Method for reducing background signal

Abstract

The invention relates to the field of in vitro detection, and provides a method for reducing background signals, which comprises the step of detecting a target after removing a carrier.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application number 202310750360.2, filed with the State Intellectual Property Office of China on June 21, 2023, and entitled “Method for Reducing Background Signals”, the entire contents of which are incorporated by reference into this application. Technical Field

[0003] The present invention relates to the field of in vitro detection, and in particular to a method for detecting a target. Background Art

[0004] The significance of detecting a target is to qualitatively or quantitatively determine the presence of a target in a sample. In traditional immunoassays, taking the double antibody sandwich method as an example, at least one antibody labeled with a signal agent (hereinafter referred to as a labeled antibody) is required. At the same time, in order to distinguish whether the labeled antibody has bound to the target in the system, another antibody fixed to a solid phase carrier (hereinafter referred to as a capture antibody) is required to bind to the target. Through, for example, a washing step, the free labeled antibody is separated outside the system, so that the labeled antibody bound to the target and the free labeled antibody can be distinguished, thereby eliminating the signal brought by the free labeled antibody. The magnetic microparticle chemiluminescence immunoassay currently popular on the market uses magnetic beads as solid phase carriers. However, the surface materials of the solid phase carriers including magnetic beads usually have a large physical adsorption capacity, which will firmly adsorb the free labeled antibodies. Even washing cannot completely remove the free labeled antibodies, so there is still undesirable background signal interference in the detection process. Moreover, the lower the concentration of the analyte in the sample, the higher the degree of interference of the background signal in the detection process, which makes it difficult to achieve high-sensitivity detection. Summary of the invention

[0005] In view of the problems of traditional immunoassay, an improved method was proposed, which has the advantage of significantly reducing background signal interference.

[0006] First, a method for detecting a target is proposed, including:

[0007] a) contacting the sample in any order or simultaneously with the following substances:

[0008] A binding agent 1 capable of binding to a target, fixed to a carrier;

[0009] A binding agent 2 that can bind to a target and is labeled with a signaling agent;

[0010] b) separating the carrier and the binding agent 1;

[0011] c) removing the carrier;

[0012] d) Detecting the presence or absence of a target.

[0013] In the second aspect, a detection reagent is proposed, using the binding agent 1 and the binding agent 2 in the aforementioned method, wherein the binding agent 1 can be fixed on a nickel carrier, and the binding agent 2 can be labeled with a signal agent. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :Use double antibody sandwich to illustrate one of the methods of the present invention and the principle of reducing background signals;

[0015] Among them, after Ab1-His (binder 1 with a His tag), Ab2-AE (binder 2 coupled with acridinium ester), nickel magnetic beads, and the target in the sample are contacted and reacted, in addition to the ternary complex to be detected, the nickel magnetic beads also have Ab2-AE directly adsorbed on their surface. If detected directly, interference signals will be generated, but if the nickel magnetic beads are removed, the interference signals will be significantly reduced. DETAILED DESCRIPTION

[0016] In view of the problems of traditional immunoassays, improved methods have been proposed. Based on limited knowledge, solid phase carriers are usually in a state where they cannot or are difficult to separate from the binder in traditional methods. Usually, the solid phase carrier is combined with the binder by chemical coupling. Because in the entire detection system, especially in the liquid phase system, the reaction has steps such as washing that are easily cut and broken up, the carrier should be considered to be firmly connected to the binder. Technical personnel in this field are limited and will not separate the solid phase carrier and the binder. However, the inventors have broken through technical knowledge and designed a scheme for separating solid phase carriers, which can effectively reduce background signal interference. Taking the nickel carrier scheme as an example, Figure 1 The illustrated principle is that after Ab1-His (binder 1 with a His tag), Ab2-AE (binder 2 coupled with acridinium ester), nickel magnetic beads, and the target in the sample are brought into contact and reacted, in addition to the ternary complex to be detected, there is also Ab2-AE directly adsorbed on the surface of the nickel magnetic beads. If detected directly, interference signals will be generated, but if the nickel magnetic beads are removed, the interference signals will be significantly reduced.

[0017] Based on the above analysis, the following improved scheme is proposed.

[0018] This embodiment provides a method for detecting a target, comprising:

[0019] a) contacting the sample in any order or simultaneously with the following substances:

[0020] A binding agent 1 capable of binding to a target, fixed to a carrier;

[0021] A binding agent 2 that can bind to a target and is labeled with a signaling agent;

[0022] b) separating the carrier and the binding agent 1;

[0023] c) removing the carrier;

[0024] d) Detecting the presence or absence of a target.

[0025] This embodiment provides a method for detecting a target, comprising:

[0026] a) contacting the sample in any order or simultaneously with the following substances:

[0027] A binding agent 1 capable of binding to a target, which is provided with a His tag and is fixed to a nickel carrier;

[0028] A binding agent 2 that can bind to a target and is labeled with a signaling agent;

[0029] b) separating the nickel carrier and the binder 1;

[0030] c) removing the nickel support;

[0031] d) Detecting the presence or absence of a target.

[0032] In some aspects, the carrier is not limited, including any insoluble material that can carry a substance. In some aspects, the carrier can be a carrier that contains no nickel or almost no nickel, such as common carboxyl magnetic beads, hydroxyl magnetic beads, amino magnetic beads, etc., or a carrier containing nickel or nickel ions, also known as a nickel carrier, such as common nickel magnetic beads. For example, BeaverBeads TM His-tag Protein Purification Magnetic Beads, Ying Ruicheng Ni-IDA Magnetic Beads. In some embodiments, the optional particle size of any magnetic bead is not limited, and in some aspects can be 0.01-100 μm, such as 0.01, 0.03, 0.05, 0.08, 0.1, 0.3, 0.5, 0.8, 1, 3, 5, 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 μm.

[0033] The order is not limited, and the substances are contacted in the most appropriate manner; in some embodiments, the sample is contacted with binding agent 1 and binding agent 2 at the same time; in one embodiment, the sample is contacted with binding agent 1 and then with binding agent 2; in one embodiment, the sample is contacted with binding agent 2 and then with binding agent 1.

[0034] In some embodiments, the contact is carried out in a liquid phase system; for example, the contact can be arbitrarily carried out in a liquid phase system; for example, the reaction is fully carried out in a liquid phase system, for example, the reaction is fully carried out under appropriate conditions.

[0035] In some embodiments, in step a), the binder 1 is fixed to a nickel support.

[0036] In some embodiments, the binding agent 1-2 specifically binds to the target. "Specifically" means that it does not substantially bind to substances other than the specific substance, or does not substantially undergo a binding reaction with substances other than the specific substance at a detectable level.

[0037] In some embodiments, a "His tag" is defined as a tag comprising oligohistidine (eg, 6His), and the tag can be provided on the target by means of chimerization, coupling, etc., for example, chimerization to the Fc end of an antibody.

[0038] In some embodiments, the signal agent labeling method includes direct labeling or indirect labeling. Direct labeling is to directly couple the signal agent to the binding agent, and indirect labeling is to achieve the coupling of the signal agent-binding agent-binding agent through a conjugate. In some aspects, the indirectly labeled conjugate is selected from an unrelated protein and a partner pair; the unrelated protein is preferably BSA; the partner pair is preferably biotin-streptavidin.

[0039] In some embodiments, in step c), the method of separating the carrier from the binding agent 1 includes presetting a dissociable tag, for example, coupling the carrier to the binding agent 1 through a cleavable agent, and then separating the carrier from the binding agent 1 through a substance that can dissociate the cleavable agent. For example, a substance that can break the direct chemical bond between the carrier and the binding agent 1 is used to achieve separation of the carrier and the binding agent 1.

[0040] In some embodiments, imidazole is used to separate the nickel support and the binding agent 1. In some aspects, the concentration of imidazole can be adaptively adjusted to a suitable level to elute the binding agent 1 with a His tag coordinated to nickel, so that the binding agent 1 is separated from the nickel support. In some embodiments, the concentration of imidazole in the eluent is 10-1000 mM, optionally 50-900 mM, 100-800 mM, 200-800 mM, 300-800 mM, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630 0, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 5 70, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860 , 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, 1000mM. In some embodiments, the elution efficiency of separating the nickel carrier and the binder 1 by imidazole is above 80%, and exemplary is above 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. In some aspects, the elution number is more than 1 time, or in some aspects, the elution number is less than 3 times, for example, elution 1 time, 2 times, 3 times.

[0041] In some embodiments, in step c), the method of removing the nickel carrier includes absorbing the liquid phase part without the nickel carrier as a new reaction system to achieve the removal of the nickel carrier.

[0042] In some embodiments, before the binding agent 1 contacts the sample, it is pre-bound to the nickel carrier. Pre-binding fixes the nickel carrier and the binding agent 1 in advance, so that the binding agent 1 contacts the target in the subsequent sample more fully. In some pre-binding reaction times, the time is 1min-12h, for example, 1min-6h, 1min-3h, 5min-3h, 10min-2h, and exemplified by 1min, 2min, 3min, 4min, 5min, 6min, 7min, 8min, 9min, 10min, 15min, 20min, 25min, 30min, 35min, 40min, 45min, 50min, 55min, 60min, 120min, 180min, 2h, 3h, 4h, 5h, 6h, 7h, 8h, 9h, 10h, 11h, 12h.

[0043] In some embodiments, a washing step is also included. For example, washing is performed after any contacting step. For example, washing is performed after the sample contacts with binder 1; for example, washing is performed after the sample contacts with binder 2; for example, washing is performed after the sample contacts with binder 1 and binder 2; in some aspects, for example, washing is performed using PB, PBS or PBST, and in some aspects, the washing solution is also allowed to include other substances. In some aspects, the number of washes can be at least 1 time and at most 5 times, for example, it can be 1 time, 2 times, 3 times, 4 times, 5 times.

[0044] In some embodiments, when only binder 1 and binder 2 are used, the presence or absence of the target can be indicated by detecting the ternary complex formed by binder 1-2 and the target; in some embodiments, for example, the indication is quantitative or qualitative; for example, the target complex containing the target is screened out by relying on a nickel carrier; and the target complex is released from the nickel carrier by imidazole elution.

[0045] In some embodiments, the target is selected from HIV p24 and CTNI.

[0046] This embodiment proposes a detection reagent, using the binding agent 1 and the binding agent 2 in the aforementioned method, wherein the binding agent 1 can be fixed on a nickel carrier, and the binding agent 2 can be labeled with a signal agent.

[0047] In some embodiments, the binding agent is fixed to a nickel carrier and is labeled with a signal agent; in some cases, the detection reagent also includes an auxiliary agent, such as a stabilizer, a preservative, such as a buffer, etc. In some cases, instructions for use are also included.

[0048] Sample refers to an isolated substance, including but not limited to, for example, whole blood, anticoagulated whole blood, plasma, serum, tissue, body fluid, cerebrospinal fluid, saliva, urine, sweat, semen, animal and plant cells, cell contents, viruses, and fungi.

[0049] Target refers to the target detection substance, for example, it can be an antigen, antibody, or nucleic acid; antigens can be proteins, peptides, small molecules, etc.; small molecules can be active factors, such as hormones, vitamins, sugar chains, etc.; proteins and peptides can be proteins of bacteria or viruses, proteins or peptides released by body diseases; as antibodies, they can be antibodies to antigens, antibodies to antibodies, etc.; as nucleic acids, they can be nucleic acids encoding disease-causing genes, nucleic acids encoding bacteria or virus genes, etc., but are not particularly limited. Exemplary detection targets are antigens, such as cardiac markers, tumor markers, inflammatory markers, small molecules, pathogen antigens, cardiac markers such as troponin, myoglobin, creatine kinase isoenzymes, N-terminal pro-brain natriuretic peptide, cardiac fatty acid binding protein, D-dimer, lipoprotein-associated phospholipase A2, myeloperoxidase or growth stimulation expressed gene 2 protein; tumor markers such as gastrin releasing peptide precursor, carbohydrate antigen, cytokeratin 19, epididymis protein 4, alpha-fetoprotein, carcinoembryonic antigen, prostate specific antigen, squamous cell carcinoma antigen or human prostate specific gene 1; inflammatory markers such as C-reactive protein The antigens include procalcitonin, interleukin 6, serum amyloid A, α-antitrypsin, α-acid glycoprotein, ceruloplasmin, haptoglobin, C1 esterase inhibitor or α2-macroglobulin; small molecules such as aldosterone, vitamin D, folic acid, vitamin B12; pathogen antigens such as new coronavirus antigen, HIV antigen, hepatitis A virus antigen, hepatitis B virus antigen, hepatitis C virus antigen, human cytomegalovirus antigen, dengue virus antigen, influenza virus antigen, Epstein-Barr virus antigen, Treponema pallidum antigen, Mycoplasma pneumoniae antigen, Mycobacterium tuberculosis antigen, Helicobacter pylori antigen and Plasmodium antigen. Exemplary detection targets are antibodies, such as pathogen antibodies and autoimmune disease antibodies; pathogen antibodies such as new coronavirus antibodies, HIV antibodies, hepatitis A virus antibodies, hepatitis B virus antibodies, hepatitis C virus antibodies, human cytomegalovirus antibodies, dengue virus antibodies, influenza virus antibodies, Epstein-Barr virus antibodies, Treponema pallidum antibodies, Mycoplasma pneumoniae antibodies, Mycobacterium tuberculosis antibodies, Helicobacter pylori antibodies, Plasmodium antibodies; preferably, the pathogen antibodies and autoimmune disease antibodies are human antibodies.

[0050] A binder refers to a substance that can bind to a target, such as an antibody, antigen, nucleic acid, aptamer, or lectin; when the target is an antibody, the binder may be an antigen, nucleic acid, or the like; when the target is an antigen, the binder may be an antibody, an aptamer, or a lectin; when the target is a nucleic acid, the binder may be a nucleic acid antibody, a paired nucleic acid, an aptamer, or the like, but is not particularly limited. In the specification, binders 1-3 are independently selected from antibodies, antigens, nucleic acids, aptamers, and lectins, and are adaptively selected depending on the target. "Antibodies" are used in the broadest sense, and include monoclonal antibodies and polyclonal antibodies, single-specific antibodies and multi-specific antibodies, full-length antibodies, and antibody fragments. Antibody fragments are part of the structure of full-length antibodies that retain the ability to specifically bind to antigens, such as half IgG, Fab, Fab', F(ab')2, Fv, scFv, or single-domain antibodies. Such antibody fragments may be produced by recombinant nucleic acid technology, or may be produced by enzymatic or chemical cleavage of full-length antibodies. An aptamer refers to an oligonucleic acid (nucleic acid aptamer) or a peptide chain (peptide aptamer) that binds to a specific target molecule. Lectins are proteins that specifically bind to sugar chains as test substances. Nucleic acids are nucleic acids that specifically bind to nucleic acids as test substances, such as DNA, RNA, peptide nucleic acids (PNA), cross-linked nucleic acids (BNA), etc., but are not particularly limited. Among these binding substances, antibodies and aptamers are preferred.

[0051] A carrier, which may be referred to as a solid phase carrier, refers to a material whose surface can directly or indirectly fix a binding agent, such as particles, microparticles, nanoparticles, metal colloids, fibers, paper, beads, membranes, filter papers, and other supports such as test tubes, microplates, chips, slides, and microarrays. Among these carriers, particles, microparticles, and nanoparticles are preferred; microspheres are preferred; and magnetic microspheres, latex microspheres, and plastic microspheres are more preferred.

[0052] Signal agent refers to any substance that can directly or indirectly generate a recognizable signal, such as any self-luminescent, photoluminescent, enzymatic luminescent, electrochemiluminescent reagent, such as enzymes, fluorescent substances, radioactive substances, chemiluminescent substances, electrochemiluminescent substances; for example, enzymes can be β-galactosidase, peroxidase, horseradish peroxidase, alkaline phosphatase, luciferase, etc.; for example, fluorescent substances can be fluorescein, fluorescein isothiocyanate, coumarin, rhodamine, fluorescein, Cy3, Cy5, Hoechst33342, 4',6-diamidine-2-phenylindole (DAPI), propidium iodide (PI), etc., radioactive substances can be 32P, 35S, etc.; chemiluminescent substances can be luminol, isoluminol, acridinium ester, acridinium, etc., and electrochemiluminescent substances can be ruthenium terpyridine, all of which are not particularly limited.

[0053] Irrelevant proteins refer to substances that have no detection interference with most detection targets. They are usually some inert proteins or substances that do not produce immunogenicity themselves; for example, bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, immunoglobulin, thyroglobulin and polylysine.

[0054] A partner pair refers to a pair of substances that bind or interact with each other, for example, antibody / antigen, antibody / hapten, enzyme / substrate, enzyme / inhibitor, enzyme / cofactor, binding protein / substrate, carrier protein / substrate, lectin / carbohydrate; specifically, for example, biotin-avidin, etc.; it can be understood that biotin-avidin includes other partner pairs modified based on biotin or avidin, such as biotin-streptavidin.

[0055] Detection reagent refers to any container that carries a substance used for detection, such as a box, tube, strip, plate, etc., such as common test kits, reagent tubes, reagent strips, etc.

[0056] In some aspects, the method of the present invention improves the occurrence of false positives by reducing background signals, while improving sensitivity to a certain extent. In some aspects, the P / N of the method of the present invention is improved by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 1 times, at least 2 times, at least 3 times compared with conventional methods.

[0057] Example 1, CTNI detection

[0058] 1. Reagent Preparation

[0059] 1.1 Preparation of AE-labeled CTNI antibody (hereinafter referred to as CTNI-Ab2-AE):

[0060] Take CTNI antibody (Phipeng Product No. CTNI-MCAB-22), add acridinium ester and mix well, react at 25°C for 15 minutes; add 0.1M glycine to the reactant and mix well; react at 25°C for 30 minutes, place the reactant in PBS buffer and dialyze at 4°C, replace the dialysis buffer every 2 hours, and dialyze 5 times; recover the dialyzed sample to obtain CTNI-Ab2-AE.

[0061] 1.2 Premixing of nickel magnetic beads and CTNI antibody (CTNI-Ab1-NI)

[0062] Wash nickel magnetic beads (purchased from BeaverBeads) with PBS buffer. TM ), after resuspending, add CTNI antibody with his tag (Phipeng catalog number CTNI-MCAB-29), incubate at room temperature for 2 hours, wash, and resuspend to obtain CTNI-Ab1-NI.

[0063] 2. Detection process 1

[0064] 2.1 Conventional mode (without removing magnetic beads)

[0065] 50ul of sample was incubated with 50ul of CTNI-Ab2-AE and 50ul of CTNI-Ab1-NI at room temperature for 15min, washed, and pre-excitation solution A and excitation solution B were added to the luminescent plate and read.

[0066] 2.2 Mode of the present invention (removal of magnetic beads)

[0067] 50ul of sample was incubated with 50ul of CTNI-Ab2-AE and 50ul of CTNI-Ab1-NI at room temperature for 15min, washed, eluted with 50ul of 750mM imidazole, magnetically separated, the eluted supernatant was aspirated and placed in another luminescent plate, pre-excitation solution A and excitation solution B were added to the luminescent plate, and the reading was taken.

[0068] 10 samples with false positives detected by conventional mode were used. 5 samples were improved by the mode of the present invention, and P / N was improved at the same time, with P / N increased by at least 10%.

[0069] 3. Detection process 2

[0070] 3.1 Conventional mode (without removing magnetic beads)

[0071] 50ul of sample was incubated with 50ul of CTNI-Ab2-AE and 50ul of CTNI-Ab1-NI at room temperature for 15min, washed three times with PBST, and pre-excitation solution A and excitation solution B were added to the luminescent plate and read.

[0072] 3.2 Mode of the present invention (removal of magnetic beads)

[0073] 50ul of sample was incubated with 50ul of CTNI-Ab2-AE and 50ul of CTNI-Ab1-NI at room temperature for 15min, washed three times with 20mM PB, 50ul of 250mM imidazole was added and eluted with shaking for 15min, magnetic separation was performed, the elution supernatant was aspirated and placed in another luminescent plate, pre-excitation solution A and excitation solution B were added to the luminescent plate, and the reading was read.

[0074] Ten CTNI clinical samples with negative Abbott values ​​were tested. Nine samples with false positive results were tested using the conventional mode, while ten samples tested negative using the mode of the present invention. The false positive problem was significantly improved, and the P / N of the calibrator was improved. The results are shown in Table 1 below.

[0075] Table 1 CTNI two modes detection results

[0076]

[0077]

[0078] Note: CTNI Abbott uses 40pg / ml as the cutoff value, and a concentration greater than 40pg / ml is considered positive.

[0079] Example 2, HIV p24 detection

[0080] 1. Reagent Preparation

[0081] 1.1 Preparation of AE-labeled HIV p24 antibody (hereinafter referred to as HIV p24-Ab2-AE):

[0082] HIV p24 antibody (Philippines catalog number HIV-REAB-C4-018) was added to acridinium ester and mixed, and the mixture was reacted at 25°C for 60 minutes; 0.1 M glycine was added to the reactant and mixed; the mixture was reacted at 25°C for 15 minutes, and the reactant was dialyzed in PBS or TBS buffer at 4°C, the dialysis buffer was replaced every 2 hours, and the dialyzation was performed 5 times; the dialyzed sample was recovered to obtain HIV p24-Ab2-AE.

[0083] 1.2 HIV-p24 antibody with his tag (HIV-p24-Ab1)

[0084] HIV p24 antibody (Philippines catalog number HIV-REAB-C4-015) is expressed with a His tag.

[0085] Among them, the competition method verified that there was no competitive relationship among the above three strains of HIV p24.

[0086] 2. Detection process 1

[0087] 2.1 Conventional mode (without removing magnetic beads)

[0088] 50ul sample and 50ul HIV p24-Ab2-AE, 50ul HIV p24-Ab1, nickel magnetic beads (purchased from BeaverBeads TM ), incubate at room temperature for 15 min, wash, add pre-excitation solution A and excitation solution B to the luminescent plate, and read.

[0089] 2.2 Mode of the present invention (removal of magnetic beads)

[0090] 50ul sample and 50ul HIV p24-Ab2-AE, 50ul HIV p24-Ab1, nickel magnetic beads (purchased from BeaverBeads TM ), incubate at room temperature for 15 min, wash, add 750 mM imidazole 50 ul for elution for 15 min, magnetic separation, aspirate the elution supernatant and place it in another luminescent plate, add pre-excitation solution A and excitation solution B to the luminescent plate, and read the reading.

[0091] Ten specimens with false positive results were detected using the conventional mode. The false positive results of five specimens were improved by using the mode of the present invention. Meanwhile, the P / N ratio was improved by at least 10%.

[0092] 3. Detection process 2

[0093] 3.1 Conventional mode (without removing magnetic beads)

[0094] 50ul sample and 50ul HIV p24-Ab2-AE, 50ul HIV p24-Ab1, nickel magnetic beads (purchased from BeaverBeads TM ), incubate at room temperature for 15 min, wash 3 times with PBST, pat the luminescent plate dry, add pre-excitation solution A and excitation solution B to the luminescent plate, and read the reading.

[0095] 3.2 Mode of the present invention (removal of magnetic beads)

[0096] 50ul sample and 50ul HIV p24-Ab2-AE, 50ul HIV p24-Ab1, nickel magnetic beads (purchased from BeaverBeads TM ), incubate at room temperature for 15 min, wash 3 times with PBST, pat the luminescent plate dry, add 250 mM imidazole 50 ul for elution for 15 min, separate magnetically, aspirate the eluted supernatant and place it in another luminescent plate, add pre-excitation solution A and excitation solution B to the luminescent plate, and read the reading.

[0097] The conventional mode was used to detect 5 false positive samples, and all of them were tested as negative using the mode of the present invention. The false positive problem was significantly improved. At the same time, the detection of the calibrator showed that the P / N was improved. The results are shown in Table 2.

[0098] Table 2 HIV p24 two modes detection results

[0099]

[0100] Note: A positive sample is judged as positive if it is more than 10 times the mean of random clinical samples, otherwise it is negative.

Claims

1. A method for detecting a target, comprising: a) contacting the sample in any order or simultaneously with the following substances: A binding agent 1 capable of binding to a target, fixed to a carrier; A binding agent 2 that can bind to a target and is labeled with a signaling agent; b) separating the carrier and the binding agent 1; c) removing the carrier; d) Detecting the presence or absence of a target.

2. Methods for detecting a target, comprising: a) contacting the sample in any order or simultaneously with the following substances: A binding agent 1 capable of binding to a target, which is provided with a His tag and is fixed to a nickel carrier; A binding agent 2 that can bind to a target and is labeled with a signaling agent; b) separating the nickel carrier and the binder 1; c) removing the nickel support; d) Detecting the presence or absence of a target.

3. The method according to claim 2, wherein: Before the binder 1 comes into contact with the sample, it is pre-bound to the nickel support.

4. The method according to any one of claims 1 to 3, wherein: Contact in a liquid phase system.

5. The method according to any one of claims 2 to 4, wherein: Imidazole was used to separate the nickel carrier and the binder 1.

6. The method according to any one of claims 1 to 5, wherein: A washing step is also included.

7. The method according to any one of claims 1 to 6, wherein: Signal agent labeling methods include direct labeling and indirect labeling.

8. The method according to any one of claims 1 to 7, wherein: Indirectly labeled conjugates are selected from unrelated protein, partner pairs.

9. The method according to any one of claims 1 to 8, wherein: The partner pair is selected from biotin-avidin; the irrelevant protein is selected from BSA.

10. The method according to any one of claims 1 to 9, wherein: The binding agents 1-2 are independently selected from antibodies, antigens, nucleic acids, aptamers, and lectins.

11. The method according to any one of claims 1 to 10, wherein: The carrier is selected from particles, microparticles, and nanoparticles.

12. The method according to any one of claims 1 to 11, wherein: The signal agent is selected from enzymes, fluorescent substances, radioactive substances, chemiluminescent substances, and electrochemiluminescent substances.

13. The method according to any one of claims 1 to 12, wherein: The target is selected from HIV p24 and CTNI.

14. A detection reagent, comprising the binding agent 1 and the binding agent 2 according to any one of claims 1 to 13; wherein the binding agent 1 can be fixed on a nickel carrier, and the binding agent 2 can be labeled with a signal agent.

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