CN115480055A - Electrochemiluminescence immunoassay kit and use method thereof - Google Patents

Abstract

Most immunoassays involve biotin/(streptavidin) coupling chemistry, and biotin in blood samples interferes with the correct output of immunoassay results. The prior art using electrochemiluminescence as the detection signal for immunoassays is, without exception, established on the biotin/(streptavidin) coupled platform with the potential to cause clinical false detection. The invention relates to an electrochemiluminescence immunoassay reagent system which is completely free of biotin and (streptoase) avidin components, so that biotin interference in a sample can be avoided. Further, the invention provides a method for capturing and separating magnetic beads in an electrochemiluminescence measurement process aiming at the change of reagent components and the structural change of immune complexes on the surfaces of the magnetic beads caused by the change of the reagent components.

Description

Electrochemical luminescence immunoassay kit and use method thereof

Technical Field

The invention belongs to the technical field of electrochemiluminescence immunoassay, and particularly relates to an electrochemiluminescence immunoassay kit and a using method thereof.

Background

Biotin (biotin), a water-soluble vitamin (B7 or H) naturally present in some foods, is involved in various metabolic processes of the human body, and its strong noncovalent interaction with avidin or streptavidin has long been used for analysis, separation and purification of bioactive substances (e.g., hormones of small molecules, etc., nucleic acids of large molecules, proteins, etc.). The small molecular weight of biotin (244.31D) and the rapid formation of biotin- (streptokinase) avidin complexes in different solvents and their high stability (dissociation constant Kd of about 10 for biotin-avidin complexes) ^-14 Whereas avidin is about 10 for biotin-streptokinase ^-15 ) Biotin- (streptoenzyme) avidin chemistry was established as a platform technology for multiple clinical immunoassay methodologies and is 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, since the pentanoic carboxyl group (FIG. 1) in the biotin molecule is reactive, it can be coupled directly or indirectly (e.g., by conversion to an active ester) to other biomolecules. When necessary, the pentanoic carboxyl group in the biotin molecule can be connected with a connecting unit (1 inker) or a spacer unit (spacer) for increasing the space distance, so that the diheterocycle composed of the imidazolidinone ring and the tetrahydrothiophene ring in the biotin molecule can be easily combined with avidin or streptavidin on the surface of the biomacromolecule in the subsequent operation process. Raw materialThe process of attaching one or more imidazolidinone/tetrahydrothiophene rings to a biomolecular moiety via the pentanoic carboxyl group of the molecule is known as biotinylation (biotinylation), while the antibody to which the biotin moiety is attached is known as a biotinylated antibody (biotinylated antibody), and in competitive immunoassays, the biotinylated moiety may also be the analyte antigen (biotinylated antigen) or an antigen derivative.

Despite the lack of strong evidence to support its benefits for hair, skin and nail growth, biotin is increasingly being ingested as a nutritional supplement by patients with alopecia, fragile hair or hirsutism. Women at this stage are also recommended to use biotin due to increased biotin metabolism during pregnancy and nursing, although the effect is controversial. Because biotin in a blood sample will compete with biotin-conjugated antibodies (or antigens) for (streptavidin) on the solid surface, the results of all immunoassays (such as the certain immunoassay platform technologies listed in fig. 2) built on the biotin-streptavidin conjugated chemical platform will be disturbed to varying degrees by biotin in the sample, causing either positive (in the competitive method) or negative (in the sandwich method) bias. The biotin obtained by ordinary people through normal diet has little content in blood, and does not influence the result of immunoassay. However, cases of Interference of Biotin, which enters the blood by ingestion of nutritional supplements, with the results of immunoassays were reported as early as 1996 (J.G.Henry, S.Sobki, N.Arafaty, interference by Biotin Therapy on Measurement of TSH and FT4 by enzyme immuno assay, biochem.1996, 33, 162-163), while those of Biotin Interference in electrochemiluminescence immunoassays have also been reported in 2009 (D.L.Meany, S.M.Jan de beer, M.J.Birata, 1and L.J.Sokoll, A.of Secondary enzyme with connected ultrasound syndrome, 17355, 1739-2009). The immunoassay of biotin Interference which has been reported so far involves NT-pro-BNP, hs-TnT, TSH, FSH, anti-Tg, anti-TPO, anti-TSHR, etc. in sandwich immunoassay, as well as T3, T4, vitamin D, etc. in competition methods (D.Li, A.Raduscu, R.T. Shrestha, association of biotin investment with performance of hormon and nonhormolone assays in health adults.JAMA2017, 318, 1150-1160, P.J.Colon 1and D.N.Greene, biotin Interference in Clinical Immunoasssays, J.Appl.Lab.Med.2018,2, 941-951, jielii Li, elizabeth A, wagar, qi H.Meng, comparative assessment of biotin interaction in immunologic assays, clinical Chia Acta,2018, 487, 293-298.. Biotin still interferes with Thyroid function testing even within 24 hours after a single 10mg oral dose (r.p.m. biscolia, m.i. chia, i.kanashiro, r.m.b. macel, j.g. h.vieira, a single 10mg oral dose of biotin intermediates with Thyroid functions tests, thyroids 2017, 27, 1099-1100.). On 28/11/2017, the U.S. Food and Drug Administration (FDA) issued warnings of biotin interference in clinical immunoassays (FDA. The FDA warns that biological proteins with their own interfaces with tags tests: FDA safety communication.). Two years later (11/5/2019), the FDA updates this warning. The purpose of the warning is to alert consumers, healthcare workers and clinical laboratory staff to the biotin that may be added to the nutritional supplement and its effect on the test results. The FDA recommended biotin dose that does not interfere with the test is no more than 0.03mg per day for adults. However, the doses of nutritional supplements on the market that claim to be beneficial to the hair, skin or nails may reach 20mg per day for adults, whereas the biotin dose prescribed by physicians for multiple sclerosis may be as high as 300mg per day for humans. Biotin levels in blood samples from patients taking high doses of biotin can be as high as 100-1200 ng/mL.

Although the metabolism of biotin is rapid and not all blood samples of people taking biotin have an effect on the results of immunoassay tests, the effect of biotin on clinical immunoassay results has become a problem that must be paid attention to in clinical immunoassays with the increasing population taking biotin and the widespread use of large doses of biotin. The american society of clinical chemistry compiles misdiagnosis cases due to Biotin Interference from various countries in their guidelines for Biotin Interference clinical immunoassays published in 2020 (d.li, a.ferguson, m.a.cervinski, k.l.lynch, p.b.kyle, AACC guide Document on Biotin Interference in Laboratory tests.j.appl.lab.med.2020, 5, 575-587.).

The following are recommended procedures for avoiding biotin interference and methods for reducing or eliminating biotin interference.

1. Precautions against biotin interference (Jieli Li, elizabeth A. Wagar, qing H.Meng, comprehensive assessment of biotin interference in immunological assays, clinical Chimica Acta,2018, 487, 293-298):

asking the patient whether to take a biotin containing drug/nutraceutical;

recording time and amount of intake; .

The patient is asked to stop taking biotin or biotin-containing drugs/nutrients 48 hours before drawing the blood sample;

if the daily dose is greater than 5mg, a blood sample is taken at least 8 hours after the last dose;

timely communication with the laboratory if the test results are inconsistent with the patient's clinical presentation and/or biotin interference is suspected.

2. The sample is pretreated prior to the immunoassay test, such as with streptavidin-coated magnetic beads to remove biotin (m. -l. Picketty, d. Prie, f. Sedel, d. Bernard, c. Hercend, p. Chanson and j. -c. Sourbnielle, high-dose biotin binding to nucleic acid biochemical profiles: evaluation of a simple method to interference biological reference, clin Chem Lab Med,2017, 55, 817-825C Trambas, Z.Lu, T.Yen and K.Sikaris, duplication of using a labeled solution to the protocol of a biological interference in a polypeptide-biological assay, ann.Clin.Biochem.,2018, 55216-226 H.M.Stiegliitz, N.Korpi-Steiner, characterization of biological interference in a polypeptide interaction 56021 organisms and analysis of biological samples simulation, simulation of biological samples 53-61, 75, 817-825C

3. The reaction sequence was changed by combining streptavidin-coated magnetic beads with biotinylated antibodies to form antibody-coated magnetic beads prior to antibody/antigen immunoreaction, (L.Johnson and D.Li, stratum to induced biological in interference in light of the FDA safety communication, J.applied. Lab.Med.,2019,3, 914-915

4. Binding biotin with a monoclonal antibody to a specific biotin; patent application WO 2018/122043Al discloses a biotin mab that binds only free biotin and not biotin conjugated to a biomacromolecule such as an antibody. The ECL immunoassay reagent modified by adding this specific biotin mab to the reagent reaction system effectively neutralizes biotin added to the blood sample against 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 control T assay with amplified free distance to biotin, clin Chem Lab Med,2021, 59, 591-597.).

Electrochemiluminescence (ECL) or electrochemiluminescence (electrochemiluminescence) immunoassays are immunoassay methods widely used in clinical examination. In ECL immunoassays, ruthenium terpyridyl (commonly expressed as Ru (bpy)) ₃ ²⁺ ) The succinimidyl ester (NHS ester, see FIG. 3A) or other luminescent metal complexes of (A) are used as labels to label the antibody or antigen of the test substance. After the antibody reacts with a detected object in a sample under certain conditions to form an antibody/antigen complex, the luminescent metal complex finally forms a luminescent excited state of the luminescent metal complex through electrochemical reaction and a series of subsequent chemical reactions in an electrochemical flow cell, and a detectable luminescent signal is generated.

ECL immunoassays involve how antibodies (in a sandwich assay) or analytes (in a competition assay) are labeledNote many technical details of how the analyte is captured, how the ECL reaction is triggered, and how the working electrode is regenerated. In a typical commercial ECL assay, using the labeling molecule shown in figure 3A, an antibody (signal antibody) is labeled at the epsilon amino site of the lysine residue of the antibody of one analyte, while the other antibody (capture antibody) is biotinylated, using the ECL sandwich immunoassay as an example. When a clinical sample is mixed with the two types of antibodies and streptavidin-coated magnetic beads at a predetermined temperature for a predetermined period of time, a sandwich immune complex is formed on the surface of the magnetic beads. The magnetic beads are then brought into an electrochemiluminescence measurement cell (flow cell) and captured at the surface of the electrochemical working electrode by a movable magnet located below the measurement cell. Containing tripropylamine (tri-N-propylamine, TPA or N (C) ₃ H ₇ ) ₃ ) The buffer solution washes out unwanted substances and provides a means for Ru (bpy) ₃ ²⁺ The luminophore undergoes the chemical environment of the ECL reaction described in reaction pathway one below.

At a specific voltage (e.g., 1.4V vs. Ag/AgCl), tripropylamine in the buffer solution is oxidized to cationic free radical N (C) ₃ H ₇ ) ₃ ^·+ (reaction 1) and further loses a proton to form a neutral radical H ₆ C ₃ ·N(C ₃ H ₇ ) ₂ (reaction 2). The neutral radical has strong reducing ability, and can react with Ru (bpy) ₃ ²⁺ Reduction to Ru (bpy) ₃ ¹⁺ (reaction 3). Cationic radical N (C) having oxidizing power ₃ H ₇ ) ₃ ^·+ Ru (bpy) ₃ ¹⁺ Oxidation to excited Ru (bpy) ₃ ^2+* (reaction 3). Ru (bpy) ₃ ^2+* Emits light with a wavelength of 620nm and returns to the original Ru (bpy) ₃ ²⁺ The ground state (reaction 5).

Reaction pathway I

N(C ₃ H ₇ ) ₃ -e ^- →N(C ₃ H ₇ ) ₃ ^·+ (1)

N(C ₃ H ₇ ) ₃ ^·+ -H ⁺ →H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ (2)

Ru(bpy) ₃ ²⁺ +H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ →Ru(bpy) ₃ ⁺ +P (3)

Ru(bpy) ₃ ⁺ +N(C ₃ H ₇ ) ₃ ^·+ →Ru(bpy) ₃ ^2+* +N(C ₃ H ₇ ) ₃ (4)

Ru(bpy) ₃ ^2+* →Ru(bpy) ₃ ²⁺ +hυ (5)

Thus, in the ECL process, ru (bpy) ₃ ²⁺ Is not consumed but undergoes a cyclic change of oxidation state, i.e., 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). This cycle is repeated during the measurement, and a long-delayed ECL signal is generated and detected. The integral of the total ECL light emission over a certain period of time (e.g., 0.5-5 seconds) can be used as a measure of ECL intensity and correlated to the amount of material being measured. After the measurement is finished, the magnetic beads and the immune complexes attached to the magnetic beads are washed away by the aqueous liquid flow, the measuring cell is cleaned, and the surface of the electrode is regenerated through an electrochemical process to be in a preparation state for the next test. Details of these chronologically conducted experiments are disclosed in U.S. Pat. Nos. 5,147,806,5,538,687 and 6,599,473.

In fact, reaction pathway one is only one that can generate ECL. Other possible reaction pathways (e.g., as follows)Reaction route two to four) Also proposed to explain the excited state Ru (bpy) under different conditions ₃ ^2+* Formation of and production of ECL (see j.k.leland 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).

Reaction pathDiameter two

Ru(bpy) ₃ ²⁺ -e ^- →Ru(bpy) ₃ ³⁺ (6)

N(C ₃ H ₇ ) ₃ -e ^- →N(C ₃ H ₇ ) ₃ ^·+ (1)

N(C ₃ H ₇ ) ₃ ^·+ -H ⁺ →H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ (2)

Ru(bpy) ₃ ³⁺ +H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ →Ru(bpy) ₃ ^2+* +P (7)

Ru(bpy) ₃ ^2+* →Ru(bpy) ₃ ²⁺ +hυ (5)

Reaction route three

Ru(bpy) ₃ ²⁺ -e ^- →Ru(bpy) ₃ ³⁺ (6)

Ru(bpy) ₃ ³⁺ +N(C ₃ H ₇ ) ₃ →Ru(bpy) ₃ ²⁺ +N(C ₃ H ₇ ) ₃ ^·+ (8)

N(C ₃ H ₇ ) ₃ ^·+ -H ⁺ →H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ (2)

Ru(bpy) ₃ ³⁺ +H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ →Ru(bpy) ₃ ^2+* +P (7)

Ru(bpy) ₃ ^2+* →Ru(bpy) ₃ ²⁺ +hυ (5)

Reaction pathway four

Ru(bpy) ₃ ²⁺ -e ^- →Ru(bpy) ₃ ³⁺ (6)

N(C ₃ H ₇ ) ₃ -e ^- →N(C ₃ H ₇ ) ₃ ^·+ (1)

N(C ₃ H ₇ ) ₃ ^·+ -H ⁺ →H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ (2)

Ru(bpy) ₃ ²⁺ +H ₆ C ₃ ^· N(C ₃ H ₇ ) ₂ →Ru(bpy) ₃ ⁺ +P (3)

Ru(bpy) ₃ ⁺ +Ru(bpy) ₃ ³⁺ →Ru(bpy) ₃ ²⁺ +Ru(bpy) ₃ ^2+* (9)

Ru(bpy) ₃ ²⁺ *→Ru(bpy) ₃ ²⁺ +hυ (5)

The above reaction paths two, three and four occur at electrode voltages high enough to hold Ru (bpy) ₃ ²⁺ Oxidation to Ru (bpy) ₃ ³⁺ (i.e., reaction 6) in the absence of Ru (bpy) in the several reactions involved in reaction pathway one ₃ ²⁺ Is oxidized to Ru (bpy) ₃ ³⁺ Reaction 6 of (3). However, under high voltage conditions that can cause reaction pathways two, three, and four to occur, the reaction in reaction pathway one also occurs simultaneously. Although researchers in the field tend to believe that the vast majority of ECL light emission comes from reaction pathway one, the operating voltage used in a practical ECL immunoassay system is 1.4V (relative to an Ag/AgCl reference electrode), at which voltage reaction pathways one, two, three and four can occur.

US 10203333 and chinese patent ZL 201480045420 disclose coordination compounds of a class of electrically Neutral metal Ruthenium (NRC). These electrically neutral ECL labels can reduce non-specific signals in immunoassays, and some electrically neutral ECL label molecules (such as the NRC of figure 3C) also produce more luminescence. U.S. patent applications US 2021/0130876 A1 and WO 2021/084472 A1 further disclose labeled molecules containing two or more ECL emitters. These better performing ECL emitters and their constituent marker molecules enrich the electrochemiluminescence immunoassay methodology-as chemiluminescence has multiple platform technologies based on different chemiluminescence emitters, electrochemiluminescence also encompasses multiple platforms based on different ECL emitters (as shown in figure 2).

In the prior art, the kit used for the clinical immunoassay of ECL consists of three parts of reagents. In the sandwich method, the three reagents are a biotin-conjugated capture antibody reagent, a luminescent label (e.g., ru (bpy) ₃ ²⁺ ) A labeled signal antibody reagent and a streptavidin-coated magnetic bead; in the competitive method, the three reagents may be biotin-conjugated antigen reagent, luminescent label (e.g., ru (bpy)) ₃ ²⁺ ) A labeled signal antibody reagent and a magnetic bead wrapped by streptavidin; alternatively, biotin conjugated capture antibody reagent, luminescent labels (e.g., ru (bpy) ₃ ²⁺ ) Labeled antigen reagent and streptavidin-coated magnetic beads.

Since the reagent system of all ECL clinical immunoassay in the prior art uses streptavidin-coated magnetic beads as a reagent component, biotin possibly present in the sample will compete with a biotin coupling component (biotin-coupled capture antibody reagent or biotin-coupled antigen reagent) in the reagent for streptavidin binding sites on the surface of the magnetic beads, resulting in unreal signal generation.

The aforementioned methods for reducing or eliminating biotin interference all have some effect on ECL immunoassays, but clearly have various problems. The solution proposed by the user side increases the complexity of the test and is not effective for all situations. The use of specific biotin mabs by ECL reagent manufacturers would greatly increase reagent costs. The present invention provides a two-component kit that is different from existing ECL immunoassay reagent systems. The kit does not contain a streptavidin component, so that interference of biotin possibly existing in a clinical ECL immunoassay sample on a test result is avoided.

Although the problem of biotin interference has long been known, the technical reasons why ECL reagent developers have not abandoned the biotin-streptavidin chemical system are several.

1. In the existing three-reagent reaction system, an immune complex [ antibody/antigen/antibody ] or [ antigen/antibody ] is formed in a homogeneous reaction, and after the immune complex is formed, the immune complex is anchored on the surface of a magnetic bead through a biotin/streptavidin reaction. Theoretically, the antibody-antigen binding reaction has better repeatability in homogeneous phase, thus leading to better result repeatability of repeated tests on the same sample.

2. Unlike other immunoassay techniques that also use magnetic beads as solid phase carriers and chemiluminescence as the detection signal, electrochemiluminescence is generated on the surface of magnetic beads deposited on two-dimensional planar electrodes, while other chemiluminescence signals are generated on magnetic beads suspended in three-dimensional solution; furthermore, the electrochemical reaction only occurs at the interface at a distance of 1-2 nm from the electrode surface, and therefore, any change in the surface chemistry of the magnetic beads will likely affect the electrochemical reaction. Abandoning the biotin-streptavidin chemical system does not guarantee that the electrochemiluminescence immunoassay established on the biotin-streptavidin chemical platform in the early development stage of the ECL technology can also have comparable analytical performance;

3. unlike chemiluminescence immunoassay, in an electrochemiluminescence immunoassay method, magnetic separation of immune complexes formed on the surfaces of magnetic beads is performed in an electrochemical flow cell. In this process, a quantity of the reaction mixture is drawn across the surface of the working electrode in the electrochemical flow cell at a certain flow rate and held at the electrode surface by the magnet located below the working electrode. In ECL immunoassays, this process is referred to as the magnetic bead capture process. Since any change in the surface chemistry of the magnetic beads may change the fluid dynamics of the magnetic beads in the fluid path, the uniform distribution of the immune complex-loaded magnetic beads on the surface of the electrode may also be affected. Therefore, abandoning the biotin-streptavidin chemical system will result in the magnetic separation process of the magnetic beads on the electrode surface being out of control, and will also change the uniform distribution of the magnetic beads on the electrode surface, resulting in the performance index of the ECL immunoassay being reduced.

Prior to the method provided by the present invention, there was a general concern based on the above-mentioned reason 1 that the two-reagent system changes the antibody-antigen binding reaction from a homogeneous (buffer solution) to a heterogeneous (magnetic bead surface) reaction, resulting in a deterioration of the precision of the assay (usually expressed by the coefficient of variation). However, surprisingly, the systems described in the examples of the present invention all give reaction repeatability comparable to the three reagent system.

Causes 2 and 3 above are unique to ECL immunoassays. Before the present invention, based on the above-mentioned reason 2, there was a fear that the absence of biotin- (streptavidin) hierarchical layers between antibodies and magnetic beads would change the signal contribution of different paths of the electrochemiluminescence reaction and also could enhance non-specific adsorption, thereby changing the established concentration response relationship and not facilitating the electrochemiluminescence technology to reach its optimal working state, however, one of the surprises in practicing the present invention is that the ECL could still reach its optimal working state after discarding biotin and streptavidin components.

The inventors of the present invention have surprisingly found that, by trying a lot of mistakes to solve the above-mentioned concerns caused by reason 3, when the magnetic beads flow over the surface of the working electrode, the electrode is 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, which not only helps to increase the signal value, but also helps to distribute the magnetic beads more uniformly over the surface of the electrode.

Disclosure of Invention

The invention provides a two-component kit which is different from the conventional ECL immunoassay three-component reagent system. The reagent consists of two components, namely antibody-coated magnetic beads and ECL luminescent marker-labeled antibodies (or antigens). The kit does not contain streptavidin-coated magnetic beads and biotin-coupled antibodies (or antigens), so that the interference of biotin possibly existing in clinical ECL immunoassay samples on test results is avoided. Further, the invention provides an improved magnetic bead capture mode aiming at the use of the reagent system without streptavidin, namely, magnetic separation is carried out under the electrode potential state higher than 0.0V.

Drawings

The present invention will be described in conjunction with the following drawing figures, wherein like numerals denote like elements, and wherein:

FIG. 1 chemical structural formula of biotin

FIG. 2 conventional immunoassay based on different detection signals

FIG. 3 ECL-labelled molecules disclosed in U.S. Pat. No.5,744,367 (A), U.S. Pat. No. 6,808,939 (B), WO 2014203067A1 (C), and U.S. patent application No. US2016/0145281A1 (D);

FIG. 4 comparison of the surface state of magnetic beads after the incubation reaction in ECL immunoassay is completed: (left) prior art; (right) the invention;

FIG. 5 the change in working electrode potential with time during a measurement cycle (the dashed line is the potential setting in the prior art);

FIG. 6 signal rise with incubation time in ECL immunoassay;

figure 7 signal versus concentration for different incubation times in ECL immunoassays.

Detailed Description

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. These terms and meanings are explained fully in the technical literature, for example in "bioconjugation technology" (g.t. hermanson, elservier, amsterdam, 2008) and "immunoassay manual" (d.wild et al, 4 th edition, elservier, amsterdam, 2013).

Within the scope of the present invention, an "analyte" includes, but is not limited to, the presence of 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, 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, enzymes, enzyme substrates, enzyme products, nucleic acid processing enzymes (e.g., polymerases, nucleases, integrases, ligases, helicases, telomerase, etc.), protein processing enzymes (e.g., proteases, kinases, protein phosphatases, ubiquitin ligase, etc.), cell metabolites, endocrine factors, paracrine factors, organometallic, cytokines, hormones, pharmacological drugs, pharmaceuticals, therapeutic drugs, synthetic organic molecules, barbitumens, biotins, salts, biotins, amino acid salts, lectins, recombinant amino acid molecules, lectins, or lectin affinity molecules.

An "analyte-specific agent" (ASR) according to the methods and reagents of the present invention is a class of molecules or biomolecules that have the ability to specifically bind to an analyte, such as antibodies, polyclonal and monoclonal antibodies, specific receptor proteins, ligands, nucleic acid sequences, and the like. They are intended for use in bioanalytical applications to identify and quantify individual chemical or biochemical substances or ligands in a biological sample by specifically binding or specifically reacting with a substance in the sample.

The term "ASR-coated magnetic beads" as used in the context of the present invention refers to a complex of a specific analyte reagent and magnetic beads via functional groups on the surface of the magnetic beads, wherein the specific analyte reagent can be covalently bound to the surface of the magnetic beads or adsorbed thereto.

The substances within the scope of the present invention, referred to as "labels", "label molecules", "ruthenium (II) labels" and "ECL labels", may be covalently bound to other substances such as a biologically active analyte or analog thereof, a bioaffinity-based analyte recognition partner or analog thereof (e.g., an analyte-specific reagent), and other binding partners for such recognition partners, or a reactive chemical capable of forming a covalent bond with the analyte, or an analog or binding partner thereof as described above. The above substances may also be combined with one or more binding partners and/or one or more combinations of reactive components. Alternatively, the above substances may be bound to the analyte or analog thereof to which the binding partner, reactive component, or combination of one or more binding partners and/or one or more reactive components is attached. It is also within the scope of the invention to bind a plurality of the above substances to the analyte or analog thereof directly or via other molecules as described above.

As used herein, the term "label" refers to any chemical or biochemical substance that, by itself or through physical/chemical interaction with other reagents, produces a detectable signal (whether a visible signal or a signal detectable by use of a suitable instrument) that can be correlated with the amount of target analyte. Labels include, but are not limited to, molecules containing radioactive atoms (radioactivity), luminescent compounds (which emit light by photoexcitation or by a chemical reaction), electroactive compounds (which generate an electrical signal by a redox reaction), magnetic particles (which generate a magnetic signal), enzymes (which generate a detectable substance or an optical signal by reaction with a substrate), enzymes, or enzymatic substrates (which catalyze chemical/biochemical reactions). A label may be composed of one or more signal producing units and one or more reactive groups.

The term "luminescence" refers to the energy released in the form of electromagnetic radiation (light emission) when an electron is converted from a low energy state to an "excited" high energy state and then falls back to a lower energy state. This emission of light typically occurs in the visible or near visible spectrum 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, but in the present invention, luminescence is electrochemiluminescence, unless specified otherwise.

In the context of the present invention, the terms "luminophore" and "luminophore" refer to the functional group of a compound which is responsible for the luminescence phenomenon. In a compound having a complex structure, for example, a structure having a plurality of functional groups (e.g., a reactive group, a hydrophilic/hydrophobic/amphiphilic group, an electron-withdrawing/electron-donating group, an electrically balancing group, a spacer group, a linking group, a branched group, etc.), a light-emitting group is the smallest structural portion (e.g., see the circled portion in fig. 1) necessary for generating a light-emitting phenomenon.

The term "luminescent label" refers to a label consisting of one or more luminescent groups and one or more reactive groups, which is susceptible to form a covalent bond with a chemical or biochemical molecule to be labeled. The luminescent label may be, for example, a fluorescent molecule, a phosphorescent molecule, a radioluminescent molecule, an electrochemiluminescent molecule in the present invention (i.e. ECL label), or a quantum dot with a reactive group on the dot surface. However, in the present invention, unless otherwise specified, the "luminescent marker" is an electrochemiluminescent marker (ECL marker). Examples of Electrochemiluminescent (ECL) labels having one luminescent group and one reactive group are most frequently disclosed in the prior art (see, e.g., the label in fig. 1and the other ruthenium complex labels in WO2003002974A2, WO 2014203067A1 and the iridium complex label in WO2014019711 A1). Examples of luminescent labels having three luminescent groups (three ruthenium complex units) and one reactive group (-COOH or NHS ester) are disclosed in US 2005/0059834 A1. Us patent 6140138 discloses examples of luminescent labels having one luminescent group (one ruthenium complex) and two reactive groups (-COOH or NHS ester). ECL labeled molecules containing two or more luminescent groups disclosed in Chinese patent application 202010983872.X, U.S. patent application 2021/0130876 A1 and WO 2021/084472 A1

According to the present application, a "detection reagent" includes an analyte-specific reagent (ASR) labeled with at least one ECL luminophore, or an analog/homolog of the analyte labeled with one ECL luminophore. As known to those skilled in the art, in an assay, the detection reagent is ultimately immobilized on a solid phase. "solid phase", also referred to as "solid support", 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; a capillary tube, which may be made of polymer, metal, glass and/or ceramic; zeolites and other porous materials; an electrode; a microtiter plate; a solid bar; and a cuvette, test tube, wafer, or other sample container of the spectrometer. The components of the solid phase during the assay differ from the inert solid surface to which the assay may be exposed in that the "solid phase" contains on its surface at least one moiety intended to interact with the capture antibody or capture molecule. The solid phase may be a fixed component, such as a test tube, strip, cuvette, sheet, or microtiter plate, or a non-fixed component, such as magnetic beads, and microparticles.

In one embodiment, the method may be performed in a sandwich assay format. In one embodiment, the method may be performed as a competitive assay. In one embodiment, the method may also be performed as a dual antigen bridging assay format (DAGS). Known immunoassay formats are described in detail in the following books: wild et al, "handbook of immunoassays, 4 th edition, elservier, amsterdam (2013) and e.p. diamondis and t.k. christopoulos," immunoassays, san diego, academic press (1996).

An "electrochemiluminescence immunoassay" or "ECL immunoassay" is an assay in which an ECL luminophore produces a luminescent signal by electrochemical excitation. A voltage between the working electrode and the reference electrode electrochemically initiates luminescence from ECL luminophores bound to the ASR or the analyte analogue/homologue. The light emitted from the ECL luminophores is measured by a photodetector and indicates the presence or quantity of the target analyte. ECL processes are described in detail in U.S. patent nos. 5,543,112, 5,935,779, and 6,316,607.

The term "operating voltage" in the present invention is a key concept and experimental parameter of the present invention. In a three-electrode electrochemical measurement system consisting of a working electrode, a counter electrode and a reference electrode, the "working voltage" is the voltage between the working electrode and the reference electrode. Unless otherwise specified, the working voltage or voltages referred to herein are +0.197V relative to a silver/silver chloride (Ag/AgCl, saturated potassium chloride) reference Electrode and the Electrode potential relative to a Standard Hydrogen Electrode (SHE). In the case of generating electrochemiluminescence or performing electrochemiluminescence immunoassay, different instrument systems may be provided with different reference electrodes, and the voltage between the reference electrode and the working electrode is different from the working voltage in the present invention, and the difference can be obtained by converting the electrode potential of the reference electrode. Those skilled in the art know that the electrode potentials of different reference electrodes relative to a Standard Hydrogen Electrode (SHE) and their scaling methods can be found in the literature and books concerning electrochemistry. One approach to the kits of the present invention is to increase the signal by adjusting the "working voltage" during the capture of the magnetic beads.

In an ECL assay procedure, magnetic beads can be suspended in the sample and detection reagents to effectively bind the analyte. The magnetic beads may have a diameter of 0.05 μm to 200 μm, 0.1 μm to 100 μm, or 0.5 μm to 10 μm, and have a surface component capable of binding biomolecules. In the ECL analysis system (Sensai diagnostics YnY 2020, ynY2050, and YnY 3030 systems) used by the present applicant, the diameter of the magnetic beads was 2.8. Mu.m. Magnetic beads may be formed from: organic polymers, polystyrene, styrene copolymers such as styrene/butadiene copolymers, acrylonitrile/butadiene/styrene copolymers, vinyl acetoacetate copolymers, vinyl chloride/acrylate copolymers, inert inorganic materials, chromium dioxide, iron oxides, silica mixtures, proteinaceous matter or mixtures thereof,

according to the present application, a "reagent component" comprises a reagent that supports ECL signal generation, such as a co-reactant (e.g. tripropylamine TPA), a buffer for pH control, a surfactant, a preservative or an antimicrobial, and optionally other components. The skilled artisan is aware of the components present in the reagent composition required to generate ECL signals in an electrochemiluminescence detection method.

As used herein, an "aqueous solution" is a homogeneous solution of particles, substances or liquid compounds dissolved in water, or a heterogeneous suspension with microparticles (from 0.05 μm to 200 μm in diameter) suspended in an aqueous solution. The aqueous solution may also contain an organic solvent. Organic solvents are known to the person skilled in the art, for example amines, methanol, ethanol, dimethylformamide or dimethyl sulfoxide. As used herein, it is also understood that the aqueous solution may comprise up to 50% organic solvent.

Substances that participate in the ECL process with ECL markers are referred to herein as ECL "co-reactants. Common co-reactants used in ECL include tertiary amines (e.g., tri-n-propylamine TPA) and their analogs/homologs (e.g., 2- (dibutylamino) ethanol, etc.), oxalates, and persulfates. Those skilled in the art are aware of co-reagents that can be used in ECL detection methods.

As used herein, "transition metal complex" relates 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, dysprosium; in another embodiment, the transition metal is ruthenium, iridium, rhenium, or osmium; in a further embodiment, the transition metal is ruthenium or iridium.

In one embodiment, the ECL luminophore is a complex of a class of electrically neutral metallic ruthenium as disclosed in US patent 10203333 and chinese patent ZL 201480045420.

In another embodiment, the ECL luminescent group is an iridium complex and is selected from the following ECL labels. Ir (6-phenylphenanthridine) ₂ Pyridine-2-carboxylic acids 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 esters, or derivatives thereof, such as iridium complexes in which the ligands are substituted with one or more sulfonic acids, or iridium complexes as described in WO2012107419 (A1), WO2012107420 (A1), WO2014019707 (A2), WO2014019708 (A1), WO2014019709 (A2), WO2014019710 (A1), WO2014019711 (A1). As is well known to those skilled in the art, the iridium (III) complex has poor solubility in aqueous solutions, and hydrophilic derivatives of the ECL compounds described above may be used. Thus, in another embodiment, the iridium (III) ECL emissive groups described above can be modified with hydrophilic substituents. In another embodiment, the ECL luminogenic group is an iridium complex with two phenylphenanthridine ligands with two sulfonylpropoxy groupsA substituent group, two sulfomethyl groups comprising 2, 9-phenanthridine dimesylate, 6-phenyl-sodium salt (CAS registry number 1554465-50-7), or two polyethylene glycol substituents, or three of the above groups per phenylphenanthridine ligand, or a combination of the above groups per phenylphenanthridine ligand.

In another embodiment, the ECL label is a multi-labeled ECL labeled molecule as disclosed in Chinese patent application 202010983872.X, U.S. patent application 2021/0130876 A1, and WO 2021/084472 A1.

As known to those skilled in the art, in ECL immunoassay, an ECL reaction is triggered by a constant operating voltage, which ultimately causes an ECL luminophore immobilized on the surface of a magnetic bead to generate an optical signal. The time for constant voltage excitation is generally between 0.2 and 10 seconds, preferably between 1.0 and 3.0 seconds. During this voltage excitation, the ECL luminescence signal gradually decays. The area under the ECL luminescence decay curve over time is the relative luminescence intensity (RLU). In a particular immunoassay, different RLUs correspond to different analyte concentrations. The signal response versus concentration in RLU is usually described by a functional relationship (linear or non-linear) that is fit. The five fitted curves in FIG. 7 conform to the four parameter logistic equation.

It is also known to those skilled in the art that in an automated ECL testing system, there is typically a pretreatment procedure for the electrode surface that is applied prior to sample testing to ensure that the electrode surface remains at the same surface chemistry prior to testing. After a test is finished, the electrochemical measuring cell and the electrode surface need to be cleaned and regenerated. These steps are disclosed in us 5147806 and us 6599473 further discloses an improved solution. In describing these steps, the prior patent uses terminology of preoperative, conditioning, cleaning. FIG. 5 depicts an electrochemiluminescence signal generation procedure comprising pre-processing and testing steps. In different ECL systems, the pre-treatment and washing procedures may be different, for example, the electrochemical procedure disclosed in us patent 6599473 adds a voltage pulse believed to improve the state of deposition of magnetic beads on the electrodes. The present invention does not involve the modification of the post-measurement cleaning procedure and post-electrode treatment, and the present invention modifies the pre-treatment phase and the capture phase by changing the negative voltage pulse from-1.2V to-0.5V and increasing the voltage of the working electrode from 0.0V to between 0.05 and 0.40V during the capture phase.

The present invention is further illustrated by the following description of specific embodiments, which are not intended to be limiting, and it will be apparent to those of skill in the art that modifications may be made to the various embodiments without departing from the spirit of the invention (i.e., the reagent composition does not contain streptavidin), but rather the embodiments are within the scope of the invention.

Examples

These and other advantages of the present invention will be apparent to those skilled in the art from the foregoing description. Accordingly, those skilled in the art will recognize that changes or modifications may be made to the above-described embodiments without departing from the broad inventive concept thereof. It is understood that the invention is not limited to the particular embodiments described herein, but is intended to include all changes and modifications that are within the scope and spirit of the invention.

Example 1 preparation of magnetic beads coated with PCT antibody Using magnetic beads having carboxyl groups on the surface

a. The magnetic beads (particle size range 2.8 μm,30 mg/mL) were suspended and mixed well, washed with 1mL of 25mM MES (pH 5.0) buffer, and vortexed for 10 minutes, followed by magnetic separation. The magnetic beads are washed repeatedly for 2-3 times.

b. Mu.g of PCT capture antibody (1 mg/mL, 600. Mu.L) was added to the beads (antibody/bead coating ratio 0.02 mg: 1 mg), and the mixture was spun at low speed at room temperature for 30 minutes.

c. 100mg/mL EDC solution was prepared in 100mM MES (pH 5.0) buffer and used as it was.

d. mu.L of EDC (i.e., 3 mg) was added to the reaction solution of the magnetic beads and the antibody and mixed, and 100. Mu.L of 25mM MES (pH 5.0) buffer was added thereto to give a final volume of 1mL (so that the magnetic beads were reacted at a concentration of 30 mg/mL), and the mixture was stirred at a low speed at 4 ℃ for 2 hours.

e. Closing and washing the coated beads: the antibody-coated magnetic beads were spun down in 50mM Tris, pH 7.4, room temperature for 15 minutes or 50mM ethanolamine, pH 8.0, room temperature for 60 minutes to block excess reactive groups. The antibody coated beads were washed four times with 1mL PBS or 50mM Tris. During the washing process, 0.1% Tween-20 or Triton X-100 can be added to reduce non-specific binding, then 0.1% -0.5% BSA or skim milk powder is added, and finally the magnetic beads are resuspended in PBS buffer to the required concentration (corresponding to 0.25mg/mL of magnetic beads).

The reagent is a PCT-M reagent and is used for subsequent immunoreactivity verification and clinical sample test.

Example 2 labeling of antibodies with electrically Neutral Ruthenium Complex (NRC)

The PCT antibody was labeled with the NRC label shown in fig. 3C, namely Ru (2, 2 '-bipyridine) (bathophenanthroline disulfonate) [4- (2, 2' -bipyridin-4-yl) butyric acid ], to form an NRC-labeled signal antibody.

2.5mg (2.5. Mu. Mol) of NRC in 5.0mmol L ^-1 The concentration of (2) was dissolved in 500. Mu.L of MES buffer (0.1 mol L) ^-1 pH = 4.7). To this solution were added 1.0mg (5.2. Mu. Mol) of EDC and 3.0mg (13.8. Mu. Mol) of sulfo NHS to obtain a concentration of about 10mmol L ^-1 EDC and 27mmol L ^-1 The sulfo-NHS of (1). The solution was shaken at room temperature for 10 minutes. To the above reaction solution was added 0.7. Mu.L (10. Mu. Mol) of 2-mercaptoethanol (final concentration: 20mmol L) ^-1 ). After 5 minutes at room temperature, 8.0. Mu.L (containing 40nmol of NRC) of this incubation solution was added to 500. Mu.L of PCT antibody (1.2 mg/mL, about 4nmol of pure PCT antibody, molar reaction ratio 10) PBS (0.1 mol L) ^-1 pH = 7.4). They were mixed and incubated at room temperature for 2 hours.

The solution obtained above (about 0.5 ml) was loaded on a PD-10 column equilibrated in advance with PBS. Two yellow bands are formed during the separation process. A first elution band (about 0.75 ml) corresponding to the labeled antibody was collected. The binding ratio of the marker NRC to the antibody was determined to be 6.1: 1.

The collected NRC-labeled antibody solution was further diluted to 1.0. Mu.g/mL for use. The reagent is a PCT-N reagent and is used for subsequent immunoreactivity verification and clinical sample test.

Example 3 immunoreactivity verification

A series of PBS solutions of different concentrations were prepared with PCT antigen as standard solutions. Immunoreactivity verification was performed on a programmable full-automatic electrochemiluminescence immunoassay analyzer (sheny anxel diagnostic technology, ltd, ynY series or procientia 2020) with different incubation times.

30. Mu.l of PCT-containing PBS solution with different concentrations and 85. Mu.l of 4. Mu.g mL of PBS solution with different concentrations were used as the test substances through a sampling needle of an immunoassay analyzer ^-1 And 85. Mu.l of PCT-N reagent. Each mixture was incubated at 37 degrees Celsius for various periods of time (2, 5, 10, 15 and 30 minutes). After the set incubation time was over, 150. Mu.l of the above reaction suspension was injected into a three-electrode measuring cell of a full-automatic immunoassay analyzer, with a photomultiplier tube above the working electrode and a movable magnet below the working electrode. When the reaction suspension is passed through the working electrode maintained at 0.2V, the immune complexes therein are retained on the electrode surface by the magnet located below the working electrode. Subsequently, the liquid path system re-sucks a phosphate buffer solution (pH 6.8,0.18mol L) containing tripropylamine ^-1 Tripropylamine) washes the magnetic beads residing on the electrode surface. Finally, the magnetically separated and washed magnetic beads coated with [ antibody/antigen/antibody ] complex were placed in a phosphate buffer (pH 6.8,0.18mol L) ^-1 Tripropylamine). ECL is generated by applying a voltage of 1.4V between the working electrode and a reference (Ag/AgCl in saturated potassium chloride), and a photomultiplier tube arranged above the measuring cell collects optical signals and obtains the relative luminescence value of ECL through mathematical processing. After each measurement, the cell was cleaned using a 3.0V voltage and the electrodes were subjected to electrochemical regeneration (post-treatment, see the manner in which the potential was varied over time as described in figure 5) in preparation for the next test.

The results of the above tests, i.e. the signal values and the relationship of signal and concentration for different incubation times, are presented in fig. 6 and 7.

Example 4 verification of anti-Biotin interference

Dissolving biotin in 10mol L ^-1 The concentration of the solution prepared in the sodium hydroxide solution is 20mg mL ^-1 The biotin aqueous solution of (1). 50 μ L of the solution was added to 950 μ L of each of the mixed serum samples containing PCT at different concentrations to obtain a biotin concentration of 1mg mL ^-1 The serum of (4). This concentration (1 mg mL) ^-1 Biotin) is 10 of the concentration possible in human blood at the maximum dose of biotin (300 mg per day) ³ -10 ⁴ And the concentration of the reagent is more than the upper limit of the anti-interference concentration of the general evaluation reagent. For comparison, 50. Mu.L of 10mol L containing no biotin ^-1 A950 μ L aliquot of the serum sample was added to the sodium hydroxide solution.

Taking the prepared sample as a sample to be tested, carrying out three repeated tests on each sample by adopting 5-minute incubation according to the measuring method of the embodiment 3, averaging the obtained results, and calculating the relative deviation.

Tables 1and 2 are the measured concentration values and ECL signal values, respectively. The results show that the concentration and signal values varied by less than an acceptable 10% after addition of very high concentrations of biotin. For people who take biotin at the maximum dose throughout the year, the result of blood immunoassay is not affected by biotin.

Table 1 concentration measurements of two concentrations of pooled serum samples after biotin addition.

Table 2 signal values of the two concentrations of pooled serum samples after biotin addition.

Claims (5)

1. An electrochemiluminescence bioassay kit consisting of only the following two reagents:

a) A buffer solution reagent suspended with magnetic beads, wherein the surfaces of the magnetic beads are coated by an antibody with specific affinity recognition capability to a detected object;

b) A buffer solution of an antibody labeled with an electrochemiluminescent substance, wherein the antibody has a specific affinity recognition ability for an analyte.

Wherein, the antibodies in the two reagents are different and respectively recognize different sites of the detected object; the diameter of the magnetic bead is 0.2-5 microns; the electrochemiluminescent substances are transition metal complexes, preferably ruthenium and iridium complexes.

2. An electrochemiluminescence immunoassay kit consisting of only the following two reagents:

a) A buffer solution reagent with suspended magnetic beads, wherein the surfaces of the magnetic beads are coated by an antibody with specific affinity recognition capability on a detected object; (ii) a

b) A test substance molecule, a test substance molecule derivative or an analogue of a test substance molecule coupled to the electrochemiluminescent substance.

Wherein the diameter of the magnetic bead is 0.2-5 microns, and the electrochemical luminescent substance is a transition metal complex, preferably a ruthenium and iridium complex.

3. An electrochemiluminescence immunoassay kit consisting of only the following two reagents:

a) A suspension reagent of magnetic beads coated with analyte molecules, analyte molecule derivatives or analogues of analyte molecules;

b) A buffer solution of an antibody labeled with an electrochemiluminescent substance, wherein the antibody has a specific affinity recognition ability for an analyte.

Wherein the diameter of the magnetic bead is 0.2-5 microns, and the electrochemical luminescent substance is a transition metal complex, preferably a ruthenium and iridium complex.

4. The transition metal complex of claims 1-3 which is a ruthenium-containing, electrically neutral complex.

5. An electrode pretreatment, magnetic bead capture and pre-magnetic bead wash procedure for an immunoassay with the electrochemiluminescence bioassay kit of claims 1-3, comprising the steps of:

a) Alternately applying a positive voltage and a negative voltage between the working electrode and the reference electrode before the magnetic beads flow with the reaction liquid through the working electrode, wherein the negative voltage is between-0.3V and-0.9V;

b) When the magnetic beads flow through the working electrode along with the reaction liquid, the voltage between the working electrode and the reference electrode is kept between 0.05V and 0.4V;

c) After the magnetic beads are retained at the working electrode, the voltage between the working electrode and the reference electrode is continuously maintained between 0.05V and 0.4V while the magnetic beads are washed with a phosphate buffer containing an organic amine, which is a tertiary amine containing a linear or branched alkyl group (e.g., tri-n-propylamine TPA) and analogs/homologs thereof.

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