Electrochemiluminescent immunoassay kit and method of use thereof

Abstract

Most of the immunoassay methods involve biotin / streptavidin conjugation chemistry, and the biotin in blood sample can interfere with the correct output of the immunoassay results. The prior art of using electrochemiluminescence as the detection signal of immunoassay is built on the biotin / streptavidin conjugation platform without exception, which can cause clinical misdiagnosis. The present application relates to an electrochemiluminescence immunoassay reagent system completely free of biotin and streptavidin components, so as to avoid the interference of biotin in the sample. Further, in view of the changes of the reagent components and the structure changes of the magnetic bead surface immune complex caused thereby, the present application provides a method for magnetic bead capture and separation in the electrochemiluminescence measurement process.

Description

TECHNICAL FIELD

[0001] The present invention is in the field of electrochemiluminescent immunoassay, and in particular to electrochemiluminescent immunoassay kits and methods of using the same. BACKGROUND

[0002] Biotin is a water-soluble vitamin (B7 or H) naturally occurring in some foods, which is involved in multiple metabolic processes in the human body. Its extremely strong non-covalent bond interaction with avidin or streptavidin has long been used for the analysis, separation and purification of bioactive substances (such as small molecule hormones, nucleic acids, proteins, etc.). The small molecular weight (244.31 D) of biotin and the rapid formation of biotin-(strept)avidin complex in different solvents and its high stability (dissociation constant Kd about 10 -14 for biotin-avidin complex and about 10 -15 for biotin-streptavidin) established biotin-(strept)avidin chemistry as a platform technology for multiple clinical immunoassay methodologies, and it has been widely used (E. P. Diamandis and T. K. Christopoulos, The biotin-(strept)avidin system: principles and applications in biotechnology, Clin. Chem. 1991, 37, 625-636). In these applications, the valeric acid carboxyl group ( Figure 1 ) in the biotin molecule is reactive, which can be directly or indirectly (such as by converting to an active ester) coupled to other biomolecules. When needed, the valeric acid carboxyl group in the biotin molecule can be first connected to a spacer or called a linker to increase the spatial distance, so that the double heterocyclic ring composed of imidazolidinone ring and tetrahydrothiophene ring in the biotin molecule can easily bind to avidin or streptavidin on the surface of a biomacromolecule in subsequent operations. The process of connecting a biomacromolecule or other measured substances to one or more imidazolidinone rings / tetrahydrothiophene rings in the biotin molecule through the valeric acid carboxyl group in the biotin molecule is called biotinylation, and the antibody connected with the biotin molecule is called biotinylated antibody. In competitive immunoassay, the measured antigen or antigen derivative may also be biotinylated.

[0003] Although there is little strong evidence to support its benefit for hair, skin and nail growth, biotin is increasingly being taken as a nutritional supplement by people suffering from hair loss, brittle hair or trichostasis syndromes. Women in the pregnancy and lactation phases are also recommended to take biotin (although its effects are controversial) due to the increased biotin metabolism during these periods. Because biotin in blood samples will compete with biotin-conjugated antibodies (or antigens) for (strept)avidin on the solid phase surface, all immunoassays based on the biotin-streptavidin coupling chemistry platform (such as ELISA, RIA, EIA, etc.) will be affected by the presence of biotin in the sample. This is especially true for the sandwich immunoassay format, where the biotin in the sample will bind to the (strept)avidin on the solid phase surface, thus preventing the biotin-conjugated antibody from binding to the (strept)avidin. This will result in a false positive result. In the competitive immunoassay format, the biotin in the sample will bind to the (strept)avidin on the solid phase surface, thus preventing the biotin-conjugated antigen from binding to the (strept)avidin. This will result in a false negative result. Figure 2The results of certain immunoassay platforms listed above will be interfered with to varying degrees by biotin in the sample, causing either a positive (in competitive assays) or negative (in sandwich assays) bias. The amount of biotin that the average person obtains from a normal diet is so small that it will not affect the results of immunoassays. However, cases of interference with immunoassay results by biotin that enters the blood through the ingestion of nutritional supplements have been reported since 1996 (J.G. Henry, S. Sobki, N. Arafaty, Interference by Biotin Therapy on Measurement of TSH and FT4 by Enzyme immunoassay on Boehringer Mannheim ES700 Analyser, Ann. Clin. Biochem. 1996, 33, 162-163), and in electrochemiluminescence immunoassays in 2009 (D.L. Meany, S.M. Jan de Beur, M.J. Bill, 1and L.J. Sokoll, A Case of Renal Osteodystrophy with Unexpected Serum Intact Parathyroid Hormone Concentrations, Clin. Chem. 2009, 55, 1737-1739.).Currently reported biotin-interferenced immunoassays involve multiple systems, including sandwich immunoassays such as NT-pro-BNP, hs-TnT, TSH, FSH, Anti-Tg, anti-TPO, and anti-TSHR, as well as competitive methods such as T3, T4, and vitamin D (D. Li, A. Radulescu, RT Shrestha, et al. Association of biotin ingestion with performance of hormone and nonhormone assays in healthy adults. JAMA 2017, 318, 1150-1160; PJ Colon1 and DN Greene, Biotin Interference in Clinical Immunoassays, J. Appl. Lab. Med. 2018, 2, 941-951; Jieli Li, Elizabeth A. Wagar, Qing H. Meng, Comprehensive assessment of biotin interference in immunoassays, Clinica Chimica). Acta, 2018, 487, 293-298. Even within 24 hours of a single 10 mg oral dose, biotin still interferes with thyroid function tests (RPMBiscolla, MIChiamolera, I. Kanashiro, RMB Maciel, JGHVieira, A single 10 mg oral dose of biotin interferes with thyroid function tests, Thyroid 2017, 27, 1099-1100.). On November 28, 2017, the U.S. Food and Drug Administration (FDA) issued a warning about the interference of biotin in clinical immunoassays (FDA. The FDA warns that biotin may interfere with lab tests: FDA safety communication.). Two years later (November 5, 2019), the FDA updated this warning. The purpose of the warning is to alert consumers, healthcare workers, and clinical laboratory personnel to biotin that may be added to nutritional supplements and its impact on test results. The FDA recommends that the biotin dose that does not interfere with tests is no more than 0.03 mg per day for adults.However, commercially available nutritional supplements claiming to benefit hair, skin, or nails may contain doses up to 20 mg per day for adults, while prescriptions for multiple sclerosis may contain up to 300 mg of biotin per day for adults. Blood samples from patients taking high doses of biotin may contain levels as high as 100-1200 ng / mL of biotin.

[0004] Although biotin is metabolized rapidly in the human body, and not all blood samples from people taking biotin affect the results of immunoassay tests, the increasing number of people taking biotin and the widespread use of high-dose biotin have made the impact of biotin on clinical immunoassay results a problem that must be addressed in clinical immunoassays. The American Association for Clinical Chemistry (AACC) compiled misdiagnosis cases due to biotin interference in its 2020 guidance document on biotin interference in clinical immunoassays from multiple countries (D. Li, A. Ferguson, M. A. C. C. D. E. M. D. E. M. D. M ...

[0005] The following are some recommended procedures to avoid biotin interference, and methods to reduce or eliminate biotin interference.

[0006] 1. Precautions to avoid biotin interference (Jieli Li, Elizabeth A. Wagar, Qing H. Meng, Comprehensive assessment of biotin interference in immunoassays, Clinica Chimica Acta, 2018, 487, 293-298):

[0007] • Ask the patient if they are taking any medications / nutritional supplements containing biotin;

[0008] Record the time and amount of intake;

[0009] • Patients are required to stop taking biotin or biotin-containing medications / nutritional supplements 48 hours before blood sampling;

[0010] • If the daily dose is greater than 5 mg, wait at least 8 hours after the last dose before taking a blood sample;

[0011] • If the test results are inconsistent with the patient's clinical presentation, and / or if biotin interference is suspected, communicate with the laboratory promptly.

[0012] 2. Sample pretreatment before immunoassay testing, such as using streptavidin-coated magnetic beads to remove biotin (M.-L. Piketty, D. Prie, F. Sedel, D. Bernard, C. Hercend, P. Chanson and J.-C. Souberbielle, High-dose biotin therapy leading to false biochemical endocrine profiles: validation of a simple method to overcome biotin interference, Clin Chem Lab Med, 2017, 55, 817-825; C. Trambas, Z. Lu, T. Yen and K. Sikaris, Depletion of biotin using streptavidin-coated microparticles: a validated solution to the problem of biotin interference in streptavidin-biotin immunoassays, Ann. Clin. Biochem., 2018, 55, 216-226; HMStieglitz, N. Korpi-Steiner, Characterization of biotin interference in 21 Vitros 5600 immunoassays and risk mitigation Clin. Biochem. 2020, 75, 53-61.)

[0013] 3. The reaction sequence was altered by first binding streptavidin-coated magnetic beads with biotinylated antibodies to form antibody-coated magnetic beads before the antibody / antigen immunoreaction. (L. Johnson and D. Li, Strategy to investigate biotin interference in light of the FDA safety communication, J. Appl. Lab. Med., 2019, 3, 914-915; Jianbo Yang, JR Wiencek, Mitigating biotin interference in two Roche immunoassays by premixing biotinylated capturing molecules with streptavidin coated beads, Clinica Chimica Acta, 2020, 505, 130-135.)

[0014] 4. A specific biotin monoclonal antibody is used to bind to biotin; patent application WO 2018 / 122043A1 discloses a biotin monoclonal antibody that binds only to free biotin and not to biotin conjugated to biological macromolecules (such as antibodies). The improved high-sensitivity cardiac troponin T (cTnT-hs) ECL immunoassay reagent, by adding this specific biotin monoclonal antibody to the reagent reaction system, effectively neutralizes biotin added to the blood sample, preventing its interference with the test results (A. von Meyer, G. Albert, S. Kunzelmann, C. Rank, R. Zerback and R. Imdahl, Evaluating the performance of an updated high-sensitivity troponin T assay with increased tolerance to biotin, Clin Chem Lab Med, 2021, 59, 591-597.).

[0015] Electrochemiluminescence (ECL) immunoassay is a widely used immunoassay method in clinical testing. In ECL immunoassay, ruthenium tripyridine (usually denoted as Ru(bpy)3) is used. 2+ ) succinimide ester (NHS ester, see Figure 3A) or other luminescent metal complexes are used as labels to label the analyte antibody or antigen. After the antibody reacts with the analyte in the sample under certain conditions to form an antibody / antigen complex, the luminescent metal complex undergoes an electrochemical reaction and a series of subsequent chemical reactions in an electrochemical flow cell, ultimately leading to the formation of the luminescent excited state of the luminescent metal complex and generating a detectable luminescent signal.

[0016] ECL immunoassay involves numerous technical details, including how antibodies (in sandwich assays) or analytes (in competitive assays) are labeled, how the analyte is captured, how the ECL reaction is triggered, and how the working electrode is regenerated. Taking ECL sandwich immunoassay as an example, in a typical commercial ECL assay, using... Figure 3 The labeled molecule shown in Figure A labels an antibody (signal antibody) at the ε-amino site of a lysine residue of an antibody in one analyte, while another antibody (capture antibody) is biotinylated. When a clinical sample is mixed with these two types of antibodies and streptavidin-coated magnetic beads at a predetermined time and temperature, a sandwich-structured immune complex forms on the surface of the magnetic beads. The beads are then introduced into an electrochemiluminescence measurement cell (flow cell) and captured on the surface of the electrochemical working electrode by a movable magnet located below the cell. A buffer solution containing tri-n-propylamine (TPA or N(C3H7)3) washes away unwanted substances and provides a Ru(bpy)3... 2+ The luminescent group undergoes a chemical environment described in the ECL reaction in the following reaction pathway one.

[0017] At a specific voltage (e.g., 1.4V relative to Ag / AgCl), tripropylamine in the buffer solution is oxidized to the cationic radical N(C3H7)3. ·+ (Reaction 1), and further loses a proton to become a neutral free radical H6C3·N(C3H7)2 (Reaction 2). This neutral free radical has strong reducing power and can reduce Ru(bpy)3. 2+ Revert to Ru(bpy)3 1+ (Reaction 3). The oxidizing cationic radical N(C3H7)3 ·+ Then Ru(bpy)3 1+ Oxidation to excited state Ru(bpy)3 2+* (Reaction 3). Ru(bpy)3 2+* After emitting light with a wavelength of 620nm, it returns to the original Ru(bpy)3. 2+ Ground state (reaction 5).

[0018] Reaction Path One

[0019] N(C3H7)3-e- →N(C3H7)3 ·+ (1)

[0020] N(C3H7)3 ·+ -H + →H6C3 · N(C3H7)2 (2)

[0021] Ru(bpy)3 2+ +H6C3 · N(C3H7)2→Ru(bpy)3 + +P (3)

[0022] Ru(bpy)3 + +N(C3H7)3 ·+ →Ru(bpy)3 2+* +N(C3H7)3 (4)

[0023] Ru(bpy)3 2+* →Ru(bpy)3 2+ +hυ (5)

[0024] Therefore, in the ECL process, Ru(bpy)3 2+ Instead of being consumed, it undergoes a cyclic change of oxidation state, namely, Ru(bpy)3. 2+ →Ru(bpy)3 1+ →Ru(bpy)3 2+* →Ru(bpy)3 2+ (See W. Miao, J.-P. Choi, A.J. Bard, J. Am. Chem. Soc. 2002, 124, 14478-14485). During the measurement, this cycle is repeated continuously, resulting in the generation and detection of a long-delayed ECL signal. The integral of all ECL light emission over a certain time period (e.g., 0.5–5 seconds) can be used as a measure of ECL intensity and correlated with the amount of the analyte. After the measurement, the magnetic beads and the attached immune complexes are washed away by an aqueous stream, the measurement cell is cleaned, and the electrode surfaces are regenerated through an electrochemical process to prepare them for the next test. Details of these time-series experiments are disclosed in U.S. Patents 5,147,806, 5,538,687, and 6,599,473.

[0025] In fact, the reaction pathway is only one possible one that can produce ECL. Other possible reaction pathways (such as the following) Reaction Paths Two to Four It has also been proposed to explain the excited state Ru(bpy)3 under different conditions. 2+*Formation and ECL generation (see J.K. Leland and M.J. Powell, J. Electrochem. Soc. 1990, 137, 3127 - 3131, and W. Miao, J.-P. Choi, A.J. Bard, J. Am. Chem. Soc. 2002, 124, 14478 - 14485).

[0026] Reaction Path Two

[0027] Ru(bpy)3 2+ -e - →Ru(bpy)3 3+ (6)

[0028] N(C3H7)3 - e - →N(C3H7)3 ·+ (1)

[0029] N(C3H7)3 ·+ -H + →H6C3 · N(C3H7)2 (2)

[0030] Ru(bpy)3 3+ +H6C3 · N(C3H7)2→Ru(bpy)3 2+* +P (7)

[0031] Ru(bpy)3 2+* →Ru(bpy)3 2+ +hυ (5)

[0032] Reaction Path Three

[0033] Ru(bpy)3 2+ -e - →Ru(bpy)3 3+ (6)

[0034] Ru(bpy)3 3+ +N(C3H7)3→Ru(bpy)3 2+ +N(C3H7)3 ·+ (8)

[0035] N(C3H7)3 ·+ -H + →H6C3 · N(C3H7)2 (2)

[0036] Ru(bpy)3 3+ +H6C3 ·N(C3H7)2→Ru(bpy)3 2+* +P (7)

[0037] Ru(bpy)3 2+* →Ru(bpy)3 2+ +hυ (5)

[0038] Reaction Path Four

[0039] Ru(bpy)3 2+ -e - →Ru(bpy)3 3+ (6)

[0040] N(C3H7)3-e - →N(C3H7)3 ·+ (1)

[0041] N(C3H7)3 ·+ -H + →H6C3 · N(C3H7)2 (2)

[0042] Ru(bpy)3 2+ +H6C3 · N(C3H7)2→Ru(bpy)3 + +P (3)

[0043] Ru(bpy)3 + +Ru(bpy)3 3+ →Ru(bpy)3 2+ +Ru(bpy)3 2+* (9)

[0044] Ru(bpy)3 2+ *→Ru(bpy)3 2+ +hυ (5)

[0045] The above reaction pathways two, three, and four occur when the electrode voltage is high enough to react Ru(bpy)3. 2+ Oxidation to Ru(bpy)3 3+ Under the conditions of (i.e., reaction 6), Ru(bpy)3 is not present in the reactions involved in reaction pathway 1. 2+ It is oxidized to Ru(bpy)3 3+Reaction 6. However, under high voltage conditions that can lead to reaction pathways two, three, and four, the reactions in reaction pathway one also occur simultaneously. Although researchers in the field tend to believe that the vast majority of ECL light emission originates from reaction pathway one, the actual operating voltage used in ECL immunoassay systems is 1.4V (relative to the Ag / AgCl reference electrode), at which reaction pathways one, two, three, and four can all occur.

[0046] US Patent 10203333 and Chinese Patent ZL 201480045420 disclose a class of electrically neutral ruthenium coordination compounds (NRCs). These electrically neutral ECL markers can reduce non-specific signals in immunoassays. Some electrically neutral ECL marker molecules (such as...) Figure 3 The NRC of C also produced stronger luminescence. US patent applications US 2021 / 0130876 A1 and WO 2021 / 084472 A1 further disclose labeled molecules containing two or more ECL luminescent agents. These higher-performance ECL luminescent agents and the labeled molecules they constitute enrich the electrochemiluminescence immunoassay methodology—just as chemiluminescence has multiple platform technologies based on different chemiluminescent agents, electrochemiluminescence also includes multiple platforms based on different ECL luminescent agents (such as...). Figure 2 (As shown).

[0047] In the prior art, the kit used for ECL clinical immunoassay consists of three reagent components. In the sandwich assay, these three components are: a biotin-conjugated capture antibody reagent, a luminescent label (such as Ru(bpy)3), and a... 2+ The reagent consists of a biotin-conjugated signal antibody and streptavidin-coated magnetic beads; in the competitive assay, these three reagents can be biotin-conjugated antigens, luminescent markers (such as Ru(bpy)3), and other similar reagents. 2+ Alternatively, it can be a biotin-conjugated capture antibody reagent or a luminescent marker (such as Ru(bpy)3). 2+ The antigen reagent is labeled with ) and magnetic beads are coated with streptavidin.

[0048] Because all ECL clinical immunoassay reagent systems in the prior art use streptavidin-coated magnetic beads as a reagent component, biotin that may be present in the sample will compete with the biotin-conjugated components in the reagent (biotin-conjugated capture antibody reagents or biotin-conjugated antigen reagents) for streptavidin binding sites on the surface of the magnetic beads, thus leading to the generation of false signals.

[0049] The aforementioned methods for reducing or eliminating biotin interference are effective for ECL immunoassays to some extent, but they also have various problems. User-proposed solutions increase the complexity of the test and are not effective in all situations. Furthermore, the use of specific biotin monoclonal antibodies by ECL reagent manufacturers would significantly increase reagent costs. This invention provides a two-component kit that differs from existing ECL immunoassay reagent systems. This kit does not contain streptavidin, thus avoiding interference from biotin that may be present in clinical ECL immunoassay samples.

[0050] Despite the long-standing problem of biotin interference, ECL reagent developers have not abandoned the biotin-streptavidin chemistry system for several technical reasons.

[0051] 1. In existing three-reagent reaction systems, immune complexes (antibody / antigen / antibody) or (antigen / antibody) are formed in a homogeneous reaction. After formation, the complexes are then anchored to the surface of magnetic beads via a biotin / streptavidin reaction. Theoretically, antibody-antigen binding reactions have good reproducibility in a homogeneous environment, resulting in good reproducibility of results when repeatedly testing the same sample.

[0052] 2. Unlike other immunoassay techniques that also use magnetic beads as a solid-phase carrier and chemiluminescence as the detection signal, electrochemiluminescence is generated on the surface of magnetic beads deposited on a two-dimensional plane electrode, while other chemiluminescence signals are generated by magnetic beads suspended in a three-dimensional solution. Furthermore, the electrochemical reaction only occurs at the interface 1-2 nanometers away from the electrode surface; therefore, any change in the surface chemistry of the magnetic beads may affect the occurrence of the electrochemical reaction. Abandoning the biotin-streptavidin chemistry system cannot guarantee that electrochemiluminescence immunoassays, which were established on a biotin-streptavidin chemistry platform in the early stages of ECL technology development, will still possess comparable analytical performance.

[0053] 3. Unlike chemiluminescence immunoassay, in electrochemiluminescence immunoassay, the magnetic separation of immune complexes formed on the surface of magnetic beads is completed in an electrochemical flow cell. In this process, a certain amount of reaction mixture flows at a specific flow rate across the surface of the working electrode in the electrochemical flow cell and is attracted by a magnet located below the working electrode, thus remaining on the electrode surface. In ECL immunoassay, this process is called the magnetic bead trapping process. Since any change in the chemical state of the magnetic bead surface can alter its hydrodynamics in the liquid path and affect the uniform distribution of the surface-loaded immune complexes on the electrode surface, abandoning the biotin-streptavidin chemical system will lead to uncontrolled magnetic separation of the magnetic beads on the electrode surface, altering the uniform distribution of the beads and resulting in a decline in the performance indicators of ECL immunoassay.

[0054] Prior to the method provided by this invention, there was a common concern based on reason 1 above: that the two-reagent system, by changing the antibody-antigen binding reaction from a homogeneous phase (buffer solution) to a heterogeneous phase (magnetic bead surface), would lead to a decrease in test precision (commonly expressed as the coefficient of variation). However, surprisingly, the systems described in the embodiments of this invention all exhibit reaction repeatability comparable to the three-reagent system.

[0055] Reasons 2 and 3 above are specific to ECL immunoassay. Prior to this invention, a concern based on reason 2 above was that the absence of the biotin-streptavidin level between the antibody and magnetic beads would alter the signal contribution of different pathways in the electrochemiluminescence reaction, and might also enhance non-specific adsorption, thereby changing the established concentration-response relationship and hindering the electrochemiluminescence technology from reaching its optimal operating state. However, an unexpected outcome of implementing this invention is that ECL can still reach its optimal operating state even after eliminating the biotin and streptavidin components.

[0056] To address the concerns raised by reason 3 above, it was necessary to develop and establish different magnetic bead capture procedures. Through extensive trial and error, the inventors of this patent unexpectedly discovered that when the magnetic beads flow across the surface of the working electrode, placing the electrode at a potential higher than 0.0V (relative to the silver / silver chloride reference electrode in a saturated potassium chloride solution) but lower than 0.45V not only helps to improve the signal value but also helps to achieve a more uniform distribution of the magnetic beads on the electrode surface. Summary of the Invention

[0057] This invention provides a two-component reagent kit that differs from existing three-component ECL immunoassay reagent systems. The kit consists of two components: antibody-coated magnetic beads and ECL luminescent marker-labeled antibodies (or antigens). This kit does not contain streptavidin-coated magnetic beads or biotin-conjugated antibodies (or antigens), thus avoiding interference from biotin that may be present in clinical ECL immunoassay samples. Furthermore, for the use of this streptavidin-free reagent system, this invention provides an improved magnetic bead capture method, namely, magnetic separation at an electrode potential higher than 0.0V. Attached Figure Description

[0058] The invention will be described in conjunction with the following drawings, in which the same reference numerals denote the same elements, and wherein:

[0059] Figure 1 Chemical structural formula of biotin

[0060] Figure 2 Conventional immunoassay methods based on different detection signals

[0061] Figure 3ECL-labeled molecules disclosed in U.S. Patent 5,744,367(A), U.S. Patent 6,808,939(B), WO 2014203067A1(C) and U.S. Patent Application US2016 / 0145281A1(D);

[0062] Figure 4 Comparison of magnetic bead surface states after incubation reaction in ECL immunoassay: (left) prior art; (right) present invention;

[0063] Figure 5 The change of the working electrode potential over time within a measurement cycle (the dashed line represents the potential setting in the prior art);

[0064] Figure 6 In the ECL immune test, the signal increases with incubation time;

[0065] Figure 7 Relationship between signal and concentration at different incubation times in ECL immunoassay. Detailed Implementation

[0066] Unless otherwise defined, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which the methods described herein pertain. These terms and meanings are well explained in technical literature, such as Bioconjugation Techniques (G. T. Hellmanson, Elservier, Amsterdam, 2008) and Handbook of Immunoassays (D. Wild et al., 4th ed., Elservier, Amsterdam, 2013).

[0067] Within the scope of this invention, "analyte" includes, but is not limited to, whole cells, cell surface antigens, protein complexes, cell signaling factors and / or components, second messengers, second messenger signaling factors and / or components, subcellular particles (e.g., organelles or membrane fragments), viruses, prions, house dust mites or fragments thereof, viroids, immune factors, antibodies, antibody fragments, antigens, haptens, fatty acids, nucleic acids (and synthetic analogs), ribosomes, proteins (and synthetic analogs), lipoproteins, polysaccharides, inhibitors, cofactors, haptens, cell receptors, receptor ligands, lipopolysaccharides, glycoproteins, peptides, polypeptides, etc. Enzymes, enzyme substrates, enzyme products, nucleic acid processing enzymes (e.g., polymerases, nucleases, integrases, ligases, helicases, telomerases, etc.), protein processing enzymes (e.g., proteases, kinases, protein phosphatases, ubiquitin-protein ligases, etc.), cell metabolites, endocrine factors, paracrine factors, autocrine factors, cytokines, hormones, pharmacological drugs, drugs, therapeutic drugs, synthetic organic molecules, organometallic molecules, sedatives, barbiturates, alkaloids, steroids, vitamins, amino acids, sugars, lectins, recombinant or derived proteins, biotin, avidin, streptavidin, or inorganic molecules present in the sample.

[0068] According to the methods and reagents of the present invention, an "analyte-specific reagent" (ASR) is a class of molecules or biomolecules with the ability to specifically bind to an analyte, such as antibodies, polyclonal antibodies and monoclonal antibodies, specific receptor proteins, ligands, nucleic acid sequences and analogues. They are intended for the identification and quantification of individual chemical or biochemical substances or ligands in biological samples through specific binding or specific chemical reactions with substances in the sample, and are used in bioanalytical applications.

[0069] Within the scope of this invention, "analyte-specific reagent-coated magnetic beads" (ASR-coated magneticbeads) refer to complexes formed between the analyte-specific reagent and the magnetic bead through functional groups on the surface of the magnetic bead. The coupling between the analyte-specific reagent and the surface of the magnetic bead can be through covalent bonding or adsorption.

[0070] Within the scope of this invention, substances referred to as "labels," "labeled molecules," "ruthenium(II) labels," and "ECL labels" can be covalently bound to other substances, such as bioactive analytes or their analogues, bioaffinity-based analyte recognition partners or their analogues (e.g., analyte-specific reagents), other binding partners of the aforementioned recognition partners, or a reactive chemical substance capable of forming a covalent bond with the analyte, or its analogues or binding partners as described above. These substances can also be bound to combinations of one or more binding partners and / or one or more reactive components. Furthermore, these substances can also be bound to analytes or their analogues that are linked to a binding partner, a reactive component, or a combination of one or more binding partners and / or one or more reactive components. The direct binding of multiple of the above substances to an analyte or its analogue, or binding to it via other molecules as described above, is also within the scope of this invention.

[0071] As used herein, the term "label" refers to any chemical or biochemical substance that, either alone or through physical / chemical interactions with other reagents, generates a detectable signal (whether a visible signal or a signal detectable by using a suitable instrument) that can be correlated with the amount of a target analyte. Labels include, but are not limited to, molecules containing radioactive atoms (radioactive), luminescent compounds (emitting light through photoexcitation or through chemical reactions), electroactive compounds (generating electrical signals through redox reactions), magnetic particles (magnetic signals), enzymes (generating detectable substances or optical signals through reactions with substrates), and enzymes or enzymatic substrates (catalyzing chemical / biochemical reactions). A label may consist of one or more signal-generating units and one or more reactive groups.

[0072] The term "luminescence" refers to the energy released in the form of electromagnetic radiation (light emission) when an electron transitions from a low-energy state to an "excited" high-energy state and then falls back to a lower-energy state. This light emission typically occurs in the visible or near-visible range of the electromagnetic spectrum. The term "luminescence" generally includes, but is not limited to, luminescence phenomena such as phosphorescence, fluorescence, bioluminescence, radioluminescence, electroluminescence, electrochemiluminescence, and thermoluminescence; however, in this invention, unless otherwise specified, luminescence refers to electrochemiluminescence.

[0073] Within the scope of this invention, the terms "luminescent group" and "luminescent body" refer to the functional group in a compound responsible for producing luminescence. In compounds with complex structures, such as those having multiple functional groups (e.g., reactive groups, hydrophilic / hydrophobic / amphiphilic groups, electron-withdrawing / electron-donating groups, electrobalancing groups, spacer groups, linking groups, branching groups, etc.), the luminescent group is the smallest structural component required to produce luminescence (e.g., see [link to other documentation]). Figure 1 (The circled part in the middle).

[0074] The term "luminescent label" refers to a label composed of one or more luminescent groups and one or more reactive groups, which readily forms covalent bonds with the chemical or biochemical molecule to be labeled. Luminescent labels can be, for example, fluorescent molecules, phosphorescent molecules, radioluminescent molecules, electrochemiluminescent molecules (i.e., ECL labels) of the present invention, or quantum dots with reactive groups on their surface. However, in this invention, unless otherwise specified, "luminescent label" refers to an electrochemiluminescent label (ECL label). Examples of electrochemiluminescent (ECL) labels having one luminescent group and one reactive group are most frequently disclosed in the prior art (see, for example, Figure 1 Examples of luminescent markers include those in WO2003002974A2, WO 2014203067A1 (other ruthenium complex markers), and WO2014019711A1 (iridium complex markers). US 2005 / 0059834 A1 discloses examples of luminescent markers having three luminescent groups (three ruthenium complex units) and one reactive group (-COOH or NHS ester). US Patent 6140138 discloses examples of luminescent markers having one luminescent group (one ruthenium complex) and two reactive groups (-COOH or NHS ester). Chinese Patent Application 202010983872.X, US Patent Application 2021 / 0130876 A1, and WO 2021 / 084472 A1 disclose ECL marker molecules containing two or more luminescent groups.

[0075] According to this application, "detection reagents" include analyte-specific reagents (ASRs) labeled with at least one ECL luminescent group, or analyte analogs / homologs labeled with one ECL luminescent group. Those skilled in the art know that, in analysis, detection reagents are ultimately immobilized on a solid phase. "Solid phase," also known as "solid carrier," refers to non-fluid substances such as magnetic beads and particles (including microparticles and beads) made of materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass, and ceramics; gel substances such as silica, alumina, and polymer gels; capillaries, which may be made of polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and sample containers for cuvettes, test tubes, films, or other spectrometers. The solid phase component in the analytical process differs from the inert solid surfaces that may be encountered during the analytical process in that the "solid phase" contains at least one structural portion on its surface designed to interact with a capture antibody or capture molecule. The solid phase can be a fixed component, such as a test tube, strip, cuvette, thin film, or microtiter plate, or a non-fixed component, such as magnetic beads, beads, and microparticles.

[0076] In one implementation, the method can be performed as a sandwich assay. In another implementation, the method can be performed as a competitive assay. In yet another implementation, the method can also be performed as a dual antigen bridging assay (DAGS). Known immunoassay formats are described in detail in the following books: D. Wild et al., Handbook of Immunoassays, 4th Edition, Elservier, Amsterdam (2013); and EPDiamondis and TK Christopoulos, Immunoassays, Santiago, Academic Press (1996).

[0077] Electrochemiluminescence immunoassay (ECL immunoassay) is an analytical method that uses electrochemical excitation to induce luminescence in ECL luminescent groups. A voltage between the working electrode and the reference electrode electrochemically activates the ECL luminescent groups bound to an ASR or analyte analog / homolog to emit light. The light emitted from the ECL luminescent groups is measured by a photodetector, indicating the presence or quantity of the target analyte. U.S. Patent Nos. 5,543,112, 5,935,779, and 6,316,607 describe ECL methods in detail.

[0078] The term "working voltage" in this invention is a key concept and experimental parameter. In a three-electrode electrochemical measurement system consisting of a working electrode, a counter electrode, and a reference electrode, "working voltage" is the voltage between the working electrode and the reference electrode. Unless otherwise specified, the working voltage or voltage mentioned in this invention refers to a silver / silver chloride (Ag / AgCl, saturated potassium chloride) reference electrode, whose electrode potential relative to the standard hydrogen electrode (SHE) is +0.197V. Different instrument systems may be configured with different reference electrodes when generating electrochemiluminescence or performing electrochemiluminescence immunoassays, and the voltage between these reference electrodes and the working electrode may differ from the working voltage in this invention. The magnitude of this difference can be obtained by converting the electrode potential of the reference electrode. Those skilled in the art will know that the electrode potentials of different reference electrodes relative to the standard hydrogen electrode (SHE) and their conversion methods can be found in literature and books related to electrochemistry. One method of the kit described in this invention involves improving the signal by adjusting the "working voltage" during the magnetic bead capture process.

[0079] In ECL analysis procedures, magnetic beads can be suspended in the sample and detection reagents to effectively bind analytes. Magnetic beads can have diameters from 0.05 μm to 200 μm, 0.1 μm to 100 μm, or 0.5 μm to 10 μm, and possess surface components capable of binding biomolecules. In the ECL analysis systems used by the applicant of this invention (Ansel Diagnostics YnY 2020, YnY 2050, and YnY 3030 systems), the diameter of the magnetic beads is 2.8 μm. Magnetic beads can be formed from the following substances: organic polymers, polystyrene, styrene copolymers such as styrene / butadiene copolymers, acrylonitrile / butadiene / styrene copolymers, vinyl acetoacrylate copolymers, vinyl chloride / acrylate copolymers, inert inorganic materials, chromium dioxide, iron oxides, silicon dioxide, mixtures of silicon dioxide, protein substances, or mixtures thereof.

[0080] According to this application, the "reagent composition" includes reagents that support the generation of an ECL signal, such as co-reactants (e.g., tripropylamine TPA), buffers for pH control, surfactants, preservatives or antibacterial agents, and optional other components. Those skilled in the art are aware of the components present in the reagent composition required to generate an ECL signal in an electrochemiluminescence detection method.

[0081] As used herein, "aqueous solution" is a homogeneous solution of a particulate, substance, or liquid compound dissolved in water, or a heterogeneous suspension having particles (from 0.05 μm to 200 μm in diameter) suspended in the aqueous solution. Aqueous solutions may also contain organic solvents. Organic solvents are known to those skilled in the art, such as amines, methanol, ethanol, dimethylformamide, or dimethyl sulfoxide. As used herein, it should also be understood that aqueous solutions may contain up to 50% organic solvent.

[0082] In this paper, substances that participate in the ECL process together with ECL markers are referred to as ECL "co-reactants". Commonly used co-reactants for ECL include tertiary amines (e.g., tri-n-propylamine TPA) and their analogs / homogeneities (e.g., 2-(dibutylamino)ethanol), oxalates, and persulfates. Those skilled in the art are familiar with co-reactants that can be used in ECL detection methods.

[0083] As used herein, "transition metal complex" refers to an ECL luminescent group comprising a transition metal ion bound to a suitable complexing or chelating agent. In one embodiment, the transition metal is selected from the group consisting of ruthenium, iridium, rhenium, osmium, europium, terbium, and dysprosium; in another embodiment, the transition metal is ruthenium, iridium, rhenium, or osmium; and in a further embodiment, the transition metal is ruthenium or iridium.

[0084] In one embodiment, the ECL luminescent group is a type of coordination compound of electrically neutral metal ruthenium disclosed in US Patent 10203333 and Chinese Patent ZL201480045420.

[0085] In another embodiment, the ECL luminescent group is an iridium complex and is selected from the following ECL markers: Ir(6-phenylphenanthridine)2-pyridine-2-carboxylic acid or derivatives thereof, including, for example, Ir(6-phenylphenanthridine)2-3-hydroxypyridine-2-carboxylic acid, Ir(6-phenylphenanthridine)2-4-(hydroxymethyl)pyridine-2-carboxylic acid, Ir(6-phenylphenanthridine)2-2-(carboxyethyl-phenyl)pyridine-2-carboxylic acid, Ir(6-phenylphenanthridine)2-5-(methoxy)pyridine-2-carboxylic acid, or Ir(6-phenylphenanthridine)2-2-(carboxyethyl-phenyl)pyridine-2-carboxylic acid ester, or derivatives thereof, such as iridium complexes in which the ligand is substituted with one or more sulfonic acids, or as in WO2012107419(A1). The iridium complexes described in WO2012107420(A1), WO2014019707(A2), WO2014019708(A1), WO2014019709(A2), WO2014019710(A1), and WO2014019711(A1). It is well known to those skilled in the art that iridium(III) complexes have poor solubility in aqueous solutions, and hydrophilic derivatives of the aforementioned ECL compounds can be used. Therefore, in another embodiment, the aforementioned iridium(III) ECL luminescent group can be modified with hydrophilic substituents. In another embodiment, the ECL luminescent group is an iridium complex having two phenylphenanthridine ligands having two sulfonylpropoxy substituents and two sulfomethyl groups, comprising 2,9-phenanthridine dimethylsulfonic acid, 6-phenyl-sodium salt (CAS Registry No. 1554465-50-7), or two polyethylene glycol substituents, or each phenylphenanthridine ligand containing three of the above groups, or each phenylphenanthridine ligand containing a combination of the above groups.

[0086] In another embodiment, the ECL marker is a multi-marker ECL marker molecule disclosed in Chinese patent application 202010983872.X, US patent application 2021 / 0130876 A1, and WO 2021 / 084472 A1.

[0087] Those skilled in the art will understand that in ECL immunoassay, the luminescence signal is triggered by a constant operating voltage, resulting in a light signal being emitted by the ECL luminescent material immobilized on the surface of a magnetic bead. The constant voltage excitation time is typically between 0.2 and 10 seconds, preferably between 1.0 and 3.0 seconds. During this voltage excitation period, the ECL luminescence signal gradually decays. The area under the ECL luminescence decay curve over a given time is the relative luminescence intensity (RLU). In a specific immunoassay, different RLUs correspond to different analyte concentrations. The relationship between the signal response, expressed in RLU, and the concentration is typically described by a fitted functional relationship (linear or nonlinear). Figure 7 The five fitted curves in the model conform to the four-parameter logistic equation.

[0088] Those skilled in the art will also know that in an automated ECL testing system, there is typically a pretreatment procedure for the electrode surface. Applying this procedure before sample testing ensures that the electrode surface maintains the same surface chemical state before testing. After a test, the electrochemical measurement cell and electrode surface need to be cleaned and regenerated. These steps are disclosed in U.S. Patent 5,147,806, and U.S. Patent 6,599,473 further discloses an improved version. In describing these steps, the prior patents use the terms preoperative, conditioning, and cleaning. Figure 5 An electrochemiluminescence signal generation procedure including pretreatment and testing steps is described. The pretreatment and cleaning procedures may differ in different ECL systems; for example, the electrochemical procedure disclosed in US Patent 6,599,473 adds a voltage pulse that is believed to improve the deposition state of the magnetic beads on the electrode. This invention does not involve improvements to the post-measurement cleaning procedure and electrode post-treatment. The improvements to the pretreatment and capture stages are changing the negative voltage pulse from -1.2V to -0.5V, and increasing the voltage of the working electrode from 0.0V to between 0.05V and 0.40V during the capture stage.

[0089] The present invention will be further described below through specific embodiments, but this is not a limitation of the present invention. Those skilled in the art can make different modifications to each embodiment according to the basic spirit of the present invention. As long as the modifications do not depart from or deviate from the basic spirit of the present invention (i.e., the reagent composition does not contain streptavidin), the embodiment is within the scope of the present invention.

[0090] Example

[0091] Based on the foregoing description, these and other advantages of the present invention will be apparent to those skilled in the art. Therefore, those skilled in the art will recognize that changes or modifications can be made to the above embodiments without departing from the broad inventive concept of the invention. It should be understood that the present invention is not limited to the specific embodiments described herein, but is intended to include all changes and modifications within the scope and spirit of the invention.

[0092] Example 1 - Preparation of PCT antibody-coated magnetic beads using magnetic beads containing carboxyl groups on their surface

[0093] a. Suspend and mix the magnetic beads (particle size range 2.8 μm, 30 mg / mL), wash the magnetic beads with 1 mL of 25 mM MES (pH 5.0) buffer, and vortex mix for 10 minutes, followed by magnetic separation. Repeat the washing of the magnetic beads 2-3 times.

[0094] b. Add 600 μg of PCT capture antibody (concentration of 1 mg / mL, add 600 μL) to the magnetic beads (antibody to magnetic bead coating ratio of 0.02 mg: 1 mg), and mix at low speed at room temperature for 30 minutes.

[0095] c. Prepare a 100 mg / mL EDC solution using 100 mM MES (pH 5.0) buffer, and use immediately after preparation.

[0096] d. Add 300 μL of EDC (i.e., 3 mg) to the reaction solution of magnetic beads and antibody and mix well. Add 100 μL of 25 mM MES (pH 5.0) buffer to a final volume of 1 mL (so that the magnetic bead reaction concentration is 30 mg / mL). Mix well by rotating at low speed at 4 °C for 2 hours.

[0097] e. Blocking and washing the coated magnetic beads: Mix the antibody-coated magnetic beads with 50 mM Tris at pH 7.4 at room temperature using a low-speed rotating motion for 15 minutes, or with 50 mM ethanolamine in PBS at pH 8.0 at room temperature using a low-speed rotating motion for 60 minutes to block excess reactive groups. Wash the antibody-coated magnetic beads four times with 1 mL of PBS or 50 mM Tris. During washing, 0.1% Tween-20 or Triton X-100 can be added to reduce non-specific binding. Then add 0.1%-0.5% BSA or skim milk powder. Finally, resuspend the magnetic beads in PBS buffer to the desired concentration (corresponding to a magnetic bead concentration of 0.25 mg / mL).

[0098] This reagent is the PCT-M reagent, used for subsequent immunoreactivity verification and clinical sample testing.

[0099] Example 2 - Antibody labeled with electrically neutral ruthenium complex (NRC)

[0100] use Figure 3 The NRC marker shown in C, namely Ru(2,2′-bipyridine)(red phenanthrene-line disulfonate) [4-(2,2′-bipyridine-4-yl)butyric acid], is used to label PCT antibodies to form NRC-labeled signal antibodies.

[0101] 2.5 mg (2.5 μmol) of NRC was administered at 5.0 mmol / L. -1 The concentration was dissolved in 500 μL of MES buffer (0.1 mol / L). -1 In a solution with pH = 4.7, 1.0 mg (5.2 μmol) of EDC and 3.0 mg (13.8 μmol) of sulfonated NHS were added to obtain a concentration of approximately 10 mmol / L. -1 EDC and 27 mmol L -1 The sulfonated NHS was added. The solution was shaken at room temperature for 10 minutes. 0.7 μL (10 μmol) of 2-mercaptoethanol was added to the above reaction solution (final concentration 20 mmol / L). -1 After incubation at room temperature for 5 minutes, 8.0 μL of this incubation solution (containing 40 nmol of NRC) was added to 500 μL of PCT antibody (1.2 mg / mL, approximately 4 nmol of pure PCT antibody, molar ratio 10) and 0.1 mol / L PBS. -1 Mix them in a solution (pH=7.4) and incubate at room temperature for 2 hours.

[0102] The solution obtained above (approximately 0.5 ml) was loaded onto a PD-10 column pre-equilibrated with PBS. Two yellow bands formed during separation. The first elution band corresponding to the labeled antibody (approximately 0.75 ml) was collected. The binding ratio of the labeled NRC to the antibody was determined to be 6.1:1.

[0103] The collected NRC-labeled antibody solution was further diluted to 1.0 μg / mL for later use. This reagent is the PCT-N reagent, used for subsequent immunoreactivity verification and clinical sample testing.

[0104] Example 3 - Validation of Immunoreactivity

[0105] A series of PBS solutions of different concentrations were prepared using PCT antigen as standard solutions. Immunoreactivity was verified using a programmable fully automated electrochemiluminescence immunoassay analyzer (Shenzhen Ansai Diagnostics Technology Co., Ltd. YnY series or ProScientia 2020) with different incubation times.

[0106] Using the sampling needle of the immunoassay analyzer, 30 μl of PBS solution with different concentrations of PCT was used as the analyte, and 85 μl of PBS solution with a concentration of 4 μg / mL was used as the analyte. -1 85 μl of PCT-M reagent and 85 μl of PCT-N reagent were mixed. Each mixture was incubated at 37°C for different times (2, 5, 10, 15, and 30 minutes). After the set incubation time, 150 μl of the above reaction suspension was injected into the three-electrode measurement cell of the automated immunoassay analyzer, with a photomultiplier tube above the working electrode and a movable magnet below it. When the reaction suspension was passed through the working electrode, which was maintained at a potential of 0.2 V, the immune complexes were retained on the electrode surface by the magnet located below the working electrode. Subsequently, the liquid circuit system aspirated phosphate buffer containing tripropylamine (pH 6.8, 0.18 mol / L). -1 Tripropylamine was used to clean the magnetic beads residing on the electrode surface. Finally, the magnetic beads, coated with antibody / antigen / antibody complexes, were placed in phosphate buffer (pH 6.8, 0.18 mol / L). -1 In tripropylamine, an ECL is generated by applying a 1.4V voltage between the working electrode and the reference (Ag / AgCl in saturated potassium chloride). A photomultiplier tube placed above the measuring cell collects the light signal, and the relative luminescence value of the ECL is obtained through mathematical processing. After each measurement, the measuring cell is cleaned with a 3.0V voltage, and the electrode is electrochemically regenerated (post-processing, see...). Figure 5 (Describe how the potential changes over time) for the next test.

[0107] The above test results, namely the signal values ​​at different incubation times and the relationship between signal and concentration, are presented in... Figure 6 and Figure 7 middle.

[0108] Example 4 - Verification of Biotin Interference Resistance

[0109] Dissolve biotin in 10 mol L -1 Prepare a sodium hydroxide solution with a concentration of 20 mg / mL -1 A biotin aqueous solution was prepared. 50 μL of this solution was added to 950 μL of mixed serum samples containing different concentrations of PCT to obtain a biotin concentration of 1 mg / mL. -1 Serum. This concentration (1 mg / mL) -1 Biotin is the concentration that may be reached in the blood of a person taking the maximum dose of biotin (300 mg daily). 3 -10 4 This is several times higher than the upper limit of the general evaluation reagent concentration for interference resistance. For comparison, 50 μL of 10 mol / L biotin-free solution was used...-1 Add 950 μL of sodium hydroxide solution to one serum sample.

[0110] The samples prepared above were used as test samples. Following the measurement method in Example 3, each sample was tested three times with a 5-minute incubation period. The average of the results was taken, and the relative deviation was calculated.

[0111] Tables 1 and 2 show the measured concentration and ECL signal values, respectively. The results indicate that even after adding extremely high concentrations of biotin, the changes in concentration and signal values ​​were less than an acceptable 10%. Blood immunoassays were unaffected by biotin in individuals taking the maximum dose of biotin year-round.

[0112] Table 1. Concentration measurements of two mixed serum samples after the addition of biotin.

[0113]

[0114] Table 2. Signal values ​​of mixed serum samples of two concentrations after the addition of biotin.

[0115]

Claims

1. The application of a reagent kit in electrochemiluminescence bioanalysis, said reagent kit being an electrochemiluminescence bioanalysis kit consisting of only the following two reagents: a) A buffer solution containing suspended magnetic beads, wherein the surface of the magnetic beads is coated with an antibody that has specific affinity recognition ability for the analyte. b) A buffer solution containing an antibody labeled with an electrochemiluminescent substance, wherein the antibody has specific affinity recognition ability for the analyte; in, The antibodies in the two reagents are different, and they recognize different sites of the analyte; the diameter of the magnetic beads is 0.2-5 micrometers; the electrochemiluminescent material is a ruthenium-containing neutral complex. In the electrochemiluminescence bioanalysis, the electrode pretreatment, magnetic bead trapping, and magnetic bead cleaning procedures before measurement include the following steps: a) Before the magnetic bead flows through the working electrode with the reaction liquid, a positive voltage and a negative voltage are alternately applied between the working electrode and the reference electrode, wherein the negative voltage is between -0.3V and -0.9V; b) While the magnetic bead flows through the working electrode with the reaction liquid, maintain the voltage between the working electrode and the reference electrode between 0.05V and 0.4V; c) After the magnetic beads are deposited on the working electrode, the voltage between the working electrode and the reference electrode is maintained between 0.05V and 0.4V, while the magnetic beads are cleaned with a phosphate buffer containing an organic amine, wherein the organic amine is a tertiary amine containing a straight-chain or branched alkyl group.

2. The application of a reagent kit in electrochemiluminescence bioanalysis, wherein the reagent kit is an electrochemiluminescence immunoassay kit consisting of only the following two reagents: a) A buffer solution containing suspended magnetic beads, wherein the surface of the magnetic beads is coated with an antibody that has specific affinity recognition ability for the analyte. b) Buffer reagents of analyte molecules or analyte molecule derivatives coupled to electrochemiluminescent substances; in, The magnetic beads have a diameter of 0.2-5 micrometers, and the electrochemiluminescent material is a ruthenium-containing electrically neutral complex. In the electrochemiluminescence bioanalysis, the electrode pretreatment, magnetic bead trapping, and magnetic bead cleaning procedures before measurement include the following steps: a) Before the magnetic bead flows through the working electrode with the reaction liquid, a positive voltage and a negative voltage are alternately applied between the working electrode and the reference electrode, wherein the negative voltage is between -0.3V and -0.9V; b) While the magnetic bead flows through the working electrode with the reaction liquid, maintain the voltage between the working electrode and the reference electrode between 0.05V and 0.4V; c) After the magnetic beads are deposited on the working electrode, the voltage between the working electrode and the reference electrode is maintained between 0.05V and 0.4V, while the magnetic beads are cleaned with a phosphate buffer containing an organic amine, wherein the organic amine is a tertiary amine containing a straight-chain or branched alkyl group.

3. The application of a reagent kit in electrochemiluminescence bioanalysis, wherein the reagent kit is an electrochemiluminescence immunoassay kit consisting of only the following two reagents: a) A magnetic bead suspension reagent encapsulated by analyte molecules or analyte molecule derivatives; b) A buffer solution containing an antibody labeled with an electrochemiluminescent substance, wherein the antibody has specific affinity recognition ability for the analyte; in, The magnetic beads have a diameter of 0.2-5 micrometers, and the electrochemiluminescent material is a ruthenium-containing electrically neutral complex. In the electrochemiluminescence bioanalysis, the electrode pretreatment, magnetic bead trapping, and magnetic bead cleaning procedures before measurement include the following steps: a) Before the magnetic bead flows through the working electrode with the reaction liquid, a positive voltage and a negative voltage are alternately applied between the working electrode and the reference electrode, wherein the negative voltage is between -0.3V and -0.9V; b) While the magnetic bead flows through the working electrode with the reaction liquid, maintain the voltage between the working electrode and the reference electrode between 0.05V and 0.4V; c) After the magnetic beads are deposited on the working electrode, the voltage between the working electrode and the reference electrode is maintained between 0.05V and 0.4V, while the magnetic beads are cleaned with a phosphate buffer containing an organic amine, wherein the organic amine is a tertiary amine containing a straight-chain or branched alkyl group.

4. The application according to any one of claims 1-3, wherein, The ruthenium-containing electrically neutral complex is Ru(2,2'-bipyridine)(bathophenanthroline disulfonate)[4-(2,2'-bipyridin-4-yl)butanoic acid].

5. The application according to any one of claims 1-3, wherein, The organic amine is tri-n-propylamine.

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