, polyvinylpyrrolidone is added to reagent 2.

[0042] In one preferred embodiment, the sodium polyacrylate has a molecular weight of 4000-5000D and an addition concentration of 0.2%-0.6% (w / v); in another preferred embodiment, the sodium polyacrylate has a molecular weight of 4500D.

[0043] In a preferred embodiment, the concentration of the analyte is 0.05 pg / mL to 2000 pg / mL, and more preferably, the concentration of the analyte is 0.2 to 1000 pg / mL; the extremely low concentration of the analyte in this invention refers to a sample with a concentration of 50 fg / mL to 2 ng / mL in blood.

[0044] Furthermore, the test items are Alzheimer's disease biomarkers, including Aβ1-42, Aβ1-40, p-tau-181, p-tau-231, p-tau-271, and GFAP.

[0045] In a second typical embodiment of the present invention, an immunoassay reagent for amplifying a signal is provided, comprising reagent 1, reagent 2, and reagent 3. Reagent 1 comprises a solid-phase carrier coated with a first ligand that specifically binds to an analyte; reagent 2 comprises a second ligand labeled with biotin that specifically binds to an analyte; reagent 3 comprises streptavidin labeled with a signal substance; the molar ratio of biotin to the second ligand is ≥2:1, the molar ratio of the signal substance to the streptavidin is ≥2:1, and the molecular weight of the streptavidin is 50-200 kD; in a preferred embodiment, the molecular weight of the streptavidin is 110-200 kD; in a preferred embodiment, the molecular weight of the signal substance is less than 1000 kD.

[0046] In a third typical embodiment of the present invention, a sample analyzer is provided, comprising a pipetting device for transferring a sample or reagent into a reaction vessel; a transfer device for transferring a reaction vessel; a detection device for detecting the sample to be tested and obtaining detection results; and a controller for controlling the pipetting device to transfer the sample to be tested, reagent 1 containing a solid-phase carrier coated with a first ligand, reagent 2 containing a biotin-labeled second ligand, and reagent 3 containing a streptavidin-labeled signal substance into the reaction vessel to form a second complex, wherein the second complex is a solid-phase carrier-first ligand-analyte-second ligand-biotin-streptavidin-signal substance complex; and controlling the transfer device to transfer the reaction vessel into the detection device, determining the content of the analyte in the sample by detecting the amount of the second complex; wherein the first ligand and the second ligand specifically bind to different structural domains of the analyte; wherein in the second complex, the molar ratio of biotin to the second ligand is ≥2:1, the molar ratio of the signal substance to streptavidin is ≥2:1, and the molar ratio of streptavidin to the second ligand is ≥1:1.

[0047] In a preferred embodiment, controlling a pipette to transfer a sample to be tested, reagent 1 containing a solid support coated with a first ligand, reagent 2 containing a biotin-labeled second ligand, and reagent 3 containing streptavidin containing a labeled signal substance into a reaction vessel to form a second complex includes: Step 1: controlling a pipette to transfer a sample to be tested, reagent 1 containing a solid support coated with a first ligand, and reagent 2 containing a biotin-labeled second ligand into a reaction vessel to form a first reaction solution containing the first complex; Step 2: controlling a pipette to transfer reagent 3 containing streptavidin containing the labeled signal substance into a reaction vessel containing the first complex to form a second reaction solution containing the second complex; Step 3: controlling a transfer device to transfer the reaction vessel containing the second complex into a detection device, and determining the content of the analyte in the sample by detecting the amount of the second complex.

[0048] The sample analyzer of this invention enables highly efficient cascaded signal amplification. Specifically, a single second ligand bound to the analyte can capture two or more biotin molecules; furthermore, each biotin can bind to streptavidin carrying two or more signal molecules. This dual amplification mechanism ultimately transforms a single analyte molecule into a complex of multiple signal molecules. The detection method of this invention, on the one hand, significantly multiplies the signal intensity through two-stage amplification, greatly improving detection sensitivity and enabling accurate detection of low-abundance analytes; on the other hand, because this process is based on specific coupling relationships and clear chemometric relationships, the amplification factor induced by each analyte molecule is more uniform, thereby ensuring excellent repeatability of the detection results.

[0049] In a preferred embodiment, the molecular weight of streptavidin is 50-200 kD; in a more preferred embodiment, the molecular weight of streptavidin is 110-200 kD. By modifying streptavidin into polystreptavidin, which has a larger molecular weight, each polystreptavidin molecule can carry significantly more signal substances than the monomer. Therefore, based on existing cascade amplification, the luminescence intensity can be multiplied, ultimately greatly improving the detection sensitivity and meeting the detection requirements of extremely low concentrations of analytes.

[0050] In a more preferred embodiment, streptavidin is an octamer or a dodecamer.

[0051] In a preferred embodiment, the molecular weight of the signal substance is less than 1000D. Using a signal substance with a molecular weight less than 1000D for streptavidin labeling allows for the labeling of more signal substances, thereby further amplifying the detection signal and improving detection sensitivity. In a more preferred embodiment, the signal substance is selected from chemiluminescent labels, and further selected from the following compounds: isoluminol, ABEI, AHEI, ABEN, ABAH, HEMA, glossin, acridine ester, ruthenium terpyridine, and derivatives containing the basic skeleton of the above compounds.

[0052] The beneficial effects of the present invention will be explained in further detail below with reference to specific embodiments.

[0053] Example 1: Raw material sources and reagent preparation

[0054] 1. Source of reagents and instruments: Raw material source: Biotin source: purchased from Thermo Fisher, product number 21336; Streptavidin (SA) Source: Purchased from Roche, catalog number 08903727103.

[0055] Preparation process of octamer streptavidin: 1) SA desalting: Desalt SA into 100mM PBS (containing 0.15M NaCl, 2mM EDTA, pH 7.5), and dilute with the buffer to 2mg / mL for later use; 2) SA-labeled SMCC: Take half of the SA and SMCC and add them to the SMCC dissolved in DMF at a molar ratio of 1:8. Stir for 2 hours, then desalt into 100mM PBS (containing 0.15M NaCl, 2mM EDTA, pH 7.5) and dilute to 1mg / mL to obtain solution A; 3) SA-labeled Traut's Reagent: While SA-labeling SMCC, take the other half of SA and add Traut's Reagent dissolved in DMF at a 1:8 molar ratio. Stir for 2 hours, then desalt to 100 mM PBS (containing 0.15 M NaCl, 2 mM EDTA, pH 7.5) and dilute to 1 mg / mL to obtain solution B; 4) Coupling: Mix equal volumes of solution A and solution B, stir for 4 hours to form SA-SMMCC-Traut'sReagent-SA polymer.

[0056] Reagent source: Polyvinylpyrrolidone (PVP) and sodium polyacrylate were purchased from Aladdin Reagents.

[0057] 2. Reagent Preparation The basic components and detection steps for test group 1 are as follows: The basic components include: Reagent 1 (Magnetic Bead Reagent): Add PBS buffer and antibody 1 that specifically recognizes Aβ1-42. The antibody is coated on magnetic beads, with a mass ratio of magnetic beads to antibody 1 of 1 mg: 10 μg, and a magnetic bead suspension concentration of 1 mg / mL. Reagent 2 (Biotin reagent): Add Tris buffer and antibody 2 that specifically recognizes Aβ1-42, wherein antibody 2 is conjugated with biotin, the molar ratio of biotin to antibody 2 is 30:1, and the concentration of antibody 2 is 500 ng / mL; Reagent 3 (luminescent reagent): Add Tris buffer and octamer SA, wherein ABEI (signal substance) is coupled to the octamer SA. The molar ratio of ABEI to octamer SA is 15:1. The concentration of octamer SA is 500 ng / mL.

[0058] Calibrators: Calibrator solutions containing Aβ1-42 concentrations of 0 pg / mL, 4 pg / mL, 40 pg / mL, and 400 pg / mL.

[0059] The testing steps are as follows: Step A: Using a fully automated chemiluminescence immunoassay analyzer, the pipettes, controlled by the controller, add 100 μL of calibrator, 20 μL of reagent 1, and 50 μL of reagent 2 to the reaction vessel. The transfer device then transfers the reaction vessel, and the reaction solution in the vessel is incubated at 37°C for 15 min without rinsing. Step B: Using a pipette, add 50 μL of reagent 3 to the reaction vessel from step A. Transfer the reaction vessel using a transfer device and incubate the reaction solution at 37°C for 15 min. Mix well and wash. Step C: Add the substrate solution (containing NaOH and H2O2) for the fully automated immunoassay system to initiate the chemiluminescence reaction and generate a light signal. The relative light intensity (RLU) is detected using a photomultiplier tube.

[0060] The calibrators and antibodies in experimental group 1 were sourced from the Aβ1-42 assay kit (catalog number 130221002M) manufactured by Shenzhen New Industries Biomedical Engineering Co., Ltd.; the detection instrument was the Maglumi X3 fully automated chemiluminescence immunoassay analyzer manufactured by Shenzhen New Industries Biomedical Engineering Co., Ltd.

[0061] Test methods and data description:

[0062] The four-gradient calibrators were repeatedly tested 10 times using a chemiluminescence analyzer with reagent components. The average detection value was calculated, and the ratio of gradient n+1 to gradient 0 was also calculated. A lower detection value for gradient 0 indicates lower non-specific binding; higher detection values ​​for gradients 1-3 indicate higher detection light intensity; and larger ratios of gradient 1 / gradient 0, gradient 2 / gradient 0, and gradient 3 / gradient 0 indicate higher detection sensitivity.

[0063] Example 2

[0064] The basic components and detection steps for test group 1 are the same as in Example 1.

[0065] Control group 1 only replaced the octamer SA in the basic component 3 of experimental group 1 with SA.

[0066] The test results are shown in Table 1. Table 1

[0067] Conclusion: The nonspecific detection signal of ABEI-labeled SA is comparable to that of octamer SA, but the detection light intensity is significantly lower than that of ABEI-labeled octamer SA. Furthermore, the detection value ratio of different gradient analytes in experimental group 1 is significantly higher than that in control group 1, indicating that the use of octamer SA can greatly improve the detection light intensity and detection sensitivity of analytes at extremely low concentrations.

[0068] Example 3 To verify the sensitivity of coating magnetic beads with octamer SA, labeling biotin with antibody 1, labeling ABEI with antibody 2, and the sensitivity of this method, the following control group 2 was set up.

[0069] Control group 2 The basic components include: Reagent A (Magnetic Bead Reagent): Add PBS buffer and octamer SA to coat the magnetic beads with octamer SA. The ratio of magnetic beads to octamer SA is 1 mg: 20 μg, and the magnetic bead suspension concentration is 1 mg / mL. Reagent B (Biotin-labeled antibody reagent): Contains Tris buffer and antibody 1, which specifically recognizes Aβ1-42, conjugated with biotin. The molar ratio of biotin to antibody is 15:1. Antibody concentration: 500 ng / mL. Reagent C (luminescent reagent): Added Tris buffer and antibody 2 that specifically recognizes Aβ1-42, wherein the antibody is conjugated with ABEI, and the molar ratio of ABEI to antibody is 15:1. Antibody concentration: 500 ng / mL.

[0070] Calibrators: Calibrator solutions containing Aβ1-42 concentrations of 0 pg / mL, 4 pg / mL, 40 pg / mL, and 400 pg / mL.

[0071] The testing steps are as follows: Step A: Incubate 100 μL of the test sample, 50 μL of luminescent label and 50 μL of Biotin-labeled antibody reagent at 37°C for 15 min without washing; Step B: Add 20 μL of magnetic beads to the system and incubate at 37°C for 15 min, mix well, and wash. Step C: Add the substrate solution (containing NaOH and H2O2) for the fully automated immunoassay system to initiate the chemiluminescence reaction and generate a light signal. The relative light intensity (RLU) is detected using a photomultiplier tube.

[0072] The calibrators, antibodies, and immunoassay analyzer were the same as those used in test group 1.

[0073] Detection method: The four-gradient calibrators were repeatedly tested 10 times using a chemiluminescence analyzer with reagent components. The average detection value was calculated, and the ratio of gradient n+1 to gradient 0 was also calculated. A lower detection value for gradient 0 indicates lower nonspecificity; higher detection values ​​for gradients 1-3 indicate higher detection light intensity; and larger ratios of gradient 1 / gradient 0, gradient 2 / gradient 0, and gradient 3 / gradient 0 indicate higher detection sensitivity.

[0074] Simultaneously, the repeatability of 10 tests for each gradient in control group 2 and experimental group 1 was calculated, and the mean CV of each gradient was calculated. The test results are shown in Table 2. Table 2

[0075] Conclusion: The detection intensity and sensitivity of the octamer SA-coated magnetic beads, antibody 1 labeled with biotin, and antibody 2 labeled with ABEI were significantly lower than those of the experimental group 1. This indicates that the signal amplification technology in the experimental group 1 can significantly improve the detection intensity and sensitivity, while the inventors speculate that the SA-Biotin process on one side of the coating in the control group may not provide a significant signal amplification effect. Furthermore, the CV value of the experimental group 1 was less than 3%, demonstrating that the detection method in the experimental group 1 has good repeatability and a controllable reaction system.

[0076] Based on the reaction principle and experimental results of Experiment Group 1, it can be inferred that in Experiment Group 1, one molecule of streptavidin binds only one molecule of biotin, that is, only one antibody. Due to the large molecular weight of streptavidin and antibody during the reaction, there is a certain steric hindrance after binding. Once streptavidin binds to the biotin labeled on an antibody, it is difficult for other biotin sites of streptavidin to be recognized by biotin labeled on other antibodies. The excellent reproducibility in the experimental data of this invention also shows that if streptavidin can bind multiple biotins, the reaction may be uncontrollable, leading to poor reproducibility.

[0077] Example 4 To investigate the effect of the fragment composition of antibody 2 on the detection intensity and sensitivity, the full-length antibody in experimental group 1 was enzymatically digested to obtain Fab and F(ab')2, and biotin was labeled according to the labeling ratio of experimental group 1. The detection intensity and sensitivity after replacement were then measured.

[0078] The preparation steps of Fab are as follows: Antibody 2 was dissolved in an enzymatic digestion buffer containing 20 mM PBS, 10 mM EDTA, and 20 mM cysteine-HCl (pH 7.0-7.4). Cysteine ​​was used to create a reducing environment and activate enzyme activity. Papain was then added at an enzyme:antibody ratio of 1:100 (w / w), and the mixture was incubated at 37°C with shaking for 2-4 hours. Immediately after the reaction was complete, 1 M Tris-HCl (pH 8.5) was added to terminate the reaction. For purification, Protein A / G affinity chromatography was used to capture and remove Fc-containing fractions. The collected flow-through was then purified by gel filtration chromatography to obtain a monovalent Fab fragment with a molecular weight of approximately 50 kDa.

[0079] The preparation steps of F(ab')2 are as follows: Antibody 2 was replaced with 100 mM sodium acetate buffer (pH 3.5-4.5), and pepsin was added at an enzyme:antibody ratio of 1:20 (w / w). The reaction was carried out at 37°C with shaking for 30 minutes to 4 hours. After digestion, the pH of the system was rapidly neutralized to neutral using 1.5 M Tris-HCl (pH 8.8) to terminate the reaction. The purification process mainly relied on gel filtration chromatography, which effectively separated the target F(ab')2 fragment (approximately 110 kDa) from the small peptides produced by enzymatic digestion and the incompletely digested components by molecular weight difference, thus obtaining the F(ab')2 fragment.

[0080] The following control group was set up:

[0081] Control group 3 used Fab antibody 2, which specifically recognizes Aβ1-42, in addition to the basic component 2 of experimental group 1.

[0082] Control group 4 used F(ab')2 antibody 2, which specifically recognizes Aβ1-42, in addition to the basic component 2 of experimental group 1.

[0083] The test results are shown in Table 3: Table 3

[0084] Conclusion: When Fab and F(ab')2 were used as antibody-2 labeled biotin for detection, the detection intensity and sensitivity were significantly lower compared to the full-length antibody method. This indicates that the full-length antibody can improve the detection intensity and sensitivity.

[0085] Example 5

[0086] Based on experimental group 1, the following experimental group was set up: Experimental group 2 only changed the molar ratio of Biotin and antibody 2 to 20:1 based on the basic component 2 of experimental group 1; Experimental group 3 only changed the molar ratio of Biotin and antibody 2 to 40:1 based on the basic component 2 of experimental group 1; Experimental group 4 only changed the molar ratio of octamer SA to luminescent marker to 10:1 based on the basic component 3 of experimental group 1; Experimental group 5 only changed the molar ratio of octamer SA to luminescent marker to 20:1 based on the basic component 3 of experimental group 1; That is, based on experimental group 1, the molar ratio of Biotin to antibody 2 was set from 30:1 to 20:1 and 40:1 for detection, and the molar ratio of octamer SA to luminescent label was set from 15:1 to 10:1 and 20:1 for detection.

[0087] Labeling efficiency verification: 5 µL of 1 mg / mL biotin-labeled antibody fraction or ABEI-labeled streptavidin fraction was loaded and analyzed by LC-MS. LC conditions were: C4 reversed-phase column, column temperature 80℃, sample temperature 8℃, 0.1% formic acid aqueous solution as mobile phase A, 0.1% formic acid acetonitrile solution as mobile phase B, and a linear gradient of 10-20 minutes. MS conditions were: ESI positive ion mode, spray voltage 2.75 kV, cone voltage 150 V, source temperature 150℃, and continuous scan acquisition mode. UNIFI software was used to analyze the LC-MS data to obtain the labeling ratio results.

[0088] The marking ratios corresponding to the above feeding ratios are: The labeling molar ratios corresponding to Biotin and Antibody 2 feed molar ratios of 1:20, 1:30, and 1:40 are 1:14, 1:21, and 1:28, respectively. The labeling molar ratios corresponding to the feed molar ratios of octamer SA to luminescent label of 1:10, 1:15, and 1:20 are 1:5, 1:9, and 1:12, respectively.

[0089] In addition, the following experimental group was set up based on experimental group 1: Experimental Group 6: Based on Experimental Group 1, the detection step A was changed to: 100 μL of calibrator, 20 μL of reagent 1 and 50 μL of reagent 2 were incubated at 37℃ for 15 min, and unbound components were washed away.

[0090] The test results are shown in Table 4: Table 4

[0091] Conclusions: When the molar ratio of Biotin to Antibody 2 was 1:20, 1:30, and 1:40, the detection intensity and nonspecific signal gradually increased. The 1:30 molar ratio showed relatively better sensitivity, and the detection intensity was close to that of the 1:40 molar ratio. This indicates that at a 1:30 labeling molar ratio, more ABEI-labeled SA binds to the antibody 1-analyte-antibody 2-biotin immune complex with minimal impact on antibody 2 activity, thereby improving detection intensity and sensitivity. When the molar ratio of SA to ABEI was 1:10, 1:15, and 1:20, both detection intensity and nonspecificity gradually increased. The 1:15 SA-ABEI labeling molar ratio showed relatively better detection sensitivity, and the intensity was close to that of 1:20. Therefore, the 1:15 molar ratio is preferred. Furthermore, according to the results of experimental group 6, the addition of washing in step A significantly decreased the detection intensity and sensitivity. The inventors speculate that this may be due to the loss of some reaction components during washing, resulting in a reduced detection signal.

[0092] Example 6 To further reduce nonspecificity and improve sensitivity, the changes in detection performance were verified by adding polyvinylpyrrolidone (PVP) of different molecular weights and concentrations to the reagent, and by further adding sodium polyacrylate (SPA) of different molecular weights and concentrations to PPVP.

[0093] Based on test group 1, polyvinylpyrrolidone and / or sodium polyacrylate were added to reagent 2 according to Table 5, and the detection method of test example 1 was used.

[0094] Table 5:

[0095] The test results are shown in Table 6: Table 6

[0097] Conclusion: Adding polyvinylpyrrolidone (PVP) at a concentration of 0.2%-0.6% (w / v) with a molecular weight of 8000D-58000D to basic component 2 significantly improves detection performance. Experimental results show that the introduction of PVP effectively reduced the detection value of gradient 0 samples, indicating that non-specific binding was significantly inhibited. The inhibition effect of PVP is molecular weight dependent; the higher the molecular weight, the more significant the reduction in non-specific signal. PVP with a molecular weight of 58000 showed the best performance in improving detection sensitivity. However, while the addition of PVP helps reduce non-specificity, it also causes a decrease in detection light intensity.

[0098] Further research revealed that introducing sodium polyacrylate into a PVP-containing system could improve the detection intensity to some extent. The results indicate that sodium polyacrylate and the PVP system have a complementary effect; their synergy can suppress non-specific binding while ensuring that the detection intensity remains unaffected.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A fully automated immunoassay method for amplifying a signal, characterized in that, a control pipetting device is used to transfer a sample to be tested, reagent 1 containing a solid phase carrier coated with a first ligand, reagent 2 containing a second ligand labeled with biotin, and reagent 3 containing streptavidin labeled with a signal substance into a reaction container, forming a second complex, the second complex being a solid phase carrier-first ligand-sample to be tested-second ligand-biotin-streptavidin-signal substance complex; the first ligand and the second ligand specifically bind to different domains of the sample to be tested; in the second complex, the molar ratio of the biotin to the second ligand is ≥ 2:1; the molar ratio of the signal substance to the streptavidin is ≥ 2:1; the molar ratio of the streptavidin to the second ligand is ≥ 1:

1.

2. The immunoassay method of claim 1, characterized in that, the control pipetting device is used to transfer a sample to be tested, reagent 1 containing a solid phase carrier coated with a first ligand, reagent 2 containing a second ligand labeled with biotin, and reagent 3 containing streptavidin labeled with a signal substance into a reaction container, forming a second complex, including: Step 1: Control the pipette to transfer the sample to be tested, reagent 1 containing a solid phase carrier coated with a first ligand, and reagent 2 containing a second ligand labeled with biotin into a reaction container, forming a first reaction liquid containing a first complex; Step 2: Control the pipette to transfer reagent 3 containing streptavidin labeled with a signal substance into the reaction container containing the first complex; form a second reaction liquid, the second reaction liquid contains a second complex, Step 3: Control the transfer mechanism to transfer the reaction container containing the second complex to the detection device, and determine the content of the sample to be tested in the sample by detecting the amount of the second complex.

3. The immunoassay method of claim 1, characterized in that, the molecular weight of the streptavidin is 50-200 kD; Optionally, the molecular weight of the streptavidin is 110-200 kD; Optionally, the molecular weight of the signal substance is less than 1000D; Optionally, the labeling molar ratio of the signal substance to the streptavidin in the reagent 3 is 5:1-12:

1.

4. The immunoassay method of claim 1, characterized in that the second ligand in the reagent 2 is a full-length antibody; Optionally, the labeling molar ratio of the biotin to the second ligand in the reagent 2 is 14:1-28:

1.

5. The immunoassay method of claim 2, characterized in that, the control pipetting device is used to transfer reagent 3 containing streptavidin labeled with a signal substance into a reaction container containing the first complex, including: controlling the pipetting device to directly transfer reagent 3 containing streptavidin labeled with a signal substance into a reaction container containing the first reaction liquid.

6. The immunoassay method of claim 1, characterized in that, at least one of the reagent 1 and the reagent 2 further comprises polyvinylpyrrolidone, Optionally, the reagent 2 further comprises polyvinylpyrrolidone, Optionally, the polyvinylpyrrolidone has a concentration of 0.2%-0.6% (w / v); Optionally, the polyvinylpyrrolidone has a molecular weight of 8000D-58000D.

7. The immunoassay method of claim 3, wherein, at least one of the reagent 1 and the reagent 2 further comprises sodium polyacrylate, Optionally, the reagent 2 further comprises sodium polyacrylate, Optionally, the sodium polyacrylate has a molecular weight of 4500D, and optionally, the sodium polyacrylate has a concentration of 0.2%-0.6% (w / v).

8. The immunoassay method of any one of claims 1-5, wherein, the concentration of the analyte is 0.05 pg / mL-2000 pg / mL; Optionally, the analyte is an Alzheimer's marker, including Aβ1-42, Aβ1-40, p-tau-181, p-tau-231, p-tau-271, GFAP.

9. An immunoassay reagent for amplifying a signal, comprising a reagent 1, a reagent 2, and a reagent 3, the reagent 1 comprises a solid phase carrier coated with a first ligand that specifically binds to the analyte; the reagent 2 comprises a second ligand that specifically binds to the analyte, labeled with biotin; the reagent 3 comprises streptavidin labeled with a signal substance; the molar ratio of the biotin to the second ligand is ≥ 2:1; the molar ratio of the signal substance to the streptavidin is ≥ 2:1, the streptavidin has a molecular weight of 50-200 kD; Optionally, the streptavidin has a molecular weight of 110-200 kD; Optionally, the signal substance has a molecular weight of less than 1000D.

10. A sample analyzer characterized by, comprising a pipetting device for transferring a sample or a reagent into a reaction cup; a transfer device for transferring a reaction vessel; a detection device for detecting the sample to be tested and obtaining a detection result; a controller for controlling the pipetting device to transfer the sample to be tested, the reagent 1 comprising a solid phase carrier coated with a first ligand, the reagent 2 comprising a second ligand labeled with biotin, and the reagent 3 comprising streptavidin labeled with a signal substance into a reaction vessel to form a second complex, the second complex being a solid phase carrier-first ligand-analyte-second ligand-biotin-streptavidin-signal substance complex, and controlling the transfer device to transfer the reaction vessel to the detection device to determine the content of the analyte in the sample by detecting the amount of the second complex; the first ligand and the second ligand specifically bind to different domains of the analyte; in the second complex, the molar ratio of the biotin to the second ligand is ≥ 2:1; the molar ratio of the signal substance to the streptavidin is ≥ 2:1; the molar ratio of the streptavidin to the second ligand is ≥ 1:1; Optionally, the controller for controlling the pipetting device to transfer the sample to be tested, the reagent 1 comprising a solid phase carrier coated with a first ligand, the reagent 2 comprising a second ligand labeled with biotin, and the reagent 3 comprising streptavidin labeled with a signal substance into a reaction vessel to form a second complex comprises: Step 1: control the pipettor to transfer the sample to be tested, reagent 1 containing solid phase carrier coated with the first ligand and reagent 2 containing the second ligand labeled with biotin into the reaction vessel to form a first reaction solution containing a first complex; Step 2: control the pipettor to transfer reagent 3 containing streptavidin labeled with signal substance into the reaction vessel containing the first complex; form a second reaction solution containing a second complex, Step 3: control the transfer device to transfer the reaction vessel containing the second complex into the detection device to determine the content of the analyte in the sample by detecting the amount of the second complex; Optionally, the molecular weight of the streptavidin is 50-200 kD. Optionally, the molecular weight of the streptavidin is 110-200 kD. Optionally, the molecular weight of the signal substance is less than 1000 D.

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