Immunoassay format for novel total antibody determination

Abstract

An immunological analysis mode of a novel total antibody assay and a kit for detecting antibodies of infectious diseases in human blood samples according to the mode, the kit comprising: a first reagent containing at least one antigen coated on a solid support and an anti-human IgM antibody coated on a solid support; and a second reagent containing at least one labeled antigen and a labeled anti-human IgG antibody; wherein at least one antigen of the at least one antigen coated on the solid support and at least one antigen of the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample. The kit can overcome the disadvantages caused by the detection principle while retaining the advantages of each detection principle. In addition, a method for detecting antibodies produced after pathogen infection in a sample is provided.

Description

Technical Field

[0001] This invention relates to the field of in vitro diagnostics, specifically to a novel immunoassay modality for total antibody assay, and more particularly to a kit and method for detecting antibodies. Background Technology

[0002] For in vitro diagnosis of infectious diseases, it is important to detect one or more classes of antibodies specific to certain antigens.

[0003] Antibodies (also known as immunoglobulins) are produced by the immune system to defend against foreign substances (also known as antigens). Immunoglobulins can be classified into five different types: IgM, IgG, IgA, IgE, and IgD.

[0004] When an antigen enters the body, the first antibody secreted in the immune response is IgM antibody, which initiates a complement cascade reaction after binding to the antigen. IgG antibody, on the other hand, is the main component of serum immunoglobulins, accounting for 75% of total immunoglobulins, and is the most persistent and important antibody in the primary immune response. By measuring IgG and IgM antibodies against certain pathogens, different stages of infection can be covered, such as acute infection, recurrent infection, chronic / persistent infection, or post-infectious disease, playing a crucial role in detecting antigens from pathogens.

[0005] Therefore, in the field of antibody in vitro diagnostics, there is a strong demand for the simultaneous detection of pathogen-specific IgG and IgM antibodies.

[0006] On the other hand, antibodies against infectious diseases can be detected using methods such as recomLine, enzyme-linked immunosorbent assay (ELISA), ELISA plate chemiluminescence assay, and magnetic microparticle chemiluminescence assay.

[0007] Currently, the detection principles of these methods are typically based on double-antigen sandwich methods, indirect methods, or capture methods. The double-antigen sandwich method involves labeling antigens on a solid-phase carrier and a tracer, respectively, allowing binding to two variable regions of the antibody to be detected in the sample. The indirect method coats the solid-phase carrier with antigens, and uses a secondary antibody as the tracer. The capture method coats the solid-phase carrier with a secondary antibody, and labels the tracer with a specific antigen. However, each of these principle-based detection methods has its own drawbacks.

[0008] In the double-antigen sandwich method, during the detection process, the analyte antibody may be bound to the antigens on the two solid phases or the antigens on the two tracers, which may lead to false negatives.

[0009] In indirect methods, the test sample contains a considerable amount of non-specific immunoglobulins (e.g., 700–1600 mg / dL IgG and 40–230 mg / dL IgM), which can bind to the solid phase to varying degrees without specificity. Therefore, when using a secondary antibody labeled with a tracer, these non-specifically bound immunoglobulins will also be recognized and bound, leading to increased background signal and decreased sensitivity.

[0010] Similar to the indirect method, the presence of the secondary antibody in the capture method may lead to non-specific binding in the reaction system, which in turn affects the detection results.

[0011] Therefore, in the field of antibody in vitro diagnostics, there is a strong need to overcome the drawbacks caused by the detection principle. Summary of the Invention

[0012] To avoid the problems existing in current detection methods, the inventors studied the mode of in vitro antibody assay and unexpectedly discovered that a new immunoassay mode that combines the principles of double antigen sandwich method, indirect method and capture method not only avoids the drawbacks of each principle, but also retains the advantages of each of them, thus completing the present invention.

[0013] In a first aspect, the present invention provides a kit comprising:

[0014] The first reagent comprises at least one antigen coated on a solid support, and an anti-human IgM antibody coated on a solid support; and

[0015] The second reagent includes at least one labeled antigen and a labeled anti-human IgG antibody;

[0016] Wherein, at least one antigen among the at least one antigen coated on the solid support and at least one antigen among the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample.

[0017] The kit of the present invention can be used for in vitro immunoassay of total antibodies (IgG+IgM).

[0018] In the kit of the present invention, the antigen in the first reagent and the antigen in the second reagent can form a sandwich structure (antigen-antibody-antigen) with the same IgG antibody or IgM antibody to be detected; the antigen in the first reagent and the anti-human IgG antibody in the second reagent can bind to the same IgG antibody to be detected; the anti-human IgM antibody in the first reagent and the antigen in the second reagent can bind to the same IgM antibody to be detected.

[0019] The antibodies detected by the kit of the present invention are the corresponding antibodies produced in the human body after infection by pathogens.

[0020] In a specific implementation, the types of antibodies detected are IgG and IgM.

[0021] Figure 1 The reaction principle of the kit of the present invention when used for detection is illustrated by way of example. When using the kit of the present invention, the following reaction may occur: such as... Figure 1 As shown in Figure A (using only one antigen as an example), one or more antigens in the first reagent can form a complex with the IgG antibody to be detected and one or more antigens in the second reagent; one or more antigens in the first reagent can form a complex with the IgM antibody to be detected and one or more antigens in the second reagent. Figure 1 As shown in Figure B (using only one antigen as an example), the antigen in the first reagent can form a complex with the IgG antibody to be detected and the anti-human IgG antibody in the second reagent. Figure 1 As shown in Figure C (taking only one antigen as an example), the anti-human IgM antibody in the first reagent can form a complex with the IgM antibody to be detected and the antigen in the second reagent.

[0022] In a specific embodiment, the anti-human IgM antibody in the first reagent may be present at a concentration of approximately 10 ng / mL to approximately 30 ng / mL, while the anti-human IgG antibody in the second reagent may be present at a concentration of approximately 100 ng / mL to approximately 300 ng / mL. In particular, the concentration of the anti-human IgG antibody in the second reagent is approximately 10 times that of the anti-human IgM antibody in the first reagent.

[0023] In one variant of the kit of the present invention, it comprises:

[0024] The first reagent comprises at least one antigen coated on a solid support, and an anti-human IgG antibody coated on a solid support; and

[0025] The second reagent includes at least one labeled antigen and a labeled anti-human IgM antibody;

[0026] Wherein, at least one antigen among the at least one antigen coated on the solid support and at least one antigen among the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample.

[0027] When this variant is used, the following reactions may occur: one or more antigens in the first reagent may form a complex with the IgG antibody to be detected and one or more antigens in the second reagent; one or more antigens in the first reagent may form a complex with the IgM antibody to be detected and one or more antigens in the second reagent; the antigen in the first reagent may form a complex with the IgM antibody to be detected and the anti-human IgM antibody in the second reagent; the anti-human IgG antibody in the first reagent may form a complex with the IgG antibody to be detected and the antigen in the second reagent.

[0028] In a specific embodiment, the anti-human IgG antibody in the first reagent may be present at a concentration of approximately 100 ng / mL to approximately 300 ng / mL, while the anti-human IgM antibody in the second reagent may be present at a concentration of approximately 10 ng / mL to approximately 30 ng / mL. In particular, the concentration of the anti-human IgG antibody in the first reagent is approximately 10 times the concentration of the anti-human IgM antibody in the second reagent.

[0029] By using the kit of the present invention, total antibodies (IgG antibodies and IgM antibodies) in a sample can be detected simultaneously, enabling the detection to cover all stages of pathogen infection, thereby facilitating the prevention and control of related infectious diseases.

[0030] Furthermore, when testing seroconversion discs, kits based solely on the double-antigen sandwich method or solely on the capture method suffer from low detection rates and unclear sample gradients. These drawbacks are overcome when using the kit of the present invention to test seroconversion discs. Additionally, when testing IgM positive samples, kits based solely on the indirect method or solely on the double-antigen sandwich method suffer from low detection rates. These drawbacks are overcome when using the kit of the present invention to test IgM positive samples. When testing negative samples, kits based solely on the indirect method or solely on the capture method suffer from poor specificity and high false-positive rates. These drawbacks are overcome when using the kit of the present invention to test negative samples.

[0031] The kit of this invention incorporates three detection principles: the double-antigen sandwich method, the indirect method, and the capture method. It is worth noting that by using the kit of this invention, the advantages of each of the three principles are essentially maintained during the detection process, while overcoming their respective shortcomings. It is generally believed that when different detection principles are used in combination, the coexistence of multiple detection systems may bring the advantages of each principle, but it may also introduce the shortcomings of each principle. As demonstrated in the following examples, when using kits based on both the double-antigen sandwich and indirect methods, compared to kits based solely on the double-antigen sandwich method, the kit exhibits worse specificity and more false positives when testing negative samples; while compared to kits based solely on the indirect method, the sensitivity for testing IgM positive samples is somewhat improved but not significantly. In other words, the kit combining the double-antigen sandwich and indirect methods does not offer a more significant advantage than a kit based on a single detection principle. While kits based on both the double-antigen sandwich and capture methods show significantly improved sensitivity for IgM-positive samples compared to kits based solely on the double-antigen sandwich method, they exhibited poorer specificity and more false positives when testing negative samples, and the sensitivity for serological transition disc samples remained unchanged. Conversely, compared to kits based solely on the capture method, although the sensitivity for serological transition disc samples was improved, it still fell short of expectations, and the specificity for negative samples remained unimproved. In other words, kits combining the double-antigen sandwich and capture methods cannot overcome the limitations of kits based on a single detection principle.

[0032] In this invention, the terms "solid support," "solid substrate," "solid carrier," and "solid carrier" are used interchangeably, referring to a solid surface on which antigens or antibodies can be attached. There are no particular limitations on the solid support used in this invention; commercially available solid supports and any solid support suitable for immunoassay can be used. Exemplary solid supports may include magnetic beads (such as superparamagnetic microspheres), ELISA plates, plastic plates, plastic tubes, latex beads, agarose beads, glass, nitrocellulose membranes, nylon membranes, silica plates, or microchips, but the invention is not limited thereto.

[0033] The markers that can be used in this invention are well known to those skilled in the art, and may include enzymes such as oxidase, microperoxidase, horseradish peroxidase, alkaline phosphatase (ALP), β-galactosidase, glucose oxidase, and glucose-6-phosphate dehydrogenase; fluorescent substances such as fluorescein isothiocyanate, tetramethylrhodamine isothiocyanate, fluorescein, rhodamine, europium, and green fluorescent protein; chemiluminescent substances such as luminol, isoluminol, phenanthrene, and acridinium ester; coenzymes such as NAD; and biotin. 35 S, 14 C32 P, 131 I and 125 Radioactive materials of class I, but the present invention is not limited thereto.

[0034] In the first reagent of the kit of the present invention, at least one antigen is coated on a solid support, and the antigen may be independently selected from one or more conserved proteins or fragments thereof of a pathogen. Preferably, the antigen may be independently selected from one or more dominant conserved fragments of the pathogen.

[0035] In some embodiments, the antigen in the first reagent may be in the form of a polymer, an antigen fragment, or a peptide.

[0036] In an exemplary embodiment, when the target being detected is an antibody against HCV, the antigen in the first reagent of the present invention may be selected from at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen, or a fusion antigen of two or more of them; preferably selected from at least one of HCV core antigen and HCV NS3 antigen; more preferably containing HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen simultaneously. In a specific embodiment, the HCV core antigen may be present in the first reagent at a concentration of about 1.5 μg / mL to about 4 μg / mL, the HCV NS3 antigen may be present at a concentration of about 0.5 μg / mL to about 2 μg / mL, the HCV NS4 antigen may be present at a concentration of about 0.05 μg / mL to about 0.2 μg / mL, and the HCV NS5 antigen may be present at a concentration of about 0.01 μg / mL to about 0.05 μg / mL.

[0037] In an exemplary embodiment, when the target of detection is an antibody against Treponema pallidum, the antigen in the first reagent of the present invention is selected from at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen, or two or more of them as fusion antigens; preferably selected from at least one of TP15 antigen, TP17 antigen and TP47 antigen; more preferably containing TP15 antigen, TP17 antigen and TP47 antigen simultaneously.

[0038] The antigen and antibody in the first reagent of the present invention can be coated onto a solid support via conventional techniques in the art (e.g., conjugation).

[0039] In this invention, the antigens and antibodies coated on the solid support can be referred to as the antigens and antibodies in the first reagent.

[0040] In this invention, the conjugate between the antigen and antibody and the solid support can be referred to as a solid-phase carrier coating. The solid-phase carrier coating of this invention can be present in a conventional diluent containing proteins and surfactants and possessing buffering capacity.

[0041] In the second reagent of the kit of the present invention, at least one antigen is labeled, and the antigen may be independently selected from one or more conserved proteins or fragments thereof of a pathogen. Preferably, the antigen may be independently selected from one or more dominant conserved fragments of the pathogen.

[0042] In some embodiments, the antigen in the second reagent may be in the form of a polymer, an antigen fragment, or a peptide.

[0043] In an exemplary embodiment, when the target of detection is an antibody against HCV, the antigen in the second reagent of the present invention may be selected from at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen, or a fusion antigen of two or more of them; preferably selected from at least one of HCV core antigen and HCV NS3 antigen; more preferably containing HCV core antigen, HCV NS3 antigen, HCV NS4 antigen, and HCV NS5 antigen simultaneously. In a specific embodiment, the HCV core antigen may be present in the second reagent at a concentration of about 0.3 μg / mL to about 1.5 μg / mL, the HCV NS3 antigen may be present at a concentration of about 0.1 μg / mL to about 1 μg / mL, the HCV NS4 antigen may be present at a concentration of about 0.01 μg / mL to about 0.1 μg / mL, and the HCV NS5 antigen may be present at a concentration of about 0.005 μg / mL to about 0.02 μg / mL.

[0044] In an exemplary embodiment, when the target being detected is an antibody against Treponema pallidum, the antigen in the second reagent of the present invention is selected from at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen, or two or more of them as fusion antigens; preferably selected from at least one of TP15 antigen, TP17 antigen and TP47 antigen; more preferably containing TP15 antigen, TP17 antigen and TP47 antigen simultaneously.

[0045] The antigen and antibody in the second reagent of the present invention can be linked to the marker via conventional techniques in the art (e.g., chemical bonding).

[0046] In this invention, the antigens and antibodies with labels can be referred to as the antigens and antibodies in the second reagent.

[0047] In this invention, the conjugate of antigen and antibody with a label can be referred to as a label conjugate. The label conjugate of this invention can be present in a conventional diluent containing protein and surfactant and having buffering capacity.

[0048] The antibodies in the kit of this invention can be monoclonal or polyclonal antibodies. In some embodiments, the antibodies in the first and second reagents can be antibody fragments, which typically include an antigen-binding region of the antibody, a light chain and / or heavy chain variable region, and at least a portion of one or more (e.g., six) CDRs. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments.

[0049] The kit of the present invention may further include a third reagent containing a blocking agent, said blocking agent being selected from one or more of the following: skim milk powder, BSA, gelatin, serum, casein, ovalbumin, animal IgG, surfactant (such as Tween-20).

[0050] By adding a third reagent containing a blocking agent, non-specific binding can be prevented, thereby enhancing the anti-interference capability of the kit of the present invention.

[0051] The kit of the present invention may further include a fourth reagent containing a reducing agent, which may be selected from one or more of the following: DTT, β-mercaptoethanol.

[0052] By adding a fourth reagent containing a reducing agent, the antigen conformation can be maintained, thereby improving the sensitivity of the kit of the present invention.

[0053] Unless otherwise specified, the terms “first,” “second,” “third,” and “fourth,” etc., used in this invention are only used to distinguish multiple similar elements and are not intended to indicate any difference in importance or order among the elements.

[0054] Those skilled in the art will understand that, in addition to the first and second reagents, the kit of the present invention may also include sample diluent, washing buffer, quality control products and / or calibrators, etc.

[0055] In a second aspect, the present invention provides an in vitro detection method for antibodies against infectious diseases, comprising:

[0056] 1) Mix and incubate the sample from the subject, the first reagent, and the second reagent.

[0057] 2) Obtain the signal values ​​associated with the markers.

[0058] 3) Determine the detection result based on the signal value;

[0059] The first reagent comprises at least one antigen coated on a solid support, and an anti-human IgM antibody coated on a solid support.

[0060] The second reagent comprises at least one labeled antigen and a labeled anti-human IgG antibody.

[0061] Wherein, at least one antigen among the at least one antigen coated on the solid support and at least one antigen among the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample. That is, at least one antigen among the at least one antigen coated on the solid support can form a double antigen sandwich complex with the antibody to be detected and at least one antigen among the at least one labeled antigen.

[0062] In one variation of the method of the present invention, it includes:

[0063] 1) Mix and incubate the sample from the subject, the first reagent, and the second reagent.

[0064] 2) Obtain the signal values ​​associated with the markers.

[0065] 3) Determine the detection result based on the signal value;

[0066] The first reagent comprises at least one antigen coated on a solid support, and an anti-human IgG antibody coated on a solid support.

[0067] The second reagent comprises at least one labeled antigen and a labeled anti-human IgM antibody.

[0068] Wherein, at least one antigen among the at least one antigen coated on the solid support and at least one antigen among the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample. That is, at least one antigen among the at least one antigen coated on the solid support can form a double antigen sandwich complex with the antibody to be detected and at least one antigen among the at least one labeled antigen.

[0069] By using the method of this invention, both IgG and IgM antibodies against infectious diseases can be detected simultaneously. IgG is a marker of past infection, while IgM is a marker of acute infection; therefore, the method of this invention can detect both chronic and acute infections. Furthermore, the method of this invention overcomes the shortcomings of traditional detection methods, balancing sensitivity and specificity.

[0070] In step 1), the sample and the first reagent can be added to the reaction vessel first, incubated for a period of time, and then washed; subsequently, the second reagent can be added to the reaction vessel, incubated for a period of time, and then washed. Alternatively, the sample, the first reagent, and the second reagent can be added to the reaction vessel simultaneously, incubated for a period of time, and then washed. Alternatively, the sample and the second reagent can be added to the reaction vessel, incubated for a period of time (without washing); subsequently, the first reagent can be added to the reaction vessel, incubated for a period of time, and then washed.

[0071] In step 2), the process of obtaining the signal value associated with the marker is well known to those skilled in the art. For example, when using alkaline phosphatase as a marker, the luminescent substrate 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane (AMPPD) can be added to the reaction vessel. The luminescent substrate is decomposed by alkaline phosphatase to produce chemiluminescence, and the number of photons produced by the reaction is finally measured by a photomultiplier tube.

[0072] In step 2), commercially available detection instruments can also be used to measure the signal value corresponding to the marker. For example, Mindray's fully automated chemiluminescence analyzer CL series.

[0073] In a preferred embodiment, step 1) includes mixing and incubating a sample from the subject, a first reagent, a second reagent, and a third reagent of the present invention. For example, the sample, the first reagent, and the third reagent can be added to the reaction vessel first, incubated for a period of time, and then washed; subsequently, the second and third reagents can be added to the reaction vessel, incubated for a period of time, and then washed. Alternatively, the sample, the first reagent, the second reagent, and the third reagent can be added to the reaction vessel simultaneously, incubated for a period of time, and then washed. Alternatively, the sample, the second reagent, and the third reagent can be added to the reaction vessel, incubated for a period of time (without washing); subsequently, the first and third reagents can be added to the reaction vessel, incubated for a period of time, and then washed.

[0074] In another preferred embodiment, step 1) includes mixing and incubating the sample from the subject, the first reagent, the second reagent, and the fourth reagent of the present invention. For example, the sample, the first reagent, and the fourth reagent can be added to the reaction vessel first, incubated for a period of time, and then washed; subsequently, the second reagent is added to the reaction vessel, incubated for a period of time, and then washed. Alternatively, the sample, the first reagent, the second reagent, and the fourth reagent can be added to the reaction vessel simultaneously, incubated for a period of time, and then washed. Alternatively, the sample, the second reagent, and the fourth reagent can be added to the reaction vessel, incubated for a period of time (without washing); subsequently, the first reagent is added to the reaction vessel, incubated for a period of time, and then washed.

[0075] In yet another preferred embodiment, step 1) includes mixing and incubating a sample from the subject, a first reagent, a second reagent, a third reagent of the present invention, and a fourth reagent of the present invention. For example, the sample, the first reagent, the third reagent, and the fourth reagent can be added to the reaction vessel first, incubated for a period of time, and then washed; subsequently, the second reagent and the third reagent are added to the reaction vessel, incubated for a period of time, and then washed. Alternatively, the sample, the first reagent, the second reagent, the third reagent, and the fourth reagent can be added to the reaction vessel simultaneously, incubated for a period of time, and then washed. Alternatively, the sample, the second reagent, the third reagent, and the fourth reagent can be added to the reaction vessel, incubated for a period of time (without washing); subsequently, the first reagent and the third reagent are added to the reaction vessel, incubated for a period of time, and then washed.

[0076] Furthermore, the in vitro detection method provided in the second aspect of the present invention can be implemented using the reagent kit provided in the first aspect of the present invention.

[0077] In this invention, the sample is a blood sample from the subject, such as blood, serum, or plasma. Attached Figure Description

[0078] Figure 1 This is a schematic diagram illustrating the reaction principle of the kit of the present invention. Detailed Implementation

[0079] The technical methods of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0080] Reagent preparation

[0081] First reagent Ra:

[0082] Use a pipette or graduated cylinder to measure V1 / D separately. x (x = 1, 2, ...) (mL) volumes of "magnetic bead coatings for each antigen" and "magnetic bead coatings for anti-human IgM antibody" were added to a magnetic bead coating tube to displace the supernatant. After magnetic separation, the supernatant was aspirated, and an equal volume of magnetic bead coating diluent was added and mixed thoroughly. The mixed magnetic beads were then added to a preparation bottle containing V1 (mL) of magnetic bead coating diluent. The mixture was stirred until the magnetic bead suspension was completely homogenized, yielding the first reagent Ra; where D... x The dilutions are for "magnetic bead coatings of various antigens" and "magnetic bead coatings of anti-human IgM antibodies"; the magnetic bead coating diluent is a conventional diluent with buffering capacity and contains protein and surfactant.

[0083] Second reagent Rb:

[0084] Measure V2 (mL) of the enzyme-labeled conjugate dilution solution using a suitable graduated cylinder and add it to the preparation bottle. Measure V2 / D using a pipette or graduated cylinder. y Add (y = 1, 2...) mL volumes of "enzyme-labeled conjugates of each antigen" and "enzyme-labeled conjugates of anti-human IgG antibodies" to the enzyme-labeled conjugate diluent; stir the solution with a stirrer to ensure complete dissolution and mixing; seal the solution and place it at 22°C for equilibration for 23-25 ​​hours; at the end of equilibration, filter the prepared solution using a suitable 0.22 μm pore size filter, collect the filtrate, and obtain the second reagent Rb; where D y These are the dilutions of "enzyme-labeled conjugates of various antigens" and "enzyme-labeled conjugates of anti-human IgG antibodies," respectively. The enzyme-labeled conjugate diluent is a conventional diluent with buffering capacity and contains protein and surfactant.

[0085] Third reagent Rc:

[0086] A buffered diluent containing a blocking agent and a surfactant.

[0087] Fourth reagent Rd:

[0088] A buffering diluent containing a reducing agent.

[0089] Pathogen detection methods

[0090] Step 1: Add the sample, third reagent, fourth reagent and first reagent to the reaction tube and incubate at 37°C for 10 minutes to allow the antigen coated on the magnetic bead solid phase and the anti-human IgM antibody to fully bind with the IgG and IgM antibodies in the sample; after incubation, place the magnetic bead solid phase in a magnetic field to be attracted, the substances bound to the magnetic bead solid phase are retained, while other unbound substances are washed away.

[0091] Step 2: Add the third and second reagents to the reaction tube; incubate at 37°C for 10 minutes. The antigen and anti-human IgG antibody on the enzyme label bind to the IgG and IgM antibodies captured on the magnetic beads, forming a sandwich complex. After incubation in the reaction tube, the complex is attracted by the magnetic field, while other unbound substances are washed away.

[0092] Step 3: Add the chemiluminescent substrate to the reaction tube to generate chemiluminescence. Then, measure the number of photons produced by the reaction using a photomultiplier tube to obtain the chemiluminescence value of the sample.

[0093] Example 1: Preparation of HCV Antibody Detection Kit

[0094] The kits used in this example were prepared according to the description in Table 1 and "Reagent Preparation".

[0095] Table 1

[0096]

[0097]

[0098]

[0099] Example 2: Serum disc detection of HCV antibodies

[0100] use" Pathogen detection methods "Three serum conversion discs (9041, 10165 and 10185, purchased from Zeptometrix) were tested, and the COI results for each sample are shown in Table 2 below."

[0101] Table 2

[0102]

[0103] In this invention, COI (Cutoff Index) is the relative luminescence value (RLU) of the measured sample, i.e., the ratio of the chemiluminescence signal value to the threshold (Cutoff). COI ≥ 1 indicates a positive sample, and COI < 1 indicates a negative sample. For qualitative detection methods, the threshold (cutoff value) is the cutoff value for determining whether a test result is positive or negative.

[0104] As shown in Table 2, Kit 1-1 (i.e., the kit of the present invention) has a comparable detection rate to Kit 1-2 (indirect method) and Kit 1-5 (double antigen sandwich + indirect method), and has a high COI value, with a relatively obvious gradient in the detected samples. However, Kit 1-3 (double antigen sandwich), Kit 1-4 (capture method), and Kit 1-6 (double antigen sandwich + capture method) detect fewer samples, and most detected samples have low COI (<5), with no obvious gradient in the detected samples. Therefore, in terms of serum plate sensitivity, the kit / method of the present invention is superior to the kits / methods based on the double antigen sandwich principle, the kits / methods based on the capture method principle, and the combination of the two (double antigen sandwich + capture method). Furthermore, Kit 1-7 (i.e., a variant of the kit of the present invention) performs similarly to Kit 1-1 in terms of serum plate sensitivity.

[0105] Example 3: Detection of HCV-IgM antibody-positive samples

[0106] use" Pathogen detection methodsThirty-one HCV-IgM antibody-positive samples (treated with IgG / RF factor adsorption reagent (purchased from Euromon GmbH, Germany) and then confirmed by Roche Anti-HCV kit) were tested, and the results are shown in Table 3 below.

[0107] Table 3

[0108] Reagent Kit 1-1 Reagent kit 1-2 Reagent kit 1-3 Reagent kit 1-4 Reagent kit 1-5 Reagent kit 1-6 Reagent kit 1-7 sample COI COI COI COI COI COI COI 1 <![CDATA[ 2.77 ]]> 0.59 <![CDATA[ 1.14 ]]> <![CDATA[ 5.33 ]]> <![CDATA[ 1.12 ]]> <![CDATA[ 2.39 ]]> <![CDATA[ 2.25 ]]> 2 <![CDATA[ 1.94 ]]> 0.82 <![CDATA[ 1.10 ]]> <![CDATA[ 2.55 ]]> <![CDATA[ 1.15 ]]> <![CDATA[ 1.05 ]]> <![CDATA[ 1.58 ]]> 3 <![CDATA[ 2.41 ]]> 0.83 <![CDATA[ 1.11 ]]> <![CDATA[ 4.36 ]]> <![CDATA[ 1.10 ]]> <![CDATA[ 1.80 ]]> <![CDATA[ 1.16 ]]> 4 <![CDATA[ 2.18 ]]> 0.62 <![CDATA[ 1.02 ]]> <![CDATA[ 3.62 ]]> <![CDATA[ 1.21 ]]> <![CDATA[ 1.62 ]]> <![CDATA[ 1.77 ]]> 5 <![CDATA[ 6.76 ]]> 0.99 1.00 <![CDATA[ 8.99 ]]> <![CDATA[ 1.13 ]]> <![CDATA[ 7.00 ]]> <![CDATA[ 5.44 ]]> 6 <![CDATA[ 1.95 ]]> 0.53 <![CDATA[ 1.26 ]]> <![CDATA[ 3.24 ]]> <![CDATA[ 1.25 ]]> <![CDATA[ 1.41 ]]> <![CDATA[ 1.59 ]]> 7 <![CDATA[ 2.73 ]]> 0.77 <![CDATA[ 1.16 ]]> <![CDATA[ 5.35 ]]> <![CDATA[ 1.12 ]]> <![CDATA[ 2.36 ]]> <![CDATA[ 2.21 ]]> 8 <![CDATA[ 2.21 ]]> 0.57 <![CDATA[ 1.23 ]]> <![CDATA[ 3.76 ]]> <![CDATA[ 1.20 ]]> <![CDATA[ 1.66 ]]> <![CDATA[ 1.80 ]]> 9 <![CDATA[ 2.36 ]]> 0.81 <![CDATA[ 1.15 ]]> <![CDATA[ 4.12 ]]> <![CDATA[ 1.11 ]]> <![CDATA[ 1.73 ]]> <![CDATA[ 1.92 ]]> 10 <![CDATA[ 1.93 ]]> 0.93 <![CDATA[ 1.11 ]]> <![CDATA[ 2.74 ]]> <![CDATA[ 1.08 ]]> <![CDATA[ 1.11 ]]> <![CDATA[ 1.57 ]]> 11 <![CDATA[ 6.80 ]]> <![CDATA[ 1.47 ]]> <![CDATA[ 1.15 ]]> <![CDATA[ 8.31 ]]> <![CDATA[ 1.21 ]]> <![CDATA[ 6.84 ]]> <![CDATA[ 5.47 ]]> 12 <![CDATA[ 2.61 ]]> <![CDATA[ 1.43 ]]> 0.95 <![CDATA[ 5.16 ]]> <![CDATA[ 1.08 ]]> <![CDATA[ 2.24 ]]> <![CDATA[ 2.12 ]]> 13 <![CDATA[ 5.59 ]]> <![CDATA[ 1.45 ]]> 0.85 <![CDATA[ 7.73 ]]> 0.96 <![CDATA[ 5.48 ]]> <![CDATA[ 4.50 ]]> 14 <![CDATA[ 1.86 ]]> 0.68 0.94 <![CDATA[ 2.81 ]]> 0.89 <![CDATA[ 1.20 ]]> <![CDATA[ 1.52 ]]> 15 <![CDATA[ 2.32 ]]> 0.81 0.88 <![CDATA[ 4.42 ]]> 0.88 <![CDATA[ 1.91 ]]> <![CDATA[ 1.19 ]]> 16 <![CDATA[ 2.36 ]]> <![CDATA[ 1.06 ]]> <![CDATA[ 1.02 ]]> <![CDATA[ 4.53 ]]> <![CDATA[ 1.04 ]]> <![CDATA[ 2.04 ]]> <![CDATA[ 1.92 ]]> 17 <![CDATA[ 1.93 ]]> <![CDATA[ 1.39 ]]> 0.93 <![CDATA[ 3.11 ]]> <![CDATA[ 1.05 ]]> <![CDATA[ 1.04 ]]> <![CDATA[ 1.57 ]]> 18 <![CDATA[ 2.55 ]]> 0.81 0.99 <![CDATA[ 4.86 ]]> 0.89 <![CDATA[ 2.11 ]]> <![CDATA[ 2.07 ]]>

[0109] 19 <![CDATA[ 3.91 ]]> 0.88 0.93 <![CDATA[ 3.32 ]]> 0.86 <![CDATA[ 1.23 ]]> <![CDATA[ 3.16 ]]> 20 <![CDATA[ 1.67 ]]> 0.88 <![CDATA[ 1.08 ]]> <![CDATA[ 2.51 ]]> <![CDATA[ 1.06 ]]> <![CDATA[ 1.01 ]]> <![CDATA[ 1.37 ]]> 21 <![CDATA[ 1.58 ]]> 0.70 0.87 <![CDATA[ 2.17 ]]> 0.82 0.83 <![CDATA[ 1.29 ]]> 22 <![CDATA[ 2.14 ]]> <![CDATA[ 1.24 ]]> 0.87 <![CDATA[ 3.74 ]]> 0.89 <![CDATA[ 1.49 ]]> <![CDATA[ 1.74 ]]> 23 <![CDATA[ 1.50 ]]> 0.62 0.97 <![CDATA[ 2.32 ]]> 0.89 0.89 <![CDATA[ 1.23 ]]> 24 <![CDATA[ 2.69 ]]> 0.70 0.88 <![CDATA[ 2.78 ]]> 0.87 <![CDATA[ 1.09 ]]> <![CDATA[ 2.18 ]]> 25 <![CDATA[ 1.66 ]]> 0.89 <![CDATA[ 1.03 ]]> <![CDATA[ 2.63 ]]> 0.98 <![CDATA[ 1.03 ]]> <![CDATA[ 1.36 ]]> 26 <![CDATA[ 2.62 ]]> 0.75 0.90 <![CDATA[ 2.39 ]]> 0.84 0.82 <![CDATA[ 2.13 ]]> 27 <![CDATA[ 1.47 ]]> 0.79 0.99 <![CDATA[ 2.28 ]]> 0.93 0.83 <![CDATA[ 1.21 ]]> 28 <![CDATA[ 4.64 ]]> 0.82 0.92 <![CDATA[ 1.73 ]]> <![CDATA[ 1.96 ]]> 0.63 <![CDATA[ 3.74 ]]> 29 <![CDATA[ 3.56 ]]> 0.72 <![CDATA[ 1.14 ]]> <![CDATA[ 2.13 ]]> <![CDATA[ 1.05 ]]> 0.92 <![CDATA[ 2.88 <!-- 10 -->]]> 30 <![CDATA[ 1.58 ]]> <![CDATA[ 1.22 ]]> 0.84 <![CDATA[ 2.61 ]]> 0.84 0.96 <![CDATA[ 1.09 ]]> 31 <![CDATA[ 2.84 ]]> <![CDATA[ 1.03 ]]> <![CDATA[ 1.03 ]]> <![CDATA[ 5.50 ]]> <![CDATA[ 1.01 ]]> <![CDATA[ 2.38 ]]> <![CDATA[ 2.30 ]]>

[0110] As shown in Table 3, when detecting HCV-IgM positive samples, kits 1-1 (the kit of this invention) and 1-4 (capture method) detected all samples. Kits 1-2 (indirect method) and 1-3 (double antigen sandwich) detected less than half of the samples, and the COI values ​​were relatively low, mostly around 1. Although kits 1-5 (double antigen sandwich + indirect method) and 1-6 (double antigen sandwich + capture method) showed improved detection rates compared to 1-2 and 1-3, some samples still failed to be detected. Therefore, in terms of sensitivity for detecting HCV IgM positive samples, the kits / methods of this invention are superior to kits / methods based on the indirect method principle, kits / methods based on the double antigen sandwich principle, and combinations of both (double antigen sandwich + indirect method). Furthermore, combining the capture method and the double antigen sandwich method actually reduces the detection rate of HCV IgM positive samples (combination 1-6 is weaker than combination 1-4). Furthermore, kits 1-7 (i.e., a variant of the kit of the present invention) performed essentially the same as kit 1-1 in terms of the detection rate of HCV-IgM antibody positive samples.

[0111] Example 4: Detection of HCV antibody-negative samples

[0112] use" Pathogen detection methods "500 HCV antibody-negative samples (derived from hospital diagnostic results) were tested, and the statistical results are shown in Table 4 below."

[0113] Table 4

[0114] Reagent Kit 1-1 Reagent kit 1-2 Reagent kit 1-3 Reagent kit 1-4 Reagent kit 1-5 Reagent kit 1-6 Total sample size 500 500 500 500 500 500 False positive sample number 1 3 1 5 2 3 False positive probability 0.2％ 0.6％ 0.2％ 1％ 0.4％ 0.6％

[0115] As shown in Table 4, when detecting HCV antibody-negative samples, the false positive rates of kit 1-1 (the kit of the present invention) and kit 1-3 (double antigen sandwich) are comparable, at 0.2%. The false positive rates of combination 1-2 (indirect method) and kit 1-4 (capture method) are higher, at 0.6% and 1%, respectively. Kits combining both detection modes—kit 1-5 (double antigen sandwich + indirect method) and combination 1-6 (double antigen sandwich + capture method)—have lower false positive rates compared to kit 1-2 (indirect method) and kit 1-4 (capture method), but are still higher than kit 1-1 (the kit of the present invention) and kit 1-3 (double antigen sandwich). Therefore, in terms of specificity for detecting HCV antibody-negative samples, the kit / method of the present invention is superior to kits / methods based on the indirect method or capture method principle, as well as kits / methods based on a combination of the indirect method or capture method and the double antigen sandwich method.

[0116] Example 5: Preparation of Treponema pallidum antibody detection kit

[0117] The kits used in this example were prepared according to the description in Table 5 and "Reagent Preparation".

[0118] Table 5

[0119]

[0120]

[0121] Example 6: Serum disc detection of Treponema pallidum antibodies

[0122] use" Pathogen detection methods "Two serum discs (0615-0017 and 0820-0300, purchased from SeraCare) were tested, and the results are shown in Table 6 below."

[0123] Table 6

[0124] Reagent Kit 2-1 Reagent Kit 2-2 Reagent kit 2-3 Reagent kit 2-4 Reagent kit 2-5 Reagent kit 2-6 sample COI COI COI COI COI COI 0615-0017-01 0.17 0.14 0.05 0.42 0.18 0.35 0615-0017-02 0.47 0.12 0.04 0.44 0.19 0.39 0615-0017-03 0.42 0.12 0.03 0.41 0.17 0.41 0615-0017-04 0.58 0.13 0.03 0.40 0.21 0.42 0615-0017-05 <![CDATA[ 1.98 ]]> 0.16 0.13 0.68 0.23 0.73 0615-0017-06 <![CDATA[ 3.07 ]]> 0.51 <![CDATA[ 1.04 ]]> <![CDATA[ 1.17 ]]> 0.78 <![CDATA[ 1.23 ]]> 0615-0017-07 <![CDATA[ 3.68 ]]> 0.80 <![CDATA[ 2.11 ]]> <![CDATA[ 1.26 ]]> <![CDATA[ 1.21 ]]> <![CDATA[ 1.31 ]]> 0615-0017-08 <![CDATA[ 4.20 ]]> 0.97 <![CDATA[ 5.10 ]]> <![CDATA[ 1.51 ]]> <![CDATA[ 1.68 ]]> <![CDATA[ 2.49 ]]> 0615-0017-09 <![CDATA[ 11.20 ]]> <![CDATA[ 1.32 ]]> <![CDATA[ 12.40 ]]> <![CDATA[ 1.87 ]]> <![CDATA[ 2.05 ]]> <![CDATA[ 3.88 ]]> 0820-0300-01 <![CDATA[ 10.14 ]]> <![CDATA[ 1.91 ]]> <![CDATA[ 5.37 ]]> 0.73 <![CDATA[ 2.19 ]]> <![CDATA[ 1.08 ]]> 0820-0300-02 <![CDATA[ 2.92 ]]> <![CDATA[ 3.16 ]]> <![CDATA[ 1.10 ]]> 0.85 <![CDATA[ 2.15 ]]> 0.98 0820-0300-03 <![CDATA[ 4.10 ]]> <![CDATA[ 4.48 ]]> <![CDATA[ 2.22 ]]> 0.77 <![CDATA[ 1.86 ]]> <![CDATA[ 1.03 ]]> 0820-0300-04 <![CDATA[ 3.31 ]]> 0.89 <![CDATA[ 2.15 ]]> 0.71 <![CDATA[ 1.36 ]]> 0.95 0820-0300-05 <![CDATA[ 3.54 ]]> <![CDATA[ 1.05 ]]> <![CDATA[ 1.33 ]]> 0.73 0.98 <![CDATA[ 1.09 ]]> 0820-0300-06 <![CDATA[ 2.90 ]]> <![CDATA[ 1.38 ]]> <![CDATA[ 1.21 ]]> 0.79 0.97 1.00 0820-0300-07 <![CDATA[ 10.20 ]]> <![CDATA[ 3.11 ]]> <![CDATA[ 7.29 ]]> 0.79 <![CDATA[ 1.04 ]]> <![CDATA[ 1.05 ]]> 0820-0300-08 <![CDATA[ 2.62 ]]> <![CDATA[ 1.05 ]]> <![CDATA[ 1.21 ]]> 0.74 <![CDATA[ 1.04 ]]> <![CDATA[ 1.17 ]]> 0820-0300-09 <![CDATA[ 8.11 ]]> <![CDATA[ 1.15 ]]> <![CDATA[ 6.41 ]]> <![CDATA[ 3.19 ]]> <![CDATA[ 1.10 ]]> <![CDATA[ 1.02 ]]> 0820-0300-10 <![CDATA[ 2.34 ]]> <![CDATA[ 1.14 ]]> <![CDATA[ 1.13 ]]> 0.96 <![CDATA[ 1.05 ]]> <![CDATA[ 1.02 ]]> 0820-0300-11 <![CDATA[ 3.28 ]]> 0.91 <![CDATA[ 1.13 ]]> 0.78 <![CDATA[ 1.02 ]]> 0.96 0820-0300-12 0.07 0.26 0.06 0.48 0.15 0.21 0820-0300-13 <![CDATA[ 4.15 ]]> <![CDATA[ 1.07 ]]> <![CDATA[ 2.26 ]]> <![CDATA[ 1.72 ]]> <![CDATA[ 1.02 ]]> <![CDATA[ 2.10 ]]> 0820-0300-14 <![CDATA[ 2.90 ]]> <![CDATA[ 1.07 ]]> <![CDATA[ 1.14 ]]> <![CDATA[ 1.69 ]]> 0.95 <![CDATA[ 2.06 ]]> 0820-0300-15 <![CDATA[ 3.61 ]]> 0.80 <![CDATA[ 1.34 ]]> <![CDATA[ 1.46 ]]> <![CDATA[ 1.02 ]]> <![CDATA[ 1.79 ]]> 0820-0300-16 <![CDATA[ 3.28 ]]> <![CDATA[ 1.29 ]]> <![CDATA[ 3.17 ]]> <![CDATA[ 1.06 ]]> <![CDATA[ 1.02 ]]> <![CDATA[ 1.33 ]]> 0820-0300-17 <![CDATA[ 2.65 ]]> 0.68 <![CDATA[ 1.28 ]]> 0.68 0.96 0.89 0820-0300-18 <![CDATA[ 2.83 ]]> 1.00 <![CDATA[ 1.20 ]]> 0.75 <![CDATA[ 2.03 ]]> <![CDATA[ 3.78 ]]> 0820-0300-19 <![CDATA[ 2.32 ]]> 0.74 <![CDATA[ 1.48 ]]> 0.73 <![CDATA[ 1.24 ]]> <![CDATA[ 1.22 ]]> 0820-0300-20 <![CDATA[ 8.16 ]]> <![CDATA[ 1.05 ]]> <![CDATA[ 5.09 ]]> 0.73 <![CDATA[ 1.00 ]]> <![CDATA[ 1.01 ]]>

[0125] The results in Table 6 show that the serum plate detection rates of kit 2-1 (i.e., the kit of this invention) and kit 2-3 (double antigen sandwich method) are comparable, but kit 2-1 exhibits a higher COI value and a more obvious sample gradient. Kits 2-2 (indirect method), 2-4 (capture method), 2-5 (double antigen sandwich + indirect method), and 2-6 (double antigen sandwich + capture method) all have lower detection rates than kits 2-1 and 2-3, and most detected samples have low COIs (around 1), indicating an indistinct sample gradient. Therefore, in terms of serum plate sensitivity, the kit / method of this invention is superior to methods based on the indirect method principle and kits / methods based on the capture method principle, as well as their individual combinations with double antigen sandwich methods (double antigen sandwich + indirect method and double antigen sandwich + capture method).

[0126] Example 7: Detection of Treponema pallidum antibody-IgM positive samples

[0127] use" Pathogen detection methods Thirty Treponema pallidum antibody-IgM positive samples (samples that were treated with IgG / RF factor adsorption reagent (purchased from Euromon GmbH, Germany) and then confirmed by Roche Treponema pallidum antibody kit) were tested, and the results are shown in Table 7 below.

[0128] Table 7

[0129] Reagent Kit 2-1 Reagent Kit 2-2 Reagent kit 2-3 Reagent kit 2-4 Reagent kit 2-5 Reagent kit 2-6 sample COI COI COI COI COI COI 1 <![CDATA[ 2.39 ]]> 0.83 0.91 <![CDATA[ 4.55 ]]> 0.91 <![CDATA[ 1.97 ]]> 2 <![CDATA[ 2.43 ]]> <![CDATA[ 1.09 ]]> <![CDATA[ 1.05 ]]> <![CDATA[ 4.67 ]]> <![CDATA[ 1.07 ]]> <![CDATA[ 2.10 ]]> 3 <![CDATA[ 1.99 ]]> <![CDATA[ 1.43 ]]> 0.96 <![CDATA[ 3.20 ]]> <![CDATA[ 1.08 ]]> <![CDATA[ 1.07 ]]> 4 <![CDATA[ 2.63 ]]> 0.83 <![CDATA[ 1.02 ]]> <![CDATA[ 5.01 ]]> 0.92 <![CDATA[ 2.17 ]]> 5 <![CDATA[ 6.96 ]]> <![CDATA[ 1.02 ]]> <![CDATA[ 1.03 ]]> <![CDATA[ 9.26 ]]> <![CDATA[ 1.16 ]]> <![CDATA[ 7.21 ]]> 6 <![CDATA[ 2.01 ]]> 0.55 <![CDATA[ 1.30 ]]> <![CDATA[ 3.34 ]]> <![CDATA[ 1.29 ]]> <![CDATA[ 1.45 ]]> 7 <![CDATA[ 2.81 ]]> 0.79 <![CDATA[ 1.19 ]]> <![CDATA[ 5.51 ]]> <![CDATA[ 1.15 ]]> <![CDATA[ 2.43 ]]> 8 <![CDATA[ 2.28 ]]> 0.59 <![CDATA[ 1.27 ]]> <![CDATA[ 3.87 ]]> <![CDATA[ 1.24 ]]> <![CDATA[ 1.71 ]]> 9 <![CDATA[ 2.43 ]]> 0.83 <![CDATA[ 1.18 ]]> <![CDATA[ 4.24 ]]> <![CDATA[ 1.14 ]]> <![CDATA[ 1.78 ]]> 10 <![CDATA[ 1.99 ]]> 0.96 <![CDATA[ 1.14 ]]> <![CDATA[ 2.82 ]]> <![CDATA[ 1.11 ]]> <![CDATA[ 1.14 ]]> 11 <![CDATA[ 7.00 ]]> <![CDATA[ 1.51 ]]> <![CDATA[ 1.18 ]]> <![CDATA[ 8.56 ]]> <![CDATA[ 1.25 ]]> <![CDATA[ 7.05 ]]> 12 <![CDATA[ 2.69 ]]> <![CDATA[ 1.47 ]]> 0.98 <![CDATA[ 5.31 ]]> <![CDATA[ 1.11 ]]> <![CDATA[ 2.31 ]]> 13 <![CDATA[ 5.76 ]]> <![CDATA[ 1.49 ]]> 0.88 <![CDATA[ 7.96 ]]> 0.99 <![CDATA[ 5.64 ]]> 14 <![CDATA[ 1.92 ]]> 0.70 0.97 <![CDATA[ 2.89 ]]> 0.92 <![CDATA[ 1.24 ]]> 15 <![CDATA[ 2.77 ]]> 0.72 0.91 <![CDATA[ 2.86 ]]> 0.90 <![CDATA[ 1.12 ]]> 16 <![CDATA[ 1.71 ]]> 0.92 <![CDATA[ 1.06 ]]> <![CDATA[ 2.71 ]]> <![CDATA[ 1.01 ]]> <![CDATA[ 1.06 ]]> 17 <![CDATA[ 2.70 ]]> 0.77 0.93 <![CDATA[ 2.46 ]]> 0.87 0.84 18 <![CDATA[ 1.51 ]]> 0.81 <![CDATA[ 1.02 ]]> <![CDATA[ 2.35 ]]> 0.96 0.85 19 <![CDATA[ 4.03 ]]> 0.91 0.96 <![CDATA[ 3.42 ]]> 0.89 <![CDATA[ 1.27 ]]> 20 <![CDATA[ 1.72 ]]> 0.91 <![CDATA[ 1.11 ]]> <![CDATA[ 2.59 ]]> <![CDATA[ 1.09 ]]> <![CDATA[ 1.04 ]]> 21 <![CDATA[ 1.63 ]]> 0.72 0.90 <![CDATA[ 2.24 ]]> 0.84 0.85 22 <![CDATA[ 2.20 ]]> <![CDATA[ 1.28 ]]> 0.90 <![CDATA[ 3.85 ]]> 0.92 <![CDATA[ 1.53 ]]> 23 <![CDATA[ 1.55 ]]> 0.64 1.00 <![CDATA[ 2.39 ]]> 0.92 0.92

[0130] 24 <![CDATA[ 4.78 ]]> 0.84 0.95 <![CDATA[ 1.78 ]]> <![CDATA[ 2.02 ]]> 0.65 25 <![CDATA[ 3.67 ]]> 0.74 <![CDATA[ 1.17 ]]> <![CDATA[ 2.19 ]]> <![CDATA[ 1.08 ]]> 0.95 26 <![CDATA[ 1.63 ]]> <![CDATA[ 1.26 ]]> 0.87 <![CDATA[ 2.69 ]]> 0.87 0.99 27 <![CDATA[ 2.93 ]]> <![CDATA[ 1.06 ]]> <![CDATA[ 1.06 ]]> <![CDATA[ 5.67 ]]> <![CDATA[ 1.04 ]]> <![CDATA[ 2.45 ]]> 28 <![CDATA[ 2.85 ]]> 0.61 <![CDATA[ 1.17 ]]> <![CDATA[ 5.49 ]]> <![CDATA[ 1.15 ]]> <![CDATA[ 2.46 ]]> 29 <![CDATA[ 2.00 ]]> 0.84 <![CDATA[ 1.13 ]]> <![CDATA[ 2.63 ]]> <![CDATA[ 1.18 ]]> <![CDATA[ 1.08 ]]> 30 <![CDATA[ 2.48 ]]> 0.85 <![CDATA[ 1.14 ]]> <![CDATA[ 4.49 ]]> <![CDATA[ 1.13 ]]> <![CDATA[ 1.85 ]]>

[0131] As shown in Table 7, when detecting Treponema pallidum antibody-IgM positive samples, Kit 2-1 (the kit of the present invention) and Kit 2-4 (capture method) detected all samples. Kit 2-2 (indirect method) and Kit 2-3 (double antigen sandwich) only detected about half of the samples, and the COI values ​​were relatively low, mostly around 1. Although Kit 2-5 (double antigen sandwich + indirect method) and Kit 2-6 (double antigen sandwich + capture method) showed improved detection rates compared to 2-2 and 2-3, some samples still failed to be detected. Therefore, in terms of sensitivity for detecting Treponema pallidum antibody-IgM positive samples, the kit / method of the present invention is superior to methods based on the indirect method principle, kits / methods based on the double antigen sandwich principle, and combinations of both (double antigen sandwich + indirect method). Furthermore, combining the capture method and the double antigen sandwich method actually reduces the detection rate of Treponema pallidum antibody-IgM positive samples (Kit 2-6 is weaker than Kit 2-4). This conclusion is consistent with the trend observed in Example 3.

[0132] Example 8: Detection of Treponema pallidum antibody-negative samples

[0133] use" Pathogen detection methods "500 samples with negative Treponema pallidum antibodies (derived from hospital diagnostic results) were tested, and the statistical results are shown in Table 8 below."

[0134] Table 8

[0135] Reagent Kit 2-1 Reagent Kit 2-2 Reagent kit 2-3 Reagent kit 2-4 Reagent kit 2-5 Reagent kit 2-6 Total sample size 500 500 500 500 500 500 False positive sample number 0 1 0 2 1 1 False positive probability 0％ 0.2％ 0％ 0.4％ 0.2％ 0.2％

[0136] As shown in Table 8, when detecting Treponema pallidum antibody-negative samples, neither Kit 2-1 (the kit of the present invention) nor Kit 2-3 (double antigen sandwich) produced false positive samples. Kit 2-2 (indirect method), Kit 2-4 (capture method), and the kits combining both detection modes: Kit 2-5 (double antigen sandwich + indirect method) and Kit 2-6 (double antigen sandwich + capture method) all had a false positive probability of 0.2% to 0.4%. Therefore, in terms of specificity for detecting Treponema pallidum antibody-negative samples, the kits / methods of the present invention are superior to those based on the indirect method or capture method, as well as those based on a combination of the indirect method or capture method and the double antigen sandwich method.

Claims

1. A kit for detecting antibodies against infectious diseases in human blood samples, comprising: The first reagent includes at least one antigen coated on a solid support and an anti-human IgM antibody coated on a solid support. as well as The second reagent comprises at least one labeled antigen and a labeled anti-human IgG antibody; Wherein, at least one antigen among the at least one antigen coated on the solid support and at least one antigen among the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample. The solid support is a magnetic bead.

2. A kit for detecting antibodies against infectious diseases in human blood, comprising: The first reagent comprises at least one antigen coated on a solid support and an anti-human IgG antibody coated on a solid support. as well as The second reagent comprises at least one labeled antigen and a labeled anti-human IgM antibody; Wherein, at least one antigen among the at least one antigen coated on the solid support and at least one antigen among the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample. The solid support is a magnetic bead.

3. The kit according to claim 1 or 2, wherein the kit further comprises a third reagent containing a blocking agent.

4. The kit according to claim 3, wherein, The sealing agent is selected from one or more of the following: skim milk powder, BSA, gelatin, serum, casein, egg white protein, animal IgG, and surfactant.

5. The kit according to claim 1 or 2, wherein the kit further comprises a fourth reagent containing a reducing agent.

6. The kit according to claim 5, wherein, The reducing agent is selected from one or more of the following: DTT, β-mercaptoethanol.

7. The kit according to any one of claims 1 to 2, wherein, The kit is used to detect antibodies produced in a subject after infection with a pathogen; wherein the pathogen is a virus or a spirochete.

8. The kit according to claim 7, wherein, The virus in question is HCV.

9. The kit according to claim 7, wherein, The spirochete is Treponema pallidum.

10. The kit according to claim 7, wherein, The kit is used to detect antibodies produced in subjects after HCV infection; the at least one antigen coated on a solid support includes at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen coated on a solid support; the at least one labeled antigen includes at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen labeled.

11. The kit of claim 10, wherein, The at least one antigen coated on the solid support includes HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen coated on the solid support respectively; the at least one labeled antigen includes HCV core antigen and HCV NS3 antigen respectively labeled.

12. The kit of claim 10, wherein, The at least one antigen coated on the solid support includes HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen coated on the solid support respectively; the at least one labeled antigen includes HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen labeled respectively.

13. The kit according to claim 7, wherein, The kit is used to detect antibodies produced in subjects after infection with Treponema pallidum; the at least one antigen coated on a solid support includes at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen coated on a solid support; the at least one labeled antigen includes at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen labeled respectively.

14. The kit of claim 13, wherein, The at least one antigen coated on the solid support includes TP15 antigen, TP17 antigen and TP47 antigen coated on the solid support respectively; the at least one labeled antigen includes TP15 antigen, TP17 antigen and TP47 antigen labeled respectively.

15. The kit according to claim 1 or 2, wherein, The antigen exists in the form of a polymer, antigen fragment, or peptide.

16. The kit according to claim 1 or 2, wherein, The second reagent uses a labeling enzyme for labeling, and the kit also includes a reaction substrate for the labeling enzyme.

17. The kit of claim 16, wherein, The reaction substrate is 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoryl)-phenyl-1,2-dioxane.

18. The kit according to claim 1, wherein, The concentration of the labeled anti-human IgG antibody in the second reagent is 100 ng / mL to 300 ng / mL, and the concentration of the anti-human IgM antibody in the first reagent is 10 ng / mL to 30 ng / mL.

19. The kit according to claim 1, wherein, The concentration of the labeled anti-human IgG antibody in the second reagent is 10 times the concentration of the anti-human IgM antibody in the first reagent.

20. The kit according to claim 2, wherein, The concentration of the anti-human IgG antibody in the first reagent is 100 ng / mL to 300 ng / mL, and the concentration of the anti-human IgM antibody in the second reagent is 10 ng / mL to 30 ng / mL.

21. The kit according to claim 2, wherein, The concentration of the anti-human IgG antibody in the first reagent is 10 times the concentration of the anti-human IgM antibody in the second reagent.

22. The use of the first and second reagents in the preparation of a kit for detecting antibodies produced after infection with a pathogen in a sample, including the following: The sample, the first reagent, and the second reagent are mixed and reacted, wherein, The first reagent comprises at least one antigen coated on a solid support and an anti-human IgM antibody coated on a solid support, and the second reagent comprises at least one labeled antigen and a labeled anti-human IgG antibody. The solution after the reaction was cleaned, and Acquire signal values; and obtain antibody detection results based on the signal values; The solid support is a magnetic bead; The at least one antigen coated on the solid support and the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample.

23. The use of the first and second reagents in the preparation of a kit for detecting antibodies produced after infection with a pathogen in a sample, including the following: The sample, the first reagent, and the second reagent are mixed and reacted, wherein, The first reagent comprises at least one antigen coated on a solid support and an anti-human IgG antibody coated on a solid support, and the second reagent comprises at least one labeled antigen and a labeled anti-human IgM antibody. The solution after the reaction was cleaned, and Acquire signal values; and obtain antibody detection results based on the signal values; The solid support is a magnetic bead; The at least one antigen coated on the solid support and the at least one labeled antigen can bind to the same IgG antibody or the same IgM antibody in the sample.

24. The use as described in claim 22 or 23, wherein, The sample is first mixed with the first reagent and incubated for a predetermined period of time, and then the second reagent is added and incubated for another predetermined period of time.

25. The use as described in claim 22 or 23, wherein, The third reagent is added together with the first and second reagents, wherein the third reagent contains a blocking agent.

26. The use as described in claim 25, wherein, The sealing agent is selected from one or more of the following: skim milk powder, BSA, gelatin, serum, casein, egg white protein, animal IgG, and surfactant.

27. The use as described in claim 22 or 23, wherein, The fourth reagent is added together with the first reagent, and the fourth reagent contains a reducing agent.

28. The use as described in claim 27, wherein, The reducing agent is selected from one or more of the following: DTT, β-mercaptoethanol.

29. The use as described in claim 22 or 23, wherein, The pathogen is a virus or a spirochete.

30. The use as described in claim 29, wherein, The virus in question is either HIV or HCV.

31. The use as described in claim 29, wherein, The spirochete is Treponema pallidum.

32. The use as described in claim 29, wherein, The pathogen is HCV; the at least one antigen coated on the solid support includes at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen coated on the solid support respectively; the at least one labeled antigen includes at least one of HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen respectively labeled.

33. The use as described in claim 32, wherein, The at least one antigen coated on the solid support includes HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen coated on the solid support respectively; the at least one labeled antigen includes HCV core antigen and HCV NS3 antigen respectively labeled.

34. The use as described in claim 32, wherein, The at least one antigen coated on the solid support includes HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen coated on the solid support respectively; the at least one labeled antigen includes HCV core antigen, HCV NS3 antigen, HCV NS4 antigen and HCV NS5 antigen labeled respectively.

35. The use as described in claim 29, wherein, The pathogen is Treponema pallidum; the at least one antigen coated on the solid support includes at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen coated on the solid support respectively; the at least one labeled antigen includes at least one of TP15 antigen, TP17 antigen, TP47 antigen and TP45 antigen respectively labeled.

36. The use as described in claim 35, wherein, The at least one antigen coated on the solid support includes TP15 antigen, TP17 antigen and TP47 antigen coated on the solid support respectively; the at least one labeled antigen includes TP15 antigen, TP17 antigen and TP47 antigen labeled respectively.

37. The use as described in claim 22 or 23, wherein, The antigen exists in the form of a polymer, antigen fragment, or peptide.

38. The use as described in claim 22 or 23, wherein, The samples were selected from whole blood, plasma, and serum.

39. The use as described in claim 22, wherein, The concentration of the labeled anti-human IgG antibody in the second reagent is 100 ng / mL to 300 ng / mL, and the concentration of the anti-human IgM antibody in the first reagent is 10 ng / mL to 30 ng / mL.

40. The use as described in claim 22, wherein, The concentration of the labeled anti-human IgG antibody in the second reagent is 10 times the concentration of the anti-human IgM antibody in the first reagent.

41. The use as described in claim 23, wherein, The concentration of the anti-human IgG antibody in the first reagent is 100 ng / mL to 300 ng / mL, and the concentration of the anti-human IgM antibody in the second reagent is 10 ng / mL to 30 ng / mL.

42. The use as described in claim 23, wherein, The concentration of the anti-human IgG antibody in the first reagent is 10 times the concentration of the anti-human IgM antibody in the second reagent.

Images