Peptides, nucleic acid, recombinant expression vector, cell, composition for detecting influenza, and kit for detecting influenza

Abstract

Disclosed are a peptide, a nucleic acid, a recombinant expression vector, a cell, a composition for detecting influenza, and a kit for detecting influenza. The peptide sequence may include CGPVPIRKVDD, CRLRLVLARDF, GDPITSRRLDY, or GTRIALPLRDV The nucleic acid may encode the peptide.

Description

Peptides, nucleic acids, recombinant expression vectors, cells, compositions for detecting influenza, and kits for detecting influenza

[0001] The present invention relates to peptides, nucleic acids, recombinant expression vectors, cells, compositions for detecting influenza, and kits for detecting influenza.

[0002] Influenza is an acute viral respiratory infection characterized by symptoms such as fever, sore throat, dry cough, and nasal congestion. It is caused by the influenza virus. Influenza infection can lead to serious or life-threatening complications in high-risk individuals. Three types of influenza viruses (influenza A, B, and C) belong to the Orthomycoviridae family and cause influenza infection. Type A is known to cause most epidemics; type B infects humans and other species and has similar biological properties to type A. Influenza viruses frequently undergo antigenic variation. This variation occurs due to significant changes in two glycoproteins on the viral surface: hemagglutinin (HA) and neuraminidase (NA). The gold standard for detecting influenza viruses includes RT-PCR and ELISA using throat swab samples. Conventional methods for detecting influenza viruses include serology, viral culture, rapid tests, and immunofluorescence assays. Biosensors based on real-time RT-PCR and various types of transducers, such as piezoelectric transducers such as quartz crystal microbalance (QCM) and surface acoustic wave (SAW), optical transducers such as surface plasmon resonance (SPR), potentiostats, amperometric transducers, impedance transducers, or field-effect transistors, have been developed for influenza virus detection, as summarized in Table 1. For real-time RT-PCR, the gold standard for influenza virus detection has been reported to be the limit of detection (LOD) in the range of threshold cycle (Ct) 37.6–39.4.

[0003] A disposable immunoassay that omits washing steps and simply mixes samples has been developed for rapid detection of influenza viruses. The present invention proposes a disposable immunoassay for influenza virus detection based on a displacement immunoassay using a nucleoprotein (NP)-like mimic. In the mimic-based disposable immunoassay, immobilized antibodies bind to a mimic displaced from an anti-NP monoclonal antibody (mAb), which occurs simultaneously with the binding of the target antigen NP to the anti-NP mAb. This is illustrated in Figure 1(a). Figure 1 illustrates a disposable immunoassay for influenza virus based on a nucleoprotein (NP)-like mimic. Figure 1(a) illustrates the procedure of the disposable immunoassay based on the NP-like mimic. Figure 2(b) illustrates the selection of NP-like mimics from an autodisplayed Fv library. Additionally, a mimic mimicking the NP of the influenza virus was selected from an Fv-antibody library using mAbs against NP immobilized on magnetic beads, as shown in Figure 1(b).

[0004] Fv antibodies represent the VH of immunoglobulin G (IgG) and are composed of three complementarity-determining regions (CDRs) and four framework regions (FRs). The CDRs interact directly with antigens, while the FRs of the middle and light chains facilitate IgG self-assembly. As shown in Figure 2(a), the 11-amino acid CDR3 was randomized to prepare an Fv antibody library through site-directed mutagenesis. The Fv antibody library was then expressed on the outer membrane of Escherichia coli using autodisplay technology. Fv antibodies could be expressed on the outer membrane with a high surface density of >10^5 Fv antibodies / E. coli and a high conversion efficiency of >90% of the Fv antibody library. Random sequencing of E. coli revealed that the diversity of the Fv antibody library was greater than 10^5 clones / library. A high-yield Fv-antibody library was used to select target clones without repeated panning of bacteriophage libraries. The autodisplayed Fv-antibody library was used to screen mAb probes mimicking dopamine, food allergens, and the spike protein of SARS-CoV-2.

[0005] In the present invention, a mimetic mimic mimicking the NP of influenza A (Inf-A) was selected from an Fv-antibody library using mAbs from an autodisplayed Fv-antibody library. After the selected Fv-antibodies (NP-like mimetic) were expressed as fusion proteins with green fluorescent protein (GFP), the binding constants (KD) of the selected clones were measured using a flow cytometer and a SPR biosensor. A disposable immunoassay for the detection of influenza viruses was constructed based on displacement analysis using anti-NP mAbs conjugated to the mimetic. Finally, the results of the disposable immunoassay were compared with those of a lateral flow immunoassay using real Inf-A and influenza B (Inf-B).

[0006] The present invention aims to provide a method for rapid and accurate detection and analysis of influenza viruses. The Mimotope-based peptide developed for this purpose mimics the structural characteristics of the influenza nucleoprotein and possesses the ability to bind to or compete with antibodies against the nucleoprotein. These peptides effectively detect the presence of influenza viruses, enabling accurate identification of their type and mutations. Furthermore, the fluorescence analysis unit included in the invention can precisely analyze the interaction between the peptide and antibody, providing more detailed information on the concentration and activity levels of the virus. The combination of these technologies enables faster and more accurate diagnosis of influenza viruses and can significantly contribute to the rapid response and management of mutating viruses. This represents a significant advance in the early detection and effective management of influenza viruses, a major challenge in public health and medicine.

[0007] In one aspect, the present invention provides a peptide having an influenza nucleoprotein mimotope function and comprising at least one peptide sequence selected from the group comprising the peptide sequence of SEQ ID NO: 1, the peptide sequence of SEQ ID NO: 2, the peptide sequence of SEQ ID NO: 3, and the peptide sequence of SEQ ID NO: 4.

[0008] [Sequence number 1]

[0009] CGPVPIRKVDD

[0010] [Sequence number 2]

[0011] CRLRLVLARDF

[0012] [Sequence number 3]

[0013] GDPITSRRLDY

[0014] [Sequence number 4]

[0015] GTRIALPLRDV

[0016] In one embodiment, the peptide may have a mimetic function capable of binding to an antibody capable of binding to an influenza nucleoprotein.

[0017] In one embodiment, the peptide can competitively bind to the antibody in the presence of influenza nucleoprotein.

[0018] In another aspect, the present invention provides a nucleic acid encoding the peptide.

[0019] In another aspect, the present invention provides a recombinant expression vector comprising the nucleic acid.

[0020] In another aspect, the present invention provides a cell transformed with the recombinant expression vector.

[0021] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells.

[0022] In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and may include one or more cells selected from the group comprising Neurospora crassa.

[0023] In another aspect, the present invention provides a composition for detecting influenza, comprising an amino acid fragment having an influenza nucleoprotein mimotope function and at least one peptide sequence selected from the group comprising a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, a peptide sequence of SEQ ID NO: 3, and a peptide sequence of SEQ ID NO: 4.

[0024] [Sequence number 1]

[0025] CGPVPIRKVDD

[0026] [Sequence number 2]

[0027] CRLRLVLARDF

[0028] [Sequence number 3]

[0029] GDPITSRRLDY

[0030] [Sequence number 4]

[0031] GTRIALPLRDV

[0032] In one embodiment, the composition for detecting influenza may have a mimotope function capable of binding to an antibody capable of binding to an influenza nucleoprotein.

[0033] In one embodiment, the composition for detecting influenza can competitively bind to the antibody in an environment in which influenza nucleoprotein is present.

[0034] In one embodiment, the amino acid fragment may further comprise a fluorescent marker.

[0035] In another aspect, the present invention provides an influenza detection kit comprising: a substrate including at least a conduit; an antibody capable of binding to an influenza nucleoprotein fixed within the conduit; and an influenza detection composition bound to the antibody; wherein the influenza detection composition competitively binds to the antibody with respect to the influenza nucleoprotein.

[0036] In one embodiment, the composition for detecting influenza may include the composition for detecting influenza according to the embodiment of the present invention.

[0037] In one embodiment, the amino acid fragment of the composition for detecting influenza may further comprise a fluorescent marker.

[0038] In one embodiment, the composition may further include a fluorescence analysis unit that performs quantitative analysis of the fluorescent marker released from the influenza detection composition when the antibody is exposed to the influenza nucleoprotein.

[0039] The effect of the present invention is to enable rapid and accurate detection and analysis of influenza viruses. The Mimotope-based peptides developed by the present invention precisely mimic the characteristics of the influenza nucleoprotein, enabling the identification of the presence and characteristics of the virus through binding and competition with antibodies. This provides a faster and more precise diagnosis of influenza viruses, and plays a particularly important role in the identification and response to mutant viruses. The fluorescence analysis unit included in the present invention can quantitatively analyze the interaction between these peptides and antibodies. This complex approach can significantly contribute to the early diagnosis and treatment of influenza and public health management. In particular, it will enable effective surveillance and management of mutant viruses, thereby bringing about significant advancements in the medical field.

[0040] Figure 1 illustrates a disposable immunoassay for influenza virus based on a nucleoprotein (NP) mimic.

[0041] Figure 2 depicts an autodisplayed Fv library with randomized CDR3 regions used for screening NP-like mimics.

[0042] Figure 3 shows the results of binding constant (KD) measurements of four selected clones.

[0043] Figure 4 shows the results of expressing the selected Fv-antibodies as soluble proteins and measuring the binding constant (KD).

[0044] Figure 5 illustrates the results of NP detection using a one-step immunoassay based on Fv antibodies. Figure 5(a) illustrates the configuration of a one-step immunoassay based on Fv antibodies.

[0045] Figure 6 illustrates a one-step immunoassay based on NP-like mimotopes (using expressed Fv antibodies and synthetic peptides) using influenza virus samples (Inf-A and Inf-B).

[0046] Figure 7 shows the results of homology analysis between NP and various influenza virus strains.

[0047] Figure 8 shows the sequence of expressed NP (amino acids 184-497) of influenza virus A (classification ID: 1038115) for immunization with monoclonal antibodies (mAb) against influenza A.

[0048] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. The present invention may be modified in various ways and may take various forms. Therefore, specific embodiments are illustrated in the drawings and described in detail in the text. However, this is not intended to limit the present invention to a specific disclosed form, but should be understood to include all modifications, equivalents, and substitutes included in the spirit and technical scope of the present invention. In describing each drawing, similar reference numerals are used to indicate similar components. In the attached drawings, the dimensions of structures are shown larger than actual size to ensure clarity of the present invention.

[0049] The terminology used in this application is only used to describe specific embodiments and is not intended to limit the present invention. The singular expression includes plural expression unless the context clearly indicates otherwise. In this application, it should be understood that the terms "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, or combination thereof described in the specification, but do not exclude in advance the possibility of the presence or addition of one or more other features, numbers, steps, operations, components, or combinations thereof. In the context of this specification, the term "about" or the like can mean about ± 1%, about ± 2%, about ± 3%, about ± 4%, about ± 5%, about ± 6%, about ± 7%, about ± 8%, about ± 9%, or about ± 10% of a numerical value described in the specification.

[0050] Additionally, the description of one aspect of the present invention may be applied identically or similarly to the same or similar configurations or terms in the description of other aspects.

[0051] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0052] A peptide according to an embodiment of the present invention provides a peptide having an influenza nucleoprotein mimotope function and comprising at least one peptide sequence selected from the group comprising a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, a peptide sequence of SEQ ID NO: 3, and a peptide sequence of SEQ ID NO: 4.

[0053] [Sequence number 1] CGPVPIRKVDD

[0054] [Sequence number 2] CRLRLVLARDF

[0055] [Sequence number 3] GDPITSRRLDY

[0056] [SEQ ID NO: 4] GTRIALPLRDV

[0057] In the context of this specification, a "mimotope" refers to a fragment that mimics the three-dimensional structural characteristics of a specific antigen and has the ability to mimic antibody binding. The peptides provided by embodiments of the present invention effectively mimic the antigenic determinants of the nucleoprotein, thereby playing a key role in the detection and diagnosis of influenza. These peptides can competitively bind to antibodies against the influenza virus nucleoprotein, thereby enabling them to detect the presence of the virus. The peptides developed by the present invention can be utilized as important components in the development of various diagnostic kits and systems for influenza detection.

[0058] In the context of this specification, the influenza nucleoprotein refers to a protein that is a crucial component of the influenza virus and plays an essential role in the viral replication and infection processes. This nucleoprotein contains the genetic information of the virus and performs a crucial function in the survival and spread of the virus. The peptides of the present invention can be utilized to detect or block viral activity by targeting specific regions of this nucleoprotein. This represents a significant advancement in the early diagnosis and treatment of influenza, and may contribute to the development of new strategies to respond to mutating viruses.

[0059] In one embodiment, the peptide may possess a mimetic function capable of binding to an antibody capable of binding to the influenza nucleoprotein. This means that the peptide can mimic the structural characteristics of the nucleoprotein, thereby functioning in a manner similar to an antibody that recognizes and binds to the nucleoprotein. This ability is crucial for the detection and analysis of influenza viruses, and may play a particularly important role in detecting and responding to viral mutations.

[0060] In one embodiment, the peptide can competitively bind to the antibody in the presence of influenza nucleoprotein. This mechanism enables rapid and precise diagnosis of influenza viruses, and may particularly enhance detection of mutant viruses.

[0061] Meanwhile, the nucleic acid according to an embodiment of the present invention can encode the peptide. In the context of this specification, peptide encoding refers to the process by which a specific nucleic acid sequence determines the amino acid sequence of the peptide. This is a crucial step in the synthesis and production of peptides, and the structure and function of a specific peptide can be precisely controlled through the nucleic acid sequence. This control ensures the precise synthesis of the specific amino acid sequence required for the peptide to mimic the structural features of the influenza nucleoprotein, thereby enabling the efficient production of mimotopes that replace the nucleoprotein. Therefore, the use of nucleic acids according to the present invention plays a key role in the development of peptide-based tools for the detection and analysis of influenza viruses, providing new strategies and methodologies in the field of influenza diagnosis and treatment.

[0062] Meanwhile, a recombinant expression vector according to an embodiment of the present invention may include the nucleic acid. In the context of this specification, a recombinant expression vector refers to a DNA or RNA structure designed to produce a specific protein, which performs the function of reading the information of a specific nucleic acid and translating it into a protein within a cell. Such a vector contains a nucleic acid sequence for peptide synthesis, thereby enabling the efficient production of a desired peptide within the cell. The use of recombinant expression vectors plays a crucial role in the development of biological agents for influenza virus detection and treatment by simplifying the complex protein production process and facilitating the mass production of specific peptides. By using such vectors, researchers can effectively produce mimotope peptides corresponding to the influenza nucleoprotein, which can contribute to the rapid response to viral mutations and the development of more precise diagnostic and therapeutic methods.

[0063] Meanwhile, cells according to embodiments of the present invention can be transformed with the recombinant expression vector. In the context of this specification, transformation refers to the process of introducing an exogenous gene into a specific cell to alter its genetic makeup. Through this process, the cell integrates the new genetic information provided by the recombinant expression vector, thereby gaining the ability to produce a specific peptide or protein. The transformed cell effectively produces the peptide of the present invention, thereby playing a significant role in the diagnosis and treatment of influenza viruses.

[0064] In one embodiment, the cell may comprise one or more cells selected from the group consisting of animal cells, plant cells, yeast, Escherichia coli, and insect cells. These various cell types each have unique characteristics and production capabilities, and can be effectively utilized for the production and research of specific peptides through the use of recombinant expression vectors. For example, animal cells are suitable for the study of human diseases because they undergo protein processing processes similar to those in humans, while simple microorganisms such as yeast or Escherichia coli can be used for rapid and economical protein production. Insect cells may also be suitable for the production of peptides with complex protein structures. The utilization of these cells can expand the development and research of mimotope peptides against influenza viruses in various directions, opening up new possibilities for the diagnosis and treatment of influenza.

[0065] In one embodiment, the cell is selected from the group consisting of monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp. and Neurospora crassa. These cells possess diverse genetic and biological characteristics, making them highly useful for the production and study of specific peptides. Each cell type possesses unique production capabilities and host characteristics, enabling the effective production of peptides with complex protein structures. In particular, cells used in human disease research undergo protein processing processes similar to those of humans, enabling more accurate studies of human diseases. On the other hand, microbial cells can be used for economical and rapid protein production, and cells such as Escherichia coli are particularly effective for mass production.

[0066] Meanwhile, an influenza detection composition according to an embodiment of the present invention provides an influenza detection composition comprising an amino acid fragment having an influenza nucleoprotein mimotope function and at least one peptide sequence selected from the group comprising a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, a peptide sequence of SEQ ID NO: 3, and a peptide sequence of SEQ ID NO: 4.

[0067] [Sequence number 1] CGPVPIRKVDD

[0068] [Sequence number 2] CRLRLVLARDF

[0069] [Sequence number 3] GDPITSRRLDY

[0070] [SEQ ID NO: 4] GTRIALPLRDV

[0071] In one embodiment, the composition for detecting influenza may have a mimotopic function capable of binding to an antibody capable of binding to the influenza nucleoprotein. In one embodiment, the composition for detecting influenza may competitively bind to the antibody in an environment in which the influenza nucleoprotein is present.

[0072] In one embodiment, the amino acid fragment may further include a fluorescent marker. By including the fluorescent marker in the amino acid fragment, the amount of the composition according to an embodiment of the present invention bound to an antibody and / or separated from the bound antibody can be quantified, thereby enabling the detection of influenza nucleoprotein. The inclusion of such a fluorescent marker plays a crucial role in the accurate detection and analysis of influenza nucleoprotein. By using a fluorescent marker, the presence and amount of nucleoprotein can be more precisely measured through the change in fluorescent signal that occurs when the composition binds to or dissociates from an antibody. This enables rapid detection and precise quantitative analysis of influenza viruses, which can significantly improve the efficiency of early diagnosis and treatment. Furthermore, methods using fluorescent markers can be more sensitive and accurate than existing diagnostic methods, which can play a crucial role in the rapid detection and response to influenza virus mutations.

[0073] Meanwhile, an influenza detection kit according to an embodiment of the present invention may include at least a substrate including a conduit; an antibody capable of binding to an influenza nucleoprotein fixed within the conduit; and an influenza detection composition bound to the antibody. In one embodiment, the influenza detection composition may competitively bind to the antibody with respect to the influenza nucleoprotein. In one embodiment, the influenza detection composition may include the influenza detection composition according to an embodiment of the present invention. In one embodiment, the amino acid fragment of the influenza detection composition may further include a fluorescent marker.

[0074] In one embodiment, the method may further include a fluorescence analysis unit that performs quantitative analysis of the fluorescent marker from the influenza detection composition released when the antibody is exposed to the influenza nucleoprotein. In one embodiment, the method may further include a fluorescence analysis unit that performs quantitative analysis of the fluorescent marker from the influenza detection composition released when the antibody is exposed to the influenza nucleoprotein. The introduction of this fluorescence analysis unit performs an important function in effectively detecting and analyzing the interaction between the antibody and the influenza nucleoprotein. The fluorescence analysis unit detects the light signal emitted by the fluorescent marker and, based on this, can quantitatively assess the presence and amount of the nucleoprotein. This technology enables accurate detection and analysis of the influenza virus, and in particular, can quickly and precisely detect even subtle changes or low-concentration presence of the virus. The use of the fluorescence analysis unit can provide more sensitive and faster results than existing methods, which provides a great advantage, especially in emergency situations or large-scale diagnostic scenarios.

[0075] Hereinafter, embodiments of the present invention will be described. However, the embodiments described below are merely some embodiments of the present invention, and the scope of the present invention is not limited to the embodiments described below.

[0076] [[Summary of the Example]]

[0077] An immunoassay based on nucleoprotein (NP)-like mimics for influenza viruses (Inf-A and Inf-B) is presented. NP-like mimics with binding affinity to an anti-NP monoclonal antibody (mAb) library of influenza A (Inf-A) virus were selected from a Fv-antibody library. The Fv-antibody library, corresponding to the VH region of immunoglobulin IgG, was expressed on the outer membrane of Escherichia coli using autodisplay technology, and the target Fv antibodies were screened using magnetic beads carrying immobilized anti-NP mAbs. The four types of selected Fv antibodies (NP-like mimics) were expressed as fusion proteins with green fluorescent protein. The binding affinities of the selected clones were measured using flow cytometry and a SPR biosensor. A disposable immunoassay for influenza virus was constructed using the expressed mimics conjugated to anti-NP mAbs. The green fluorescent protein-labeled mimic was quantitatively separated when the NP bound to the mAb, and thus the NP concentration in the sample was determined by fluorescence measurement. The disposable immunoassay based on the NP-like mimic of Inf-A showed higher sensitivity for detecting Inf-A along with influenza B (Inf-B) than the conventional lateral flow immunoassay. This sensitivity was derived from the similarity (>70%) in the amino acid sequence between the probe NP used for screening and the target NPs of Inf-A and Inf-B. This disposable immunoassay has a significantly improved detection limit compared to the conventional lateral flow immunoassay, and can be used for the medical diagnosis of influenza.

[0078] [[Materials and Methods]]

[0079] [ingredient]

[0080] High-salt Luria-Bertani (LB) medium and LB agar were purchased from Duchefa Co. (Haarlem, Netherlands). Bovine serum albumin (BSA) and Tween® 20 were purchased from Sigma-Aldrich (Seoul, Korea). Dynabeads™ Protein G, high-fidelity Phusion polymerase, and PCR reagents were purchased from Thermo Fisher Scientific (Waltham, MA, USA). PCR Clean-up and NucleoSpin Gel kits were purchased from Macherey-Nagel Co. (Duren, Germany). Primers were synthesized by BIONICS Co. (Seoul, Korea). Klenow (exo) polymerase was purchased from New England Biolabs (Ipswich, MA, USA). Inactivated Inf-A and Inf-B viruses (A / New Caledonia / 20 / 99 and B / Tokio / 53 / 99) were purchased from Meridian Biosciences (San Jose, CA, USA). Monoclonal antibodies against Inf-A were purchased from Gyeongsang National University (Jinju, Korea). Fv-antibody peptides were synthesized at 90% purity by Peptron Co. (Daejeon, Korea). Amicon® Ultra 0.5 mL filters with a cutoff of 10 kDa were purchased from Millipore Co. (Darmstadt, Germany). Phosphate-buffered saline (PBS) (20×) was purchased from CUREBIO Co. (Seoul, Korea). Commercial lateral flow immunoassay kits for detecting Inf-A and Inf-B were purchased from SD Biosensors (Gyeonggi-do, Korea).

[0081] [Production of monoclonal antibodies against Inf-A nuclear protein]

[0082] The Inf-A component was isolated by extraction and filtration. Eight female BALB / c mice were immunized with Inf-A. For the first boost, 100 μL of Inf-A at a concentration of 1.0 mg / mL was emulsified with an equal volume of Freund's complete adjuvant, and this emulsified immunogen solution was injected into the peritoneal cavity of the mice. The second and third booster injections were administered at 2-week intervals. Three days after the third injection, blood was collected from the tail vein of each mouse, and the titers of each antisera were determined by indirect ELISA (Shim et al., 2006). The final booster injection was administered with the immunogen (200 μL) without adjuvant to mice that showed high antisera titers. Three days after the final injection, spleen cells from the immunized mice were collected and used for cell fusion with SP2 / 0 myeloma cells. Cell fusion was performed as previously described (Kohler and Milstein 1975). Spleen cells (2.0 × 10^8 cells) were fused with SP2 / 0 myeloma cells (2.0 × 10^7 cells) using a 50% PEG 1500 solution (1 mL). After selection in hypoxanthine-aminopterin-thymidine medium (HAT), the supernatant of the fused cells was analyzed by indirect ELISA to determine the antibody titer. Fused cells that were positive for the NP of Inf-A by indirect ELISA were selected. Monoclonal hybridoma cells producing mAb specific for the corresponding Inf-A were selected using indirect ELISA (Kim et al., 2023b; Kim et al., 2023c; Sung et al., 2022a).

[0083] [Preparation and screening of Fv antibody libraries]

[0084] As shown in Fig. 2(a), a single-stranded forward primer containing a randomized CDR3 sequence (75 bp) was mixed with the corresponding reverse primer (22 bp) to synthesize an Fv-antibody library through site-directed mutagenesis in the CDR3 region. Table 1 presents the specific primer sequences used in the Fv-antibody library and indicates the primer sequences for the randomized CDR3 region of the Fv-antibody library.

[0085] Primer typeOligonucleotide sequenceRandomized forward primer (75 bases)5'-GTCTATTATTGCGCTCGT 1 KRYVNN 7 VNNVNN 13 VNNVNN 19 VNNVNN 25 VNNGAT 31 KWYTGGGGTCAAGGTACTACGGTTACG-3'Corresponding reverse primer (22 bases)3'-CCCAGTTCCATGATGCCAATGC-5'Composition of nucleotidesat each positionN = A, C, G, T / R = A, G / K = G, T / Y = C, T / W = A, T / V = A, C, G

[0086] A PCR machine was used to construct the Fv-antibody library. The forward primer (2 μL) and reverse primer (2 μL) containing the randomized CDR3 sequence were combined with NEB buffer (4 μL) and deionized water (32 μL) by heat treatment (heating at 95 °C for 5 min, then cooling to 36 °C at a cooling rate of 0.3 °C / s). For the extension reaction, Klenow (exo-) polymerase enzyme (3 μL), NEB buffer (16 μL), 10 mM dNTPs (8 μL), and deionized water (133 μL) were added to the product (40 μL), and the reaction was performed at 37 °C for 15 min. After inactivating the polymerase enzyme reaction at 75 °C for 20 min, the product containing the double-stranded primers of the Fv-antibody library generated at this step was purified using a PCR Clean-up kit and NucleoSpin® gel. The Fv-antibody library plasmid was prepared by mixing pST009 template plasmid (150 ng), double-stranded primer of purified Fv-antibody library (140 ng), 10 mM dNTPs (1 μL), HF buffer (10 μL), Fusion High-Fidelity Polymerase (0.5 μL), and DW to a total volume of 50 μL. Then, the PCR steps were performed as follows: (1) initial denaturation at 98 °C for 1 min, (2) denaturation at 98 °C for 30 s, (3) binding at 68 °C for 1 min, (4) extension at 72 °C for 5 min, (5) repeating steps (2)-(4) 30 times, and (5) termination at 72 °C for 10 min. After PCR, the template plasmid (pST009) was digested with DpnI restriction enzyme at 37 °C for 16 h. Finally, the Fv-antibody library plasmid was filtered using an Amicon Ultra filter with a cutoff of 100 kDa. Fv-antibodies with various CDR3 region sequences were electroporated into E.Autodisplay on the outer membrane of E. coliBL21(DE3) was achieved by transfecting the prepared Fv-antibody library plasmid into electrocompetent cells (Bong et al., 2018; Dower et al., 1988; Jose and Meyer 2007; Jung et al., 2021b; Jung et al., 2023; Lee et al., 2021; Lopez-Rojas et al., 2023; Sung et al., 2022a; Sung et al., 2022b).

[0087] [Autodisplay of Fv antibodies on the outer membrane of E. coli]

[0088] Autodisplay of Fv antibodies on the outer membrane of E. coli was performed using a previously reported method. Transformed E. coli cells were cultured in high-salt LB medium containing 50 μg / mL kanamycin antibiotic at 37 °C for 16 h with shaking at 200 rpm. Then, 50 μL of the cultured E. coli cells were inoculated into 5 mL of LB medium containing 4 μL of 99% β-mercaptoethanol, kanamycin (50 μg / mL), and ethylenediaminetetraacetic acid (5 μM) and cultured with shaking at 150 rpm until the optical density at 600 nm reached 0.5. Afterwards, 1 mM isopropylthio-β-galactoside (IPTG) was added to the E. coli culture to induce Fv antibody expression and cultured at 30 °C with shaking at 150 rpm for 3 h.

[0089] [Screening of target Fv-antibody variants (clones)]

[0090] Target E. coli with autodisplayed Fv-antibodies with specific binding affinity to mAb against Inf-A NP (A / Hong Kong / H090-701-V10 / 2009) were selected as follows:

[0091] (1) Dynabeads™ protein G (10 μL) and mAb (200 μg) against Inf-A NP were mixed with PBS containing 0.1% Tween 20 (0.1% PBST) for 1 hour.

[0092] (2) The magnetic beads bound to mAb were sorted using a magnet, and the unbound mAb was washed five times with 0.1% PBST and PBS.

[0093] (3) The magnetic beads bound to mAb were mixed with E. coli (100 μL, OD600 = 1.0) containing the autodisplayed Fv-antibody library and rotated at 20 rpm for 1 h at 23 °C.

[0094] (4) The magnetic beads bound to E. coli were separated using a magnet and washed ten times with 0.1% PBST.

[0095] (5) The separated magnetic beads bound to E. coli were resuspended in LB medium (50 μL), and the separated magnetic beads were spread on an agar plate containing 50 μg / mL kanamycin to obtain E. coli clones (Jung et al., 2021b; Kim et al., 2023d; Mair et al., 2019).

[0096] The binding affinity of mAb to Inf-A NP of E. coli harboring autodisplayed Fv-antibodies was assessed using a FACSCalibur flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). Selected E. coli were mixed with antibodies to Inf-A NP under gentle rotation at 20 rpm for 1 h. After centrifugation (3000 × g, 3 min), E. coli were washed twice with PBS, and antibodies to Inf-A NP were labeled with a FITC-labeled secondary antibody against a mouse monoclonal antibody. Quantitative binding of E. coli harboring autodisplayed Fv-antibodies to antibodies to Inf-A NP was performed using a flow cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA).

[0097] [Expression of influenza A NP]

[0098] A plasmid encoding the open reading frame of Inf-A NP was synthesized at Gyeongsang National University (Jinju, Korea). Inf-A NP was expressed in Escherichia coli by transforming the custom plasmid into competent cells. The transformed E. coli were cultured in 100 mL of high-salt LB medium containing 1 mM IPTG and 30 μg / mL carbenicillin at 30 °C for 16 h. The E. coli pellet was collected by centrifugation (3,000 × g for 3 min) and resuspended in 20 mL of binding buffer (5 mM Tris-HCl, 0.5 mM EDTA, and 1 M NaCl) containing 6 M urea. The resuspended E. coli cells were sonicated in an ultrasonic reactor (Vibracell VCX-130, Sonics, USA). The lysate was centrifuged at 25,000 × g for 10 min. The Fv antibody in the supernatant was purified using an elution buffer containing 6 M urea and 500 mM imidazole on a His-tag purification column (Roche, Basel, Switzerland). The purified Fv antibody was dialyzed at 50 rpm at 4 °C for 16 h to remove urea and imidazole.

[0099] [Expression of Fv antibodies]

[0100] Figure 4 shows the results of expressing the selected Fv antibodies as soluble proteins and measuring the binding constants (KD). Figure 4(a) shows the expression of the screened Fv antibodies. Figure 4(b) shows the binding constants (KD) of the Fv antibodies (Fv-NP-1, Fv-NP-2, Fv-NP-3, Fv-NP-4) from four screened clones against immobilized anti-antibodies using an SPR biosensor.

[0101] As shown in Fig. 4(a), a plasmid encoding the open reading frame of an Fv antibody having binding affinity for antibodies to Inf-A NP was synthesized by Peptron Co. (Daejeon, Korea). The amino acid sequence of the Fv antibody includes CDRs and FRs, which are summarized in Table 2. In Table 2, the amino acid sequence of the Fv antibody was composed of CDRs and FRs (N-terminal-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-C-terminal).

[0102] RegionAmino acid (N -> C-term)Oligonucleotide sequence (5' → 3')Frame-1EVQLVESAAEVRRPGASVKITCKASGYSFSGAA GTG CAG CTC GTG GAA AGC GCT GCC GAA GTT CGG CGT CCT GGG GCT AGC GTG AAG ATC ACC TGC AAA GCG TCC GGC TAT TCA TTC AGCCDR1TYGIQACC TAT GGG ATT CAGFrame-2WMRQAPGQRPEWLGTGG ATG CGC CAA GCG CCA GGC CAG CGT CCG GAA TGG CTT GGGCDR2WIHAGTGGTKYSRKFQGTGG ATA CAT GCA GGC ACA GGT GGG ACT AAG TAC TCG CGC AAA TTT CAG GGTFrame-3RITITRDTSANTVYLDLNSLTSEDTAVYYCARCGC ATT ACT ATC ACC CGT GAT ACC AGC GCG AAT ACC GTC TAT CTG GAT CTG AAC TCT CTG ACA TCG GAG GAT ACG GCC GTC TAT TAT TGC GCT CGTCDR3(Template)DKVTVWACQDNGAC AAA GTT ACA GTC TGG GCT TGT CAG GAT AATFrame-4WGQGTTVTVSSTGG GGT CAA GGT ACT ACG GTT ACG GTC AGC AGT

[0103] Fv antibodies were expressed in E. coli by transducing a custom plasmid into competent cells. The transformed E. coli were cultured in 100 mL of high-salt LB medium containing 1 mM IPTG and 30 μg / mL carbenicillin at 30 °C for 16 h. The E. coli pellet was collected by centrifugation (3,000 × g for 3 min) and then resuspended in 20 mL of binding buffer (5 mM Tris-HCl, 0.5 mM EDTA, and 1 M NaCl) containing 6 M urea. The resuspended E. coli cells were sonicated in an ultrasonic reactor (Vibracell VCX-130, Sonics, USA). The lysate was centrifuged at 25,000 × g for 10 min. The Fv antibody in the supernatant was purified using an elution buffer containing 6 M urea and 500 mM imidazole on a His-tag purification column (Roche, Basel, Switzerland). The purified Fv antibody was dialyzed at 50 rpm at 4 °C for 16 h to remove urea and imidazole (Spriestersbach et al., 2015).

[0104] [Measurement of binding affinity of Fv antibodies]

[0105] Surface plasmon resonance (SPR) measurements of autodisplayed Fv antibodies obtained from antibodies against Inf-A NP were performed using an SPR biosensor (i-Cluebio, Seongnam, Korea). SPR analyses were performed using Fv antibodies on the outer membrane of selected E. coli autodisplayed on gold-coated BK-7 glass. The gold-coated BK-7 glass was prepared as follows:

[0106] 1) BK-7 glass (1 × 1 cm2) was washed with ethanol, isopropanol, and DW.

[0107] 2) Titanium was coated with a thickness of 2 nm as an adhesive layer, and gold was coated with a thickness of 48 nm using a K575X sputter from Quorum Emitech (Kent, UK) (Bong et al., 2018).

[0108] Figure 3(c) shows that the SPR chip was incubated with the outer membrane of E. coli (20 μg / mL) containing autodisplayed Fv antibodies at 23 °C for 1 h. After washing with PBS, blocking was performed with BSA (1 mg / mL) for 30 min. After washing again with PBS, antibodies against Inf-A NPs (8.2–66.6 nM) were introduced for 20 min (10 μL / min) as the binding step. Finally, PBS was introduced for 20 min (10 μL / min) as the dissociation step. SPR measurements were performed by processing the sample under flow conditions, and the SPR signal corresponding to the amount of bound analyte after the dissociation step was recorded.

[0109] The binding affinity of purified Inf-A NPs and purified Fv antibodies to anti-Inf-A NP antibodies was measured using a SPR biosensor. SPR analysis was performed using antibodies against Inf-A NPs covalently immobilized on gold-coated BK-7 glass, modified with 11-mercaptodecanoic acid (MUA) (Kim et al., 2022c). Figure 4(b) shows the SPR chip incubated with anti-NP mAb (10 g / mL) using EDC / NHS reagent at 23 °C for 2 h. After washing with PBS, the antibody-immobilized gold chip was blocked with 1 M ethanolamine for 30 min. After washing again with PBS, purified NPs (5–100 nM) or Fv antibodies (5–200 nM) were flowed through the chip for 15 min (30 μL / min) as a binding step. Finally, PBS was flowed for 15 minutes (30 μL / min) as a dissociation step. SPR measurements were performed by processing the sample under flow conditions, and the SPR signal corresponding to the amount of bound analyte after the dissociation step was recorded. The SPR signal was calculated from the dose-response curve of the sample containing the solution protein using the ideal model expressed by the following equation:

[0110] R = (Rmax [Ag]) / (Kd+[Ag])

[0111] Here, R represents the SPR signal, Rmax represents the maximum SPR signal, [Ag] represents the solution protein concentration, and Kd represents the affinity constant. The SPR signal was applied using Hill's equation.

[0112] One-Step Immunoassay for Influenza Virus

[0113] Figure 2 depicts an autodisplayed Fv library with randomized CDR3 regions used for screening NP-like mimetics. Figure 2(a) depicts the preparation of an Fv library with randomized CDR3 regions in the VH using site-directed mutagenesis. Expression of the Fv library in the outer membrane of E. coli using an autodisplay vector (autodisplayed Fv variants, a control strain with CDR1 and CDR2, and the original E. coli, analyzed by SDS-PAGE) is depicted. Figure 2(b) depicts the selection procedure of target clones using magnetic beads with immobilized anti-NP mAb. Figure 2(c) depicts the selection of target clones through genetic sequence analysis of the CDR3 region.

[0114] A disposable immunoassay for quantitative analysis of influenza virus was performed using Fv antibodies. For the disposable immunoassay, anti-NP mAb (10 μg / mL, 100 μL) was immobilized on a microplate, and the corresponding Fv antibody (labeled with GFP) was bound to the paratope of the immobilized mAb. The Fv antibody (fluorescently labeled) was bound by incubating the Fv antibody (fluorescently labeled) (1 μM, 100 μL) with antibodies against the modified NP for 2 hours. When a positive sample reacts with the corresponding antigen, the pre-bound mimic with a fluorescent label is released depending on the antigen concentration in the sample. For negative samples without the corresponding antigen, the fluorescence remains unchanged. The fluorescence signal was measured using a Victor5 fluorometer (PerkinElmer Co, Massachusetts, USA) at an excitation wavelength of 488 nm and an emission wavelength of 512 nm. The limit of detection (LOD) was determined as the average intensity of the blank sample (PBS) plus three standard deviations (3σ). The analytical results were compared with a commercial lateral flow immunoassay. A commercial lateral flow immunoassay kit detecting Inf-A and Inf-B was purchased from SD Biosensors.

[0115] [[Results and Discussion]]

[0116] [Screening of NP-like mimotopes from Fv antibody libraries]

[0117] NP-like mimics of Inf-A were selected from an autodisplayed Fv-antibody library using magnetic beads immobilized with anti-NP mAb, as shown in Figure 1(b). Fv-antibodies represent the VH of IgG and are composed of three CDRs and four FRs (Marillet et al., 2017). The Fv-antibody library was prepared through site-directed mutagenesis of the 11-amino acid CDR3 (Dao-Pin et al., 1987; Fortier et al., 2005), as shown in Figure 2(a). Subsequently, the Fv-antibody library was expressed on the outer membrane of Escherichia coli using autodisplay technology (Park et al., 2015; Yoo et al., 2015), as shown in Figure 2(b). The modified E. Random sequence analysis of E. coli revealed that the diversity of the Fv-antibody library was greater than 105 clones / library. The Fv-antibody library was used to screen NP-like mimetics with binding affinity for anti-NP mAb. For immunization with this antibody, the modified NP of Inf-A, consisting of amino acids 184-498 (UniProt ID: HQU4F3, red region) with a molecular weight of 38 kDa, was used as an antigen, as shown in Figure 8. Figure 8 depicts the sequence of the expressed NP (amino acids 184-497) of influenza virus A (classification ID: 1038115) for immunization with a monoclonal antibody (mAb) against influenza A. To screen the Fv-antibody library, the anti-NP mAb was immobilized on Dynabeads magnetic beads from Thermo Fisher Scientific (Waltham, MA, USA). As shown in Fig. 1(b), antibodies were immobilized on magnetic beads and the amino groups of the antibodies using protein G. After allowing the magnetic beads to react with the Fv-antibody library, the subject E with the autodisplayed Fv-antibodies on the outer membrane was exposed to an external magnet.E. coli cells were isolated, and the magnetic beads bound to E. coli were cultured on agar plates. More than 50 clones were selected from the colonies cultured on the agar plates, and their binding affinity to the anti-NP mAb was measured by sequential treatment with a monoclonal antibody and a fluorescently labeled secondary antibody. Figure 2(c) shows that E. coli with autodisplay of CDR1 and CDR2 (but no CDR3) was used as a control strain. Fluorescence signal analysis using flow cytometry showed that several clones exhibited higher fluorescence than the control strain.

[0118] These results indicate that the selected clones exhibited autodisplayed Fv antibodies with binding affinity for anti-NP mAb. The CDR3 oligonucleotide sequences were analyzed to determine which clones had valid CDR3 sequences that differed from the template sequence before site-directed mutagenesis. From oligonucleotide sequence analysis, four selected clones (NP-1, NP-2, NP-3, and NP-4) were observed to have valid genetic sequences, while the other clones were observed to have nucleotide sequences identical to the template or had nucleotides deleted in the CDR3. Finally, the four selected clones were identified as target clones. The oligonucleotide sequences of the selected CDR3 and their corresponding amino acid sequences are summarized in Table 3. Table 3 shows the oligonucleotides and corresponding amino acid sequences of the CDR3 of the screened clones and their binding constants (KD) to anti-NP mAb measured by flow cytometry and SPR.

[0119] Screened cloneCDR3 sequenceK D value(Flow cytometer)K D value (SPR)Clone NP-1 1 CGPVP 6 IRKVD 11 D40 nM19 nMClone NP-2 1CRLRL 6 VLARD 11 F34 nM12 nMClone NP-3 1 GDPIT 6 SRRLD 11 Y69 nM64 nMClone NP-4 1 GTRIA 6 LPLRD 11 V38 nM29 nM

[0120] Figure 3 shows the binding constant (KD) measurements of four selected clones. Figure 3(a) shows the results of flow cytometry analysis after treating the Fv-antibody library, a control strain (a strain with CDR1 and CDR2), and the original E. coli with anti-NP mAb and FITC-labeled secondary antibody. Figure 3(b) shows the results of binding constant (KD) measurements in a quantitative binding analysis using flow cytometry. Figure 3(c) shows the SPR analysis procedure after layering the outer membrane of the Fv-antibody library E. coli onto the Au chip of the SPR biosensor. Figure 3(d) shows the determination of binding constant (KD) in a quantitative binding analysis using the SPR biosensor. To investigate the binding properties of Inf-A with mAb to NP, the selected clones were treated with various concentrations of mAb. After treatment with a fluorescently labeled secondary antibody, the fluorescence intensity was measured using a flow cytometer. As shown in Fig. 3(a), the four selected clones exhibited higher fluorescence signal areas than the control strain with only CDR1 and CDR2 or the original E. coli (BL21). These strong fluorescence signals indicated that the anti-NP mAb specifically bound to the autodisplayed Fv antibody of the selected clones. Furthermore, specific binding of the anti-NP mAb via CDR3 was achieved in the selected clones. As shown in Fig. 3(b), the four selected clones reacted with anti-NP mAb concentrations ranging from 1.1 to 270 nM, and the fluorescence signals increased in a concentration-dependent manner. The control strain with autodisplayed CDR1 and CDR2 (but no CDR3) maintained the fluorescence signal at the baseline. From the dose-response curves of these four clones, the KD values ​​were calculated as 40 nM (clone NP-1), 34 nM (clone NP-2), 69 nM (clone NP-3), and 38 nM (clone NP-4) using the ideal model (Bong et al., 2020b; Kim et al., 2022c).These results demonstrate that the four clones harbor autodisplayed Fv antibodies with binding affinity for anti-NP mAb, and that specific binding occurs through CDR3 of the selected Fv antibodies, unlike the control strain with only CDR1 and CDR2. The binding properties of the selected Fv antibodies were investigated using an SPR biosensor with immobilized outer membranes of the four selected clones. The outer membrane of E. coli was isolated as previously described (Bong et al., 2014; Park et al., 2010; Park et al., 2015). The outer membrane layer was immobilized on the Au surface of the SPR biosensor as shown in Figure 3(c) (Jung et al., 2023). After treating various concentrations of mAb against the modified NP, the SPR signals were measured after the binding and dissociation steps. Figure 3(d) shows that the control strain with only CDR1 and CDR2 (but no CDR3) maintained the SPR signal at the baseline level. A quantitative increase in the fluorescence signal was observed when the four selected clones were reacted with anti-NP mAb at concentrations ranging from 0.2 to 67 nM. From the dose-response curves of these four clones, the KD was determined to be 19 nM (clone NP-1), 12 nM (clone NP-2), 64 nM (clone NP-3), and 29 nM (clone NP-4). These results demonstrate that the four clones possess autodisplayed Fv antibodies with binding affinity for anti-NP mAb. The binding constants (KD) for the four selected clones, determined using flow cytometry and SPR biosensor, are summarized in Table 3.

[0121] One-Step Immunoassay for Influenza Virus

[0122] The selected Fv antibodies with binding affinity for the modified NP were expressed as fusion proteins with GFP, as shown in Figure 4(a). The fusion proteins with GFP were prepared for the following reasons: (1) Fv antibodies have limited solubility, whereas GFP has higher solubility (Liu et al., 2019; Pedelacq et al., 2006), and (2) GFP can be effectively immobilized on the metal surface of a biosensor. The immobilization efficiency of GFP was measured to be similar to that of BSA, a blocking protein frequently used in immunoassays. The four expressed Fv antibodies contained three CDRs and four FRs, including the selected CDR3 sequence (13 kDa). In addition, GFP (at the proband, 27 kDa) and His-tags (at both ends) were added to the fusion proteins. Four types of Fv antibodies with molecular weights of approximately 40 kDa were expressed as fusion proteins with GFP, as confirmed by SDS-PAGE. The KD values ​​of the four expressed Fv antibodies against anti-NP mAb were measured using an SPR biosensor. As shown in Figure 4(b), anti-NP mAb was immobilized on the Au surface of the SPR biosensor, and the four expressed Fv antibodies were applied at various concentrations. The KD values ​​were calculated to be 423 nM (Fv-NP-1), 210 nM (Fv-NP-2), 976 nM (Fv-NP-3), and 380 nM (Fv-NP-4). For the modified NP of Inf-A (A / Hong Kong / H090-701-V10 / 2009), the KD was calculated to be 26 nM using the same SPR biosensor with immobilized anti-NP mAb. To realize a single-use immunoassay based on a mimic, several prerequisites were necessary: ​​(1) the Fv antibody (mimic) must remain bound to the monoclonal antibody until antigen treatment, and (2) the Fv antibody must be quantitatively separated from the monoclonal antibody immediately after antigen treatment.The difference in binding constants (KD) of the NP-like mimic (KD = 210 - 976 nM) and the target antigen (NP, KD = 26 nM) indicated that Fv-antibody could be quantitatively released from the immobilized mAb when NP in the sample bound to the immobilized mAb.

[0123] The disposable immunoassay for influenza virus detection is a displacement immunoassay. In this immunoassay, upon binding of the target antigen to a monoclonal antibody, the immobilized antibody binds to a mimic released from the monoclonal antibody.

[0124] Figure 5 illustrates the results of NP detection using a one-step immunoassay based on Fv antibodies. Figure 5(a) illustrates the configuration of a one-step immunoassay based on Fv antibodies. Figure 5(b) illustrates the results of optimizing the Fv antibody concentration for the one-step immunoassay. Figure 5(c) illustrates the detection results of a standard NP sample using the one-step immunoassay.

[0125] Fluorescently labeled Fv antibodies (fluorescently labeled) were bound to immobilized anti-NP mAb, as shown in Figure 5(a). The bound Fv antibodies were released from the monoclonal antibodies when the modified NP (or influenza virus extract) was made to react, and the fluorescent signal from the released Fv antibodies was detected in the reaction solution. As the first step in constructing a single-use immunoassay based on the mimic, the optimal Fv antibody concentration was determined. Figure 5(b) shows that four types of Fv antibodies were treated with anti-NP mAb at various concentrations ranging from 20 to 500 nM. The target antigen (modified NP at a concentration of 200 nM) was added to the monoclonal antibodies, and the fluorescent signal from the released Fv antibodies (fluorescently labeled) was measured. The fluorescent signal increased with increasing Fv antibody concentration. The fluorescence signal of the blank sample without the target antigen and the monoclonal antibody conjugated to the Fv antibody (mimetic) remained at the baseline level, unlike the fluorescence signal of the sample with the target antigen. This result indicates that the optimal concentration of the mimetic (Fv antibody) for the disposable immunoassay is 200 nM. The LOD of the disposable immunoassay was determined using the concentration of the mimetic (Fv antibody) determined based on the optimized concentration of the Fv antibody for binding to the monoclonal antibody. As shown in Fig. 5(c), the fluorescence signal was quantitatively increased when the target antigen (modified NP) in the concentration range of 1.4-1 μM was treated with the monoclonal antibody conjugated to the mimetic. Additionally, the LOD values ​​for the detection of modified NPs were measured as 39 nM (Fv-NP-1), 24 nM (Fv-NP-2), 77 nM (Fv-NP-3), and 22 nM (Fv-NP-4), which were three times the standard deviation of the fluorescence signal of the blank sample (n = 3). These values ​​were three times the standard deviation of the fluorescence signal of the blank sample (n = 3). These results demonstrate that the modified NPs (or influenza virus extracts) can be detected using a disposable immunoassay based on a mimic (Fv-antibody).

[0126] Figure 6 illustrates a one-step immunoassay based on NP-like mimotope (using expressed Fv antibodies and synthetic peptides) using influenza virus samples (Inf-A and Inf-B). Figure 6(a) illustrates the results of a lateral flow immunoassay for an influenza virus sample (Inf-A). Figure 6(b) illustrates a one-step immunoassay based on Np-like mimotope (using expressed Fv antibodies) for an Inf-A sample. Figure 6(c) illustrates a one-step immunoassay based on Np-like mimotope (using synthetic peptides) for an Inf-A sample. Figure 6(d) illustrates the results of a lateral flow immunoassay for an influenza virus sample (Inf-B). Figure 6(e) illustrates a one-step immunoassay based on Np-like mimotope (using expressed Fv antibodies) for an Inf-B sample. Figure 6(f) illustrates a one-step immunoassay based on Np-like mimotope (using synthetic peptide) for Inf-B samples.

[0127] A disposable immunoassay based on NP-like mimics was performed for Inf-A and Inf-B. The analytical results were compared with those of a conventional lateral flow immunoassay for Inf-A and Inf-B. Figure 6(a) shows a conventional lateral flow assay performed in the concentration range of 10 - 5 × 10^4 ng / mL for Inf-A, where a separate signal band for Inf-A was observed on the test strip depending on the antigen concentration. The same antigen sample was used in the disposable immunoassay based on mimics. As shown in Figure 6(b), the disposable immunoassay provided quantitative results based on the Inf-A concentration. The LOD values ​​for Inf-A detection were measured as 0.1 (Fv-NP-1), 7.5 (Fv-NP-2), 9.3 (Fv-NP-3), and 0.02 ng / mL (Fv-NP-4), which are three times the standard deviation of the fluorescence signals of the blank samples (n = 3). Compared with the LOD of the conventional lateral flow immunoassay of 1,024 ng / mL, the LOD values ​​for Inf-A detection were measured to be improved by 10,240 (Fv-NP-1), 136.8 (Fv-NP-2), 109.8 (Fv-NP-3), and 51,200-fold (Fv-NP-4). In addition, a disposable immunoassay based on fluorescently labeled peptides containing the CDR3 sequence using Inf-A was performed. The amino acid sequences of four peptides synthesized from the CDR3 of the screened Fv-antibodies are summarized in Table 4. Table 4 shows the amino acid sequences of four peptides synthesized from the screened CDR3 of the Fv-antibodies.

[0128] The analytical results were compared with a conventional lateral flow immunoassay for Inf-A. Figure 6(c) shows that the disposable immunoassay based on fluorescently labeled peptides showed quantitative results depending on the Inf-A concentration. The LOD values ​​for Inf-A detection were measured as 0.1 (peptide-NP-1), 90 (peptide-NP-2), 3.4 (peptide-NP-3), and 0.01 ng / mL (peptide-NP-4), which are three times the standard deviation of the fluorescence signals of the blank samples (n = 3).

[0129] Selected peptidePeptide sequencePeptide-NP-1 1 ARCGP 6 VPIRK 11 VDDWG 16 KPeptide-NP-2 1 ARCRL 6 RLVLA 11 RDFWG 16 KPeptide-NP-3 1 ARGDP 6 ITSRR 11 LDYWG 16 KPeptide-NP-4 1 ARGTR 6 IALPL 11 RDVWG 16 K

[0130] The same disposable immunoassay was performed for Inf-B detection, and the analytical results were compared with the conventional lateral flow immunoassay for Inf-B in Fig. 6(d). Fig. 6(e) shows that the disposable immunoassay provided quantitative results depending on the Inf-B concentration. The LOD values ​​for Inf-B detection were measured as 0.4 (Fv-NP-1), 13.8 (Fv-NP-2), 6.0 (Fv-NP-3), and 0.14 (Fv-NP-4), which are three times the standard deviation of the fluorescence signal of the blank samples (n = 3). Compared with the LOD of the traditional lateral flow immunoassay of 426 ng / mL, the LODs for Inf-B detection were measured to be significantly improved at 990.7 (Fv-NP-1), 30.8 (Fv-NP-2), 71.2 (Fv-NP-3), and 3,042-fold (Fv-NP-4). Compared with the analysis results for Inf-A using the same disposable immunoassay, the LODs for Inf-B showed similar trends depending on the Fv antibody, and the disposable immunoassay based on Fv-NP-4 showed the most sensitive detection of both Inf-A and Inf-B. In addition, the same disposable immunoassay based on a fluorescently labeled peptide was performed for Inf-B detection. As shown in Fig. 6(f), the disposable immunoassay provided quantitative results depending on the Inf-B concentration. The LODs of Inf-B were measured as 0.6 ng / mL (peptide-NP-1), 72 ng / mL (peptide-NP-2), 2.8 ng / mL (peptide-NP-3), and 0.50 ng / mL (peptide-NP-4), which are three times the standard deviation of the fluorescence signal of the blank samples (n = 3). Compared with the analytical results for Inf-A using the same disposable immunoassay, the LODs of Inf-B showed a similar trend depending on the peptide, and the disposable immunoassay based on peptide-NP-4 showed the most sensitive detection of both Inf-A and Inf-B.These results indicate that a disposable immunoassay based on NP-like mimics (or peptides) can achieve more sensitive detection of Inf-A than a traditional lateral flow immunoassay. Furthermore, the disposable immunoassay based on NP-like mimics (or peptides) can be applied to the detection of Inf-A and Inf-B. The sensitivity of the disposable immunoassay indicates that the monoclonal antibodies recognize these two types of influenza viruses with higher binding affinity than the mimics. Fv antibodies (mimetic) were selected from an Fv antibody library using anti-NP mAb as a probe. The amino acid sequences of the NP and NP of Inf-A and Inf-B were compared to investigate the sensitivity of the two types of influenza viruses using the probes. The amino acid sequence of NP for screening the Fv-antibody library obtained from Inf-A of the Hong Kong variant (A / Hong Kong / H090-701-V10 / 2009) consisted of 315 amino acids (positions: 184-498 in the sequence, 38 kDa). Inf-A NPs of the Singapore (A / Singapore / 63 / 04) and Florida (B / Florida / 02 / 06) variants were analyzed for amino acid sequence similarity. BLAST sequence analysis using BLAST software (https: / blast.ncbi.nlm.nih.gov) determined amino acid sequence identity (exact identity of amino acid sequences) and similarity (identity of chemical properties of amino acid sequences).

[0131] Figure 7 illustrates the results of homology analysis of NP with various influenza virus strains. Figure 7(a) illustrates the homology analysis of the NP amino acid sequence between the NP of the A / Hong Kong / H090-701-V10 / 2009 strain and the NP of A / Singapore / 63 / 04. Figure 7(b) illustrates the results of homology analysis of the NP amino acid sequence between the NP of the A / Hong Kong / H090-701-V10 / 2009 strain and the NP of B / Florida / 02 / 06 strain.

[0132] Figure 7(a) shows that the NP of the Singapore variant (sequence number: 498; code: A / Singapore / 63 / 04) has 93.8% identity (314 of 316 amino acids) and 95.5% similarity. The amino acid sequence of the NP of the Inf-A variant was identical to the amino acid sequence of the NP used as a probe. Figure 7(b) shows that the Inf-B NP of the Florida variant (sequence number: 560; code: B / Florida / 02 / 06) has 35.3% identity (118 of 318 amino acids) and 60.8% similarity (177 of 318 amino acids). These results indicate that the amino acid sequence of Inf-A is identical to the amino acid sequence of the NP used as a probe. Therefore, the sensitivity of the disposable immunoassay stems from the amino acid sequence similarity between the target NPs of Inf-A and Inf-B and the probe NPs used to screen for mimics. Finally, the disposable immunoassay based on these four types of Fv antibodies can be considered applicable to the medical diagnosis of influenza viruses, compared to the LOD of conventional lateral flow immunoassays.

[0133] [[conclusion]]

[0134] NP-like mimics of Inf-A were screened from an autodisplayed Fv-antibody library. Four types of NP-like mimics with binding affinity to an anti-NP mAb library were screened from the Fv-antibody library. An Fv-antibody library corresponding to the VH of IgG with randomized CDR3 regions was expressed on the outer membrane of E. coli using autodisplay technology. The target Fv-antibodies were screened using magnetic beads containing immobilized anti-NP mAb. Four clones were screened from the Fv-antibody library, and the KDs were calculated to be 40, 34, 69, and 38 nM for clones NP-1, NP-2, NP-3, and NP-4, respectively, from dose-response curves using the ideal model. A control strain with only CDR1 and CDR2 (but no CDR3) and the original E. Compared to E. coli, these results demonstrate that the four clones harbored autodisplayed Fv antibodies with binding affinity for anti-NP mAb, indicating that specific binding occurred through the CDR3 of the selected Fv antibodies. The selected Fv antibodies (NP-like mimetics) were expressed as fusion proteins with GFP. The KD of the selected clones (selected Fv antibodies on the outer membrane of E. coli) toward anti-NP mAb was measured using a SPR biosensor. The calculated KD values ​​were 423 nM (Fv-NP-1), 210 nM (Fv-NP-2), 976 nM (Fv-NP-3), and 380 nM (Fv-NP-4). A disposable immunoassay for influenza virus was constructed using the expressed mimetics conjugated to anti-NP mAb. The mimic (labeled GFP) was quantitatively isolated when NP (in the sample) was bound to a monoclonal antibody, and the NP concentration in the sample was determined by fluorescence measurement.

[0135] A disposable immunoassay based on a mimic achieved higher sensitivity for the detection of Inf-A and Inf-B than a conventional lateral flow immunoassay. The sensitivity of the disposable immunoassay based on an NP-like mimic of Inf-A stems from the similarity (approximately 56%) in amino acid sequence between the target NPs of Inf-A and Inf-B and the probe NPs used to screen the mimics. The mimic-based disposable immunoassay has the potential to be applied to the medical diagnosis of influenza based on the LOD of the conventional lateral flow immunoassay.

[0136] Although the present invention has been described above with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various modifications and changes may be made to the present invention without departing from the spirit and scope of the present invention as set forth in the claims below.

[0137] National Research and Development Project that supported this invention

[0138] - Assignment ID (10 digits): 2710001146

[0139] - Project number: RS-2023-00209053

[0140] - Ministry name: Ministry of Science and ICT

[0141] - Project Management (Professional) Organization Name: National Research Foundation of Korea

[0142] - Research Project Name: Individual Basic Research (Ministry of Science and ICT)

[0143] - Research Project Name: Development of Fv-antibodies and a one-step diagnostic kit for virus detection using an Fv-antibody library.

[0144] - Contribution rate: 1 / 2

[0145] - Project implementation organization name (host organization): Yonsei University Industry-Academic Cooperation Foundation

[0146] Research period: March 1, 2024 - February 28, 2025

[0147]

[0148] - Assignment ID (10 digits): 1465038753

[0149] - Assignment number: RS-2022-KH128357

[0150] - Ministry name: Ministry of Health and Welfare

[0151] - Project Management (Professional) Organization Name: Korea Health Industry Development Institute

[0152] - Research Project Name: Rapid Universal Vaccine Technology Development

[0153] Research Project Name: Research on a Vaccine Screening Platform Capable of Rapidly Responding to Mutant Viruses

[0154] - Contribution rate: 1 / 2

[0155] - Project implementation organization name (host organization): Yonsei University Industry-Academic Cooperation Foundation

[0156] Research period: January 1, 2023 - December 31, 2023

Claims

1. It has the function of influenza nucleoprotein mimotope, Comprising at least one peptide sequence selected from the group consisting of a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, a peptide sequence of SEQ ID NO: 3, and a peptide sequence of SEQ ID NO:

4. Peptide: [Sequence number 1] CGPVPIRKVDD [Sequence number 2] CRLRLVLARDF [Sequence number 3] GDPITSRRLDY [Sequence number 4] GTRIALPLRDV.

2. In paragraph 1, The above peptide has a mimotopic function that can bind to an antibody that can bind to the influenza nucleoprotein. Peptide.

3. In paragraph 2, The above peptide competitively binds to the antibody in the presence of influenza nucleoprotein. Peptide.

4. Coding a peptide according to paragraph 1; Nucleic acid.

5. Containing nucleic acid according to Article 4, Recombinant expression vector.

6. Transformed with a recombinant expression vector according to Article 5, cell.

7. In paragraph 6, The above cell comprises one or more cells selected from the group including animal cells, plant cells, yeast, E. coli and insect cells. cell.

8. In paragraph 7, The above cells are monkey kidney cells 7 (COS7), NSO cells, SP2 / 0 cells, Chinese hamster ovary (CHO) cells, W138, baby hamster kidney (BHK) cells, MDCK, myeloma cell lines, HuT 78 cells, and HEK293 cells, Escherichia coli, Bacillus subtilis, Streptomyces sp, Pseudomonas sp, Proteus mirabilis or Staphylococcus sp, Aspergillus sp, Pichiapastoris, Saccharomyces cerevisiae, Schizosaccharomyces sp, and Comprising one or more cells selected from the group comprising Neurospora crassa; cell.

9. Has the function of influenza nucleoprotein mimotope, An amino acid fragment comprising at least one peptide sequence selected from the group consisting of a peptide sequence of SEQ ID NO: 1, a peptide sequence of SEQ ID NO: 2, a peptide sequence of SEQ ID NO: 3, and a peptide sequence of SEQ ID NO:

4. Composition for detecting influenza: [Sequence number 1] CGPVPIRKVDD [Sequence number 2] CRLRLVLARDF [Sequence number 3] GDPITSRRLDY [Sequence number 4] GTRIALPLRDV.

10. In paragraph 9, The composition for detecting influenza above has a mimotope function capable of binding to an antibody capable of binding to an influenza nucleoprotein. A composition for detecting influenza.

11. In paragraph 10, The composition for detecting influenza above competitively binds to the antibody in an environment where influenza nucleoprotein exists. A composition for detecting influenza.

12. In paragraph 9, The above amino acid fragment further comprises a fluorescent marker, Composition for detecting influenza 13. A substrate including at least a conduit; An antibody capable of binding to the influenza nucleoprotein immobilized within the above-mentioned conduit; and A composition for detecting influenza, comprising: The composition for detecting influenza above competitively binds to the antibody and the influenza nucleoprotein, The composition for detecting influenza comprises an influenza detecting composition according to any one of claims 9 to 11. Kit for detection of influenza.

14. In paragraph 13, The amino acid fragment of the composition for detecting influenza further comprises a fluorescent marker. Kit for detection of influenza.

15. In paragraph 14, Further comprising a fluorescence analysis unit for performing quantitative analysis of the fluorescent marker from the influenza detection composition released when the antibody is exposed to the influenza nucleoprotein. Kit for detection of influenza.

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