Binding molecule for acute infection diagnosis, acute infection diagnosis system and kit

Abstract

The invention belongs to but not limited to the technical field of human and animal health diagnostics, and discloses a binding molecule for acute infection diagnosis adapted to a point-of-care (POC) scene, an acute infection diagnosis system and a kit. The binding molecule is an antibody, a nano antibody or an aptamer (preferably not combined with IgM) which is specifically combined with a J chain of dIgA, steric hindrance and conformation rearrangement are avoided by combining the J chain, and the flexibility of a compound is kept so as to efficiently combine with an antigen; the diagnostic system and the kit are adapted to POC scenarios (such as lateral flow test strips). The problems that POC deployment is difficult, RAT sensitivity is low and dIgA detection efficiency of CSC is low in existing NAT are solved, and the kit has the advantages of being low in cost, high in specificity and good in stability, can be used for rapid diagnosis of multiple acute infections such as measles and syphilis, fills up related technical gaps and has high commercial value.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of human and animal health diagnostics, and in particular relates to a binding molecule for acute infection diagnosis adapted to point-of-care (POC) settings, an acute infection diagnosis system and a kit. BACKGROUND

[0002] Nucleic acid detection tests (NATs), such as polymerase chain reaction (PCR), are considered the “gold standard” for most acute infection diagnoses, but are limited by equipment, operational complexity, and cost, and cannot be deployed on a large scale in point-of-care (POC) settings, making it difficult to meet the needs of rapid diagnosis at the grassroots or on-site. Rapid antigen detection tests (RATs) have been widely used for diseases such as SARS-CoV-2 and hepatitis B virus, but have low sensitivity and are not suitable for detection of diseases such as syphilis, which have very low antigen content, and are prone to false negative results.

[0003] Serological testing (antibody testing): Disease-specific immunoglobulin M (IgM) and dimeric immunoglobulin A (dIgA) have high potential in the diagnosis of acute infection, especially dIgA as an acute infection marker is more specific than IgM (e.g. in patients with hepatitis C, IgM / IgA responses can last for months to years, while dIgA responses are transient). However, in the prior art, when detecting dIgA through polymeric immunoglobulin receptor (pIgR) or secretory component, the detection efficiency is extremely low due to steric hindrance between antigen and antibody binding. Further, the dIgA detection system based on chimeric secretory component (CSC) has a key defect: after CSC or pIgR binds to dIgA, a significant conformational rearrangement occurs, forming a rigid complex, which can cause a significant decrease in antigen capture efficiency (e.g. in measles diagnosis, CSC can only detect trace amounts of measles nucleoprotein (NP)-specific dIgA, but can detect large amounts of measles virus lysate-specific dIgA, proving that CSC has incompatibility with dIgA detection of specific antigens such as NP: Mohd Hanafiah K, Hiebert J, Zubach V, Severini A, Anderson DA, Drummer HE. Microbiol Spectr. 2024 Jan 11;12(1):e0343723. doi: 10.1128 / spectrum.03437-23. Published 2023 Dec 11. PMID: 38078716). In addition, the CSC technology also has the problem of too long reaction time: in enzyme-linked immunosorbent assay (ELISA) and other experiments, overnight incubation is required to obtain results, which is incompatible with routine diagnostic applications; at the same time, CSC cannot effectively bind to dIgA that has already bound to antigen, and vice versa, antigen cannot effectively bind to dIgA that has already bound to CSC (see Wei et al., Immun Cell Biol. 2023 Oct;101(9):857-866. doi: 10.1111 / imcb.12682).

[0004] Furthermore, while existing anti-J chain antibodies (such as Thermo Fisher's Mc19-9 monoclonal antibody) can bind to the J chain (both dIgA and pentamer IgM contain one J chain), Thermo Fisher's Mc19-9 monoclonal antibody only fails to detect IgM in enzyme-linked immunosorbent assay (ELISA), but can detect IgM in Western blot. It is unclear whether other existing anti-J chain monoclonal antibodies can distinguish between dimeric immunoglobulin A (dIgA) and pentamer immunoglobulin M (IgM), and none of the existing anti-J chain antibodies (including the aforementioned selective anti-J chain antibodies) have been documented for the diagnosis of acute infections; their industrial applicability in acute infection detection has not been explored.

[0005] Based on the above analysis, the technical problems that urgently need to be solved in the existing diagnostic techniques for acute infections are: (1) Existing NAT technology is difficult to deploy on a large scale in POC scenarios and cannot meet the needs of rapid diagnosis; (2) RAT has low sensitivity and cannot be used to diagnose acute infections (such as syphilis) with extremely low antigen content; (3) Because IgM antibodies have low affinity but high affinity, the sensitivity and / or specificity of detecting IgM antibodies for diagnosing acute infections are often low. (4) When CSC detects dIgA, the antigen capture efficiency is low and the compatibility is poor due to conformational rearrangement and steric hindrance. (5) Existing anti-J chain antibodies are not used for the diagnosis of acute infection and lack the ability to specifically detect dIgA, so the diagnostic specificity cannot be improved by dIgA. Summary of the Invention

[0006] To address the problems existing in the prior art, this invention provides a binding molecule, an acute infection diagnostic system, and a reagent kit adapted for point-of-care testing (POC) scenarios, which can be used to detect acute infections in humans or other mammals (such as measles, syphilis, hepatitis C, etc.).

[0007] This invention is achieved by providing a binding molecule for the diagnosis of acute infection, wherein the binding molecule is an antibody, nanobody, or aptamer that specifically binds to the J chain of the target species dimeric immunoglobulin A (dIgA), and the binding molecule does not bind to the J chain of the target species pentamer immunoglobulin M (IgM); the binding molecule maintains the structural flexibility of the dIgA-binding molecule complex by binding to the J chain of dIgA, thereby efficiently binding acute infection-related antigens.

[0008] Furthermore, the target species is a human or a mammal.

[0009] Furthermore, the binding molecules are obtained through the following screening method: recombinant dIgA is bound to an acute infection-associated antigen to form an antigen-dIgA complex, and the antigen-dIgA complex is used as a screening target to screen for molecules that can specifically bind to the J chain of the dIgA.

[0010] The binding molecules are obtained through the following specific screening method: recombinant dIgA targeting a specific acute infection-associated antigen (such as recombinant human dIgA against syphilis TP0453) is bound to the corresponding acute infection-associated antigen (such as syphilis TP0453 antigen) to form an antigen-dIgA complex. Using this complex as a screening target, molecules that can specifically bind to the J chain in dIgA and do not bind to the J chain in the target species IgM are screened.

[0011] Another object of the present invention is to provide an acute infection diagnostic system, characterized in that it includes the aforementioned binding molecule and a detection component; the detection component includes an acute infection-associated antigen conjugated with a marker, wherein the marker is colloidal gold, a fluorescent substance, or a visible biomarker.

[0012] Furthermore, the detection method of the detection unit is immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or fluorescence immunoassay.

[0013] Furthermore, the system is used in point-of-care medical testing (POC) scenarios.

[0014] Another object of the present invention is to provide an acute infection diagnostic kit comprising the aforementioned binding molecule and the aforementioned detection component; the kit is a lateral flow test strip kit, the test strip comprising a test line and a control line, the test line being coated with the binding molecule and the control line being coated with a quality control antibody.

[0015] Furthermore, the binding molecules are fixed on a solid support, which is a nitrocellulose membrane, a microporous plate, or magnetic particles.

[0016] Furthermore, the acute infection is measles infection, syphilis infection, or hepatitis C infection; the acute infection-related antigen is measles nucleoprotein (NP) antigen, syphilis TP0453 antigen, or hepatitis C virus-related antigen, or antigen derived from human immunodeficiency virus (HIV) or other viruses that are significant to human or animal health.

[0017] Based on the above technical solutions and the technical problems solved, the advantages and positive effects of the technical solution to be protected by this invention are as follows: First, this invention features high antigen binding efficiency and strong compatibility; by binding to the J chain of dIgA (rather than the entire molecule), it avoids conformational rearrangement and steric hindrance caused by CSC or pIgR. The dIgA-binding molecule complex can effectively bind to a variety of acute infection-related antigens (such as measles NP antigen, syphilis TP0453 antigen, and HIV antigen), solving the problem that CSC is only compatible with some antigens.

[0018] Compared to CSC / HSC, diagnostic detection of dIgA / IgM using anti-J chain or other J chain binding agents is more economical and performs better because: (1) the anti-chain / antibody complex has less steric hindrance when binding to the relevant antibody / antigen complex, thus being compatible with more antigens and applicable to more diseases; (2) recombinant expressed antibodies / nanobosomes may have advantages in stability and production cost compared to CSC reagents; (3) reliable conversion to other animal species (preparing monoclonal antibodies or cross-reactive polyclonal antibodies, rather than relying on species cross-reactivity of polyimmunoglobulin receptors and polyimmunoglobulins, which may or may not be effective). The diagnostic specificity and stability of the present invention are significantly improved by including: Specificity: The preferred binding molecules detect only dIgA (not IgM), and dIgA has better specificity as a marker of acute infection than IgM (e.g. in hepatitis C, the dIgA response is short-lived and can accurately distinguish between acute and recovery infections). Stability and cost: Recombinant expressed antibodies / nanobosomes may have higher stability and lower production costs compared to CSC reagents, due to the many different service experiences in antibody production versus recombinant CSC production and purification. Cross-species adaptability: By preparing monoclonal or cross-reactive polyclonal binding molecules, it is possible to adapt to the diagnosis of acute infections in different mammalian species (without relying on species cross-reactivity of pIgR, resulting in stronger compatibility).

[0019] Secondly, as supplementary evidence of the inventive step of the claims of this invention, it is also reflected in the following important aspects: (1) The expected benefits and commercial value of the technical solution of this invention after transformation are as follows: It is adapted to POC scenarios and can be used for rapid diagnosis in primary healthcare, on-site epidemic prevention and other scenarios, meeting the clinical need for "immediate detection" of acute infections; It is low in cost, has good stability, is easy to industrialize, and can cover a variety of acute infections such as measles, syphilis, hepatitis C, and HIV, with a wide range of market applications.

[0020] (2) The technical solution of this invention fills a technical gap in the industry both domestically and internationally: This invention fills the technological gap in "selective detection of dIgA using anti-J chain binding molecules for the diagnosis of acute infection POC". Existing anti-J chain antibodies either cannot distinguish between dIgA and IgM, or have not been applied to the diagnosis of acute infection. This invention solves the problem that CSC requires long-term incubation and cannot effectively bind to dIgA that has already bound antigens by screening selective binding molecules and diagnostic applications, thereby improving diagnostic specificity and detection efficiency.

[0021] (3) The technical solution of the present invention solves a technical problem that people have long wanted to solve but have never been able to solve successfully: The technical challenge of "low antigen capture efficiency due to steric hindrance in dIgA detection" has been overcome, solving the problem of "inability to adapt to the short-time detection needs of routine diagnosis" in CSC technology, and adapting to the rapid detection needs of POC scenarios. At the same time, the industrial applicability of anti-J chain antibodies in the diagnosis of acute infections has been explored.

[0022] The technical solution of this invention overcomes technical bias: Breaking away from the technical bias of "relying on pIgR / CSC to detect dIgA", this paper proposes a new approach of "achieving efficient detection of dIgA through J chain specific binding", providing a brand-new technical path for the serological diagnosis of acute infections. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the side-flow instantaneous test strip provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the point-of-care (POC) test for measles nucleoprotein-specific dIgA plus / minus IgM provided in an embodiment of the present invention. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0025] This invention specifically binds to the J chain of dIgA (rather than pIgR / CSC binding the entire dIgA molecule), avoiding conformational rearrangement and steric hindrance of the complex, thus maintaining the structural flexibility of the dIgA-binding molecule complex and achieving efficient binding of acute infection-related antigens. Simultaneously, by screening, binding molecules that bind only dIgA (without IgM) can be obtained, avoiding the low specificity (and correspondingly low sensitivity) problems associated with IgM detection, further improving diagnostic accuracy.

[0026] The product structure and composition provided in this invention embodiment, whose core binding molecule includes antibodies, nanobodies, or aptamers, are characterized by: It specifically binds to the J chain of dIgA in the target species (human or other mammals); Preferably, the binding molecule does not bind to the J chain in the pentameric IgM of the target species (achieved through screening). The following screening method can be used: Recombinant dIgA (such as recombinant dIgA against syphilis TP0453) is combined with acute infection-associated antigen (such as recombinant syphilis TP0453) to form an "antigen-dIgA complex," which can be screened and verified using enzyme-linked immunosorbent assay (ELISA): Acute infection-associated antigen (such as syphilis TP0453 antigen) is coated onto a detection plate, reacts with the corresponding recombinant dIgA to form an antigen-dIgA complex, and then the molecule to be screened is added. The binding ability of the molecule to be screened with the complex is detected, and the binding activity with IgM is excluded by reaction test. Using this complex as a target, molecules that can efficiently bind to the J chain of dIgA are screened to ensure that the binding molecule is compatible with the dIgA that has already bound the antigen.

[0027] The acute infection diagnostic system provided in this embodiment of the invention consists of the above-mentioned binding molecules and detection components. The detection components include acute infection-related antigens (such as measles NP antigen, syphilis TP0453 antigen, and various HIV antigens) coupled with markers (colloidal gold, fluorescent substances, etc.) and are adapted for POC scenarios (such as lateral flow test strips).

[0028] The acute infection diagnostic kit provided in this invention comprises the above-mentioned binding molecule and detection component, preferably a lateral flow test strip kit, wherein the test strip comprises: Detection line: coated with the bound molecules; Control line: coated with quality control antibody (such as anti-IgG antibody); Gold labeling zone: Acute infection-associated antigens of adsorbed and coupled markers.

[0029] This invention uses an antibody targeting the J chain of a dimeric IgA antibody to selectively bind dIgA, and optionally selectively binds the J chain of IgM. While both dIgA and pentamer IgM contain a J chain, some anti-J chain antibodies are known to be selective only for dIgA and not for IgM (e.g., ThermoFisherMc19-9). By binding the antibody to the J chain, rather than to the entire dIgA molecule via pIgR / CSC, this complex retains greater structural flexibility, thus enabling more efficient antigen binding.

[0030] Conversely, when dIgA binds to the target antigen for the first time, the anti-J chain antibody can bind to the dIgA antibody without requiring structural rearrangement of the dIgA / antigen binding complex, which is less efficient if the antibody has already bound to the antigen.

[0031] This invention provides an antibody or other binding molecule (such as a nanobody or aptamer) that specifically binds to the J chain of dimeric IgA and pentameric IgM in species of interest (humans or other mammals), thereby enabling the simultaneous detection of both dIgA and IgM, both of which serve as biomarkers for acute infection in many diseases. More preferably, the antibody or other binding molecule binds only to the J chain of dimeric IgA and not to the J chain of pentameric IgM, thereby enabling selective detection of dIgA instead of IgM. Detecting dIgA instead of IgM avoids the common low specificity problem of IgM-based detection, as dIgA is a more specific biomarker for acute infection. For example, in patients infected with hepatitis C virus, IgM and IgA responses can persist for months or years after infection, while dIgA responses are transient (BMC Research Notes, October 1, 2018; 11(1):688. doi:10.1186 / s13104-018-3799-2. Detection of virus-specific polyimmunoglobulin A in serum samples of acute hepatitis A, C, and E virus using novel chimeric secretory components. Khayriyya hMohd Hanafiah, Mary L Garcia, Nadine C Barnes, David A Anderson PMID:30285838DOI:10.1186 / s13104-018-3799-2) Side-flow instantaneous test strips, such as Figure 1 As shown, Figure 1 A: Established technologies (specifically targeting dimeric IgA / polymeric IgA) can efficiently bind dimeric IgA (and can be detected by anti-IgA gold labeling), but the structural arrangement of dimeric IgA bound to CSCs limits its effective binding to antigens. Figure 1 B: The anti-J chain binds to both dimer IgA / dimer IgA and IgM, and can also efficiently capture dimer IgA (and IgM), but the antibody structure does not rearrange, thus it can effectively bind to the antigen. Figure 1 C: The anti-J chain binds only to dimer IgA / merger IgA but not to IgM, and can effectively bind to antigens like (B).

[0032] This invention provides a lateral flow point-of-care (POC) test for diagnosing measles infection; measles nucleoprotein is the only measles antigen that can be used as a highly purified, recombinant protein product for laboratory (ELISA) or POC diagnosis. ELISA against measles nucleoprotein has shown good performance, although POC tests against measles nucleoprotein-specific IgM have been described, but their performance is poor (PMID: 38078716). In ELISA, measles nucleoprotein-specific dIgA was detected using CSC reagent, but its level was much lower than that of nucleoprotein-specific IgM, indicating that a sensitive method for detecting measles dIgA cannot be prepared using CSC reagent and nucleoprotein antigen. In contrast, we anticipate that the structural flexibility of dIgA achieved through anti-streptolysin antibodies will enable efficient binding of recombinant NPs (as well as NPs bound to colloidal gold or other visible, fluorescent, or other labeled substances), potentially leading to the development of an efficient, sensitive, and specific point-of-care (POC) assay for dIgA or dIgA plus IgM for measles detection. A lateral flow point-of-care (POC) assay targeting measles nucleoprotein-specific dIgA plus / minus IgM is described below. Figure 2 As shown; Figure 2 A: CSC (binding only dIgA / polymeric IgA) is an established technology that can effectively bind dIgA, but the structural arrangement of dIgA bound to CSC limits its effective binding to the antigen. Figure 2 B: The anti-J chain binds to both dIgA / polymeric IgA and IgM, effectively capturing dIgA (and IgM), and the captured dIgA / IgM can effectively bind to the NP-gold complex. Figure 2 C: The anti-J chain binds only dIgA / polymeric IgA but not IgM. The captured dIgA can effectively bind to the NP-gold complex, the same as B.

[0033] Evidence related to the technical effects obtained by the embodiments of the present invention.

[0034] POC tests, especially lateral POC tests used to detect antibodies as markers of acute infection, typically use anti-IgM antibodies to capture IgM in a subject's blood or plasma sample. Then, a disease-specific antigen bound to the marker (such as colloidal gold as a visible marker) is reacted with the captured sample. If the captured sample is present, it is disease-specific IgM. For example, this method was used to detect hepatitis E virus (HEV) IgM antibodies (Chen HY, Lu Y, Howard T, Anderson D, Fong PY, Hu WP, Chia CP, Guan m). Clinical Diagnostic Laboratory Immunology. May 2005; 12(5):593-8. doi:10.1128 / cdli.12.5.593-598.2005. PMID: 15879020;PMCID: PMC1112076). The capture of one antibody by another antibody (or a similar binding agent such as a nanobody) generally does not limit the conformational flexibility and efficient reactivity of the capturing antibody (such as IgM or dIgA).

[0035] Conversely, capturing dIgA via CSC reagents (or capturing dIgA or IgM via pIgR) involves a complex rearrangement of CSC / pIgR with dIgA / IgM, forming what is equivalent to secretory IgA or IgM, where the secretory component is encapsulated in a significant proportion of the Fc portion of the dIgA molecule. This conformational rearrangement may reduce the structural flexibility of the bound dIgA / IgM and decrease its ability to react effectively with homologous antigens to provide diagnostic test results.

[0036] Some antigens are expected to bind efficiently to CSC dIgA / IgM / pIgR complex, depending on having an easily accessible antigenic epitope. POC and lateral flow assays for SARS-CoV-2 dIgA have been described, which is observed (Drummer, TA, NYK, E; Zheng, N.S.; Wei, Z.S.; Hugh, RJ; Li, F.S.; Bhat, P.; F.F., R.S.; Liu, JS.; McMahon, J.; Laeyendecker, O.; Fernandez Reinsurance, Manabe, YC.; Klein, SL.; Quinn, TC.; Anderson, D.S.). Dimeric IgA is a specific biomarker for recent SARS-CoV-2 infection. medRxiv preprint. July 1, 2021. 2021.06.28.21259671. doi: 10.1101 / 2021.06.28.21259671. PMID: 34230936; PMCID: PMC8259913). However, as mentioned above, the measles ribonucleoprotein-linked immunosorbent assay (Mohd Hanafiah K, Hiebert J, Zubach V, Severini A, Anderson DA, Drummer HE. Dimeric immunoglobulin A as a novel diagnostic marker for measles infection Microbial Spectrum, January 11, 2024; 12(1):e0343723. doi:10.1128 / spectrum.03437-23. Electronic version December 11, 2023. PMID: 38078716; PMCID:PMC10783017) and ELISA intermediate SARS-CoV-2 antigen (Wei Z, Angrisano F, Eriksson EM, Mazhari R, Van H, Zheng S, Center RJ, Boo I, McMahon J, Lau J, Kiernan-Walker N, Ruybal-Pesántez S, Mueller I, Robinson LJ, Anderson DA, Drummer HE). Serological detection method for SARS-CoV-2 infected patients with dimeric IgA antibodies. Immunocyte Biology, October 2023; 101(9):857-866. doi: 10.1111 / imcb.12682. Electronic version August 18, 2023. PMID: 37593973; PMCID: PMC10952984). The detection reactivity of dIgA may require a longer incubation period for the detection component (e.g., overnight instead of 1 hour), which is incompatible with point-of-care or side-flow applications.

[0037] By using antibodies or other binding components, such as nanobodies targeting dIgA (or dIgA and IgM), which bind to the J-chain component in a manner similar to any other anti-ig antibody, it is expected that assays including POC and lateral flow assays will maintain the efficient binding of dIgA or dIgA / IgM to the homologous specific antigen, thereby increasing the flexibility of antibody binding and resulting in more efficient assays with higher sensitivity and specificity compared to assays using CSC or pIgR to bind dIgA or dIgA / IgM.

[0038] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any modifications, equivalent substitutions, and improvements made by those skilled in the art within the scope of the technology disclosed in the present invention, and within the spirit and principles of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A binding molecule for the diagnosis of acute infection, characterized in that, The binding molecule is an antibody, nanobody, or aptamer obtained through a specific screening method that specifically binds to the J chain of the target species' dimeric immunoglobulin A (dIgA), and the binding molecule does not bind to the J chain of the target species' pentamer immunoglobulin M (IgM). The specific screening method involves binding recombinant dIgA targeting an acute infection-associated antigen to the acute infection-associated antigen to form an antigen-dIgA complex. Using this complex as a screening target, molecules that specifically bind to the J chain of dIgA but do not bind to the J chain of IgM are screened. The binding molecule maintains the structural flexibility of the dIgA-binding molecule complex by binding to the J chain of dIgA, thereby binding to the acute infection-associated antigen.

2. The binding molecule according to claim 1, characterized in that, The target species is a human or a mammal.

3. An acute infection diagnostic system, characterized in that, The invention includes the binding molecule as described in claim 1 or 2, and a detection component; the detection component includes an acute infection-associated antigen with a conjugated marker, said marker being colloidal gold, a fluorescent substance, or a visible marker.

4. The diagnostic system according to claim 3, characterized in that, The detection unit employs immunochromatography, enzyme-linked immunosorbent assay (ELISA), chemiluminescent immunoassay, or fluorescence immunoassay as its detection method.

5. The diagnostic system according to claim 3, characterized in that, The system is used for point-of-care medical testing (POC) scenarios.

6. An acute infection diagnostic kit, characterized in that, The kit comprises the binding molecule as described in claim 1 or 2, and the detection component as described in claim 3; the kit is a lateral flow test strip kit, the test strip comprising a test line and a control line, the test line being coated with the binding molecule, and the control line being coated with a quality control antibody.

7. The diagnostic kit according to claim 6, characterized in that, The binding molecules are fixed on a solid support, which is a nitrocellulose membrane, a microporous plate, or magnetic particles.

8. The reagent kit according to claim 6, characterized in that... The acute infection is measles infection, syphilis infection, hepatitis C infection, or HIV infection; the acute infection-related antigen is measles nucleoprotein (NP) antigen, syphilis TP0453 antigen, hepatitis C virus-related antigen, or HIV-related antigen.

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