Preparation of a diagnostic monoclonal antibody against a new epitope of HNL and its application

Abstract

The application provides preparation and application of a diagnostic monoclonal antibody for a new epitope of human neutrophil lipocalin (HNL). Two new HNL protein conformation epitopes and one new HNL linear epitope are designed and provided, and an anti-HNL antibody with higher binding affinity and better binding specificity is screened to combine the new epitopes. The anti-HNL antibody of the application can be used for preparation of an HNL detection reagent and kit, and has higher sensitivity and specificity when used for detecting HNL protein in a sample, and can be applied to distinguishing bacterial infection and viral infection.

Description

Technical Field

[0001] This invention relates to the field of biodiagnostic technology, specifically to the preparation of a diagnostic monoclonal antibody targeting a novel epitope of human neutrophil lipid transport protein (HNL) and its application in the differential diagnosis of bacterial and viral infections. Background Technology

[0002] Human neutrophil lipocalin (HNL), also widely known as neutrophil gelatinase-associated lipocarrier protein (NGAL), is a glycoprotein widely distributed in organisms. This important protein was initially isolated from human neutrophils and found to be primarily located in specific granular structures of these cells. From a molecular structural perspective, HNL can exist in two forms: a monomeric form with a relative molecular mass of 25 kDa and a 45 kDa homodimer form linked by disulfide bonds. Furthermore, it can further form a more complex 135-kDa heterodimer through intermolecular disulfide bridges, covalently conjugating with gelatinase (i.e., matrix metalloproteinase 9, MMP-9).

[0003] As a major component of the secondary granules of human neutrophils, HNL is an important member of the lipid transporter protein family. It is mainly produced specifically by activated neutrophils and stored in the secondary granules of neutrophils, serving as one of the important weapons in the body's innate immune defense.

[0004] The core physiological function of HNL lies in its powerful iron-scavenging ability. By chelating siderophore-iron complexes, it deprives bacteria of the iron essential for growth, thereby directly inhibiting bacterial proliferation and acting as a natural chemical defense against bacterial invasion. This direct antibacterial activity provides the molecular basis for the rapid increase in HNL levels during bacterial infection.

[0005] Under normal physiological conditions, HNL is expressed at low levels in organs such as the kidneys, lungs, prostate, and gastrointestinal tract. Studies have shown that when a person experiences a bacterial infection, neutrophils release HNL into the peripheral blood, causing a significant increase in HNL levels. The levels rise as early as 6-8 hours after infection, peaking at 24-48 hours, and can be used for the diagnosis of early bacterial infections. However, in healthy individuals or during viral infections, HNL levels do not show a significant increase. When using HNL detection to differentiate between acute bacterial and viral infections, the sensitivity and specificity are 97% and 96%, respectively. Therefore, HNL can serve as a diagnostic biomarker for bacterial / viral infections.

[0006] Antibodies available for HNL detection exist in the prior art, such as the anti-HNL antibodies disclosed in patents CN107001455B and EP0756708B1. However, there are currently no commercially available antibodies in China, and existing antibodies suffer from problems such as low titer, low antigen-binding efficiency, and insufficient detection specificity.

[0007] Therefore, there is an urgent need in this field to develop an anti-HNL antibody with higher binding affinity, detection sensitivity, and specificity. Summary of the Invention

[0008] In a first aspect of the invention, an anti-neutrophil apolipoprotein (HNL) antibody or an antigen-binding fragment thereof is provided, said antibody or antigen-binding fragment comprising a heavy chain variable region (VH) and a light chain variable region (VL), said heavy chain variable region and light chain variable region comprising CDRs selected from the group consisting of:

[0009] (1) VH-CDR1 shown in SEQ ID NO: 11, VH-CDR2 shown in SEQ ID NO: 12, VH-CDR3 shown in SEQ ID NO: 13, VL-CDR1 shown in SEQ ID NO: 14, VL-CDR2 with the amino acid sequence WNI, and VL-CDR3 shown in SEQ ID NO: 15;

[0010] (2) VH-CDR1 shown in SEQ ID NO: 16, VH-CDR2 shown in SEQ ID NO: 17, VH-CDR3 shown in SEQ ID NO: 18, VL-CDR1 shown in SEQ ID NO: 19, VL-CDR2 with the amino acid sequence NNN, and VL-CDR3 shown in SEQ ID NO: 20; or

[0011] (3) VH-CDR1 shown in SEQ ID NO: 21, VH-CDR2 shown in SEQ ID NO: 22, VH-CDR3 shown in SEQ ID NO: 23, VL-CDR1 shown in SEQ ID NO: 24, VL-CDR2 with the amino acid sequence SCQ, and VL-CDR3 shown in SEQ ID NO: 25.

[0012] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 5, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 6, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 6.

[0013] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 7, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 8, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0014] In another preferred embodiment, the amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 9, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 9; and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 10, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 10.

[0015] In another preferred embodiment, the antibody further comprises a heavy chain constant region and a light chain constant region.

[0016] In another preferred embodiment, the heavy chain constant region and the light chain constant region are of mouse or human origin.

[0017] In another preferred embodiment, the heavy chain constant region is derived from mouse heavy chain IgG1 or IgG2a.

[0018] In another preferred embodiment, the light chain constant region is derived from the mouse kappa (κ) chain.

[0019] In another preferred embodiment, the antibody is a monoclonal antibody.

[0020] In another preferred embodiment, the antigen-binding fragment includes: scFv, Fv, Fab, Fab', F(ab')2.

[0021] In another preferred embodiment, the antibody binds to a conformational epitope of the amino acid sequence as shown in SEQ ID NO: 2.

[0022] In another preferred embodiment, the antibody binds to a conformational epitope of the amino acid sequence as shown in SEQ ID NO: 3.

[0023] In another preferred embodiment, the antibody binds to a linear epitope of the amino acid sequence as shown in SEQ ID NO: 4.

[0024] In another preferred embodiment, the antibody has the following characteristics:

[0025] (1) It can specifically bind to the conformational epitope shown in SEQ ID NO: 2, the conformational epitope shown in SEQ ID NO: 3, or the linear epitope shown in SEQ ID NO: 4, with an OD450 value ≥ 0.8 for the binding reaction;

[0026] (2) The dissociation constant of HNL is KD ≤ 1 × 10 -8 M; and

[0027] (3) It does not cross-react with epitopes at positions 82-102 and 141-156 of HNL, and the cross-reaction rate is ≤5%.

[0028] In a second aspect of the invention, a polynucleotide molecule is provided that encodes an anti-HNL antibody or an antigen-binding fragment thereof as described in the first aspect of the invention.

[0029] In another preferred embodiment, the polynucleotide includes DNA and RNA.

[0030] In a third aspect of the invention, an expression vector is provided containing a polynucleotide molecule as described in the second aspect of the invention.

[0031] In another preferred embodiment, the vector includes a eukaryotic cell expression vector and a prokaryotic cell expression vector.

[0032] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0033] In a fourth aspect of the invention, a host cell is provided which contains an expression vector as described in the third aspect of the invention, or whose genome is integrated with a polynucleotide molecule as described in the second aspect of the invention.

[0034] In another preferred embodiment, the host cell includes eukaryotic cells (such as mammalian cells) and prokaryotic cells.

[0035] In a fifth aspect of the invention, an antibody conjugate is provided, the antibody conjugate comprising:

[0036] (a) an anti-HNL antibody or an antigen-binding fragment thereof as described in the first aspect of the invention; and

[0037] (b) The coupling motif selected from the following groups: detectable markers, enzymes that can produce detectable products, radionuclides, nanoparticles / nanorobars.

[0038] In another preferred embodiment, the coupling portion is a colloidal gold marker.

[0039] In a sixth aspect of the invention, the use of an anti-HNL antibody or antigen-binding fragment thereof as described in the first aspect of the invention, or an antibody-drug conjugate as described in the fifth aspect of the invention, in the preparation of a detection reagent or kit for detecting HNL is provided.

[0040] In another preferred embodiment, the method for detecting HNL includes immunochromatography, chemiluminescence, and ELISA.

[0041] In another preferred embodiment, the detection is used for: 1) differential diagnosis of bacterial and viral infections; and / or 2) monitoring of antibiotic use.

[0042] In a seventh aspect of the invention, a detection reagent is provided, the detection reagent comprising an anti-HNL antibody or an antigen-binding fragment thereof as described in the first aspect of the invention, or an antibody conjugate as described in the fifth aspect of the invention, and a detection-acceptable carrier.

[0043] In another preferred embodiment, the detection reagent is a detection reagent for HNL detected by immunochromatography, chemiluminescence, or ELISA.

[0044] In an eighth aspect of the present invention, a colloidal gold immunochromatographic test strip is provided, wherein the colloidal gold immunochromatographic test strip is coated with an anti-HNL antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention.

[0045] In another preferred embodiment, the colloidal gold immunochromatographic test strip comprises: a PVC base plate, a sample pad, a gold-labeled pad, a detection line, a control line, a nitrocellulose membrane, and an absorbent pad; wherein the gold-labeled pad is coated with a mixture of colloidal gold-labeled anti-HNL antibody II and mouse IgG antibody, the detection line is coated with anti-HNL antibody I, and antibody II and antibody I are selected from the anti-HNL antibodies described in the first aspect of the present invention, and antibody II and antibody I are different antibodies.

[0046] In another preferred embodiment, the heavy chain variable region and light chain variable region of antibody II comprise the following CDRs:

[0047] VH-CDR1 shown in SEQ ID NO: 11, VH-CDR2 shown in SEQ ID NO: 12, VH-CDR3 shown in SEQ ID NO: 13, VL-CDR1 shown in SEQ ID NO: 14, VL-CDR2 with the amino acid sequence WNI, and VL-CDR3 shown in SEQ ID NO: 15;

[0048] Preferably, the amino acid sequence of the heavy chain variable region of antibody II is as shown in SEQ ID NO: 5, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region of antibody II is as shown in SEQ ID NO: 6, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 6; and

[0049] The heavy chain variable region and light chain variable region of antibody I contain the following CDRs:

[0050] VH-CDR1 shown in SEQ ID NO: 16, VH-CDR2 shown in SEQ ID NO: 17, VH-CDR3 shown in SEQ ID NO: 18, VL-CDR1 shown in SEQ ID NO: 19, VL-CDR2 with the amino acid sequence NNN, and VL-CDR3 shown in SEQ ID NO: 20;

[0051] Preferably, the amino acid sequence of the heavy chain variable region of antibody I is as shown in SEQ ID NO: 7, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 7; and the amino acid sequence of the light chain variable region of antibody I is as shown in SEQ ID NO: 8, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 8.

[0052] In another preferred embodiment, the control line is coated with goat anti-mouse polyclonal antibody.

[0053] In another preferred embodiment, the coating amount of the mixture of colloidal gold-labeled anti-HNL antibody II and mouse IgG antibody on the gold-labeled pad is 3-6 μL / cm, preferably 4-5 μL / cm, and more preferably 4.5 μL / cm.

[0054] In another preferred embodiment, the coating amount of anti-HNL antibody I on the detection line is 0.5-1.5 μL / cm, preferably 0.8-1.2 μL / cm, and more preferably 0.8 μL / cm.

[0055] In another preferred embodiment, the coating amount of the goat anti-mouse polyclonal antibody on the control line is 0.5-1.5 μL / cm, preferably 0.8-1.2 μL / cm, and more preferably 0.8 μL / cm.

[0056] In a ninth aspect of the invention, a kit is provided comprising the detection reagents as described in the seventh aspect of the invention, and / or the colloidal gold immunochromatographic test strips as described in the eighth aspect of the invention, and instructions.

[0057] In another preferred embodiment, the specification states that the kit is used for non-invasive detection of HNL levels in a test subject.

[0058] In another preferred embodiment, the test subject includes a human or a non-human mammal.

[0059] In a tenth aspect of the present invention, a method for preparing an anti-HNL antibody or an antigen-binding fragment thereof as described in the first aspect of the present invention is provided, the method comprising:

[0060] (i) Under conditions suitable for antibody production, host cells as described in the fourth aspect of the invention are cultured to obtain a culture containing the anti-HNL antibody or its antigen-binding fragment;

[0061] (ii) Isolating or recovering the anti-HNL antibody or its antigen-binding fragment from the culture; and

[0062] (iii) Purification and / or modification of the anti-HNL antibody or its antigen-binding fragment obtained in step (ii).

[0063] In an eleventh aspect of the present invention, a method for in vitro detection of whether a sample contains HNL is provided, the method comprising:

[0064] (S1) Contact the sample with the anti-HNL antibody or its antigen-binding fragment as described in the first aspect of the present invention, or the antibody conjugate as described in the fifth aspect of the present invention;

[0065] (S2) Detect whether an antigen-antibody complex is formed, where the formation of an antigen-antibody complex indicates the presence of HNL in the sample.

[0066] In another preferred embodiment, the sample includes: an isolated tissue sample or an isolated cell sample.

[0067] In another preferred embodiment, the sample is whole blood, plasma, or serum.

[0068] In another preferred embodiment, the sample is a peripheral blood sample.

[0069] In another preferred embodiment, the method is a non-diagnostic and non-therapeutic method.

[0070] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0071] Figure 1: Specificity verification diagram of monoclonal antibody (cross-reaction results with existing patented epitope antigens and homologous proteins).

[0072] Figure 2 ROC curves for clinical sample testing, with AUC, sensitivity, and specificity marked on the graph.

[0073] Figure 3 : Schematic diagram of the colloidal gold immunochromatographic test strip prepared in this invention. Detailed Implementation

[0074] Through extensive and in-depth research, and through numerous experimental screenings and verifications, the inventors have, for the first time, proposed novel HNL protein epitopes and prepared anti-HNL antibodies that specifically bind to these new epitopes. This invention designs and provides two novel HNL protein conformational epitopes and one novel HNL linear epitope. These epitopes differ from existing HNL protein epitopes, and the screened anti-HNL antibodies binding to these new epitopes exhibit relatively higher binding affinity and better binding specificity. Therefore, the anti-HNL antibodies with novel binding epitopes screened in this invention can be used in the preparation of HNL detection reagents and kits, exhibiting higher sensitivity and specificity when detecting HNL protein in samples, and can be applied to differentiate between bacterial and viral infections.

[0075] Based on this, the present invention was completed.

[0076] the term

[0077] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0078] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0079] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0080] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0081] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting examples include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0082] The new HNL epitope of the present invention

[0083] This invention proposes novel epitopes for HNL proteins for antibody development. Specifically, this invention utilizes intelligent bioinformatics tools such as AlphaFold2, AutoDock Vina 1.2.5 (antibody-antigen docking), GROMACS 2023.2 (50ns molecular dynamics), PRODIGY (interfacial binding energy calculation), AbYsis (antibody sequence analysis), BepiPred-3.0 (linear epitope prediction), PyMOL2.5 (structural modeling), RosettaDock 4.0 (molecular docking), and MM-GBSA (binding free energy calculation) to identify and develop novel linear and conformational epitopes.

[0084] This invention provides a novel conformational epitope 1 for the HNL protein: the amino acid sequence is SEQ ID NO: 2 (corresponding to positions 19-45 of HNL (SEQ ID NO: 1): QAQDSTSDLIPAPPLSKVPLQQNFQD), which is a conformational epitope with a solvent-accessible surface area ≥50 Å. 2 .

[0085] The present invention also provides another novel HNL protein conformational epitope 2: the amino acid sequence is SEQ ID NO: 3 (corresponding to positions 58-97 of HNL: GNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCD), which is a conformational epitope containing an α-helix and a flexible loop structure.

[0086] This invention also provides a novel linear epitope 3 of the HNL protein: the amino acid sequence is SEQ ID NO:4 (corresponding to positions 169-189 of HNL: KENFIRFSKSLGLPENHIVFP).

[0087] Antibody

[0088] As used herein, the term "antibody" or "immunoglobulin" is a heterotetramer composed of two light chains (L) and two heavy chains (H). The N-terminus of each heavy chain is a variable region (VH) connecting to the constant region of the heavy chain. The N-terminus of each light chain is a variable region (VL) connecting to the constant region of the light chain.

[0089] As used herein, the term "variable" refers to the fact that the variable regions of an antibody differ in a specific sequence, resulting in the affinity and specificity of a particular antibody for a specific antigen. Antibody variable regions include complementarity-determining regions (CDRs) or hypervariable regions, as well as more conserved framework regions (FRs). The primary sequence of the heavy and light chain variable regions consists of four FR sequences and three CDR sequences spaced apart (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The sequence and spatial conformation of the heavy and light chain variable regions determine the specific binding of the antibody to the antigenic epitope. Antibody constant regions do not directly participate in antibody-antigen binding, but they affect the performance of antibody capture and detection.

[0090] The "light chain" of vertebrate antibodies (immunoglobulins) can be classified according to the amino acid sequence of their constant region. and Immunoglobulins are a class of proteins. Based on the amino acid sequence of their heavy chain constant region, immunoglobulins can be divided into different types, mainly five classes: IgA, IgD, IgE, IgG, and IgM, as well as antibody subtypes (isotypes), such as mouse IgG, which includes IgG1, IgG2a, and IgG2b subtypes. The subunit structures and three-dimensional conformations of different classes of immunoglobulins are well known to those skilled in the art.

[0091] As used herein, the term "monoclonal antibody (MABS)" refers to an antibody obtained from a largely homogeneous population, meaning that the individual antibodies contained in that population are identical. Monoclonal antibodies target a single antigenic determinant (epitope) with high specificity. The modifier "monoclonal" indicates the antibody's characteristic of being obtained from a largely homogeneous population of antibodies, which should not be interpreted as requiring any special methods to produce the antibody.

[0092] The present invention also includes monoclonal antibodies having the corresponding amino acid sequence of the anti-HNL monoclonal antibody, monoclonal antibodies having the variable region chain of the anti-HNL monoclonal antibody, and other proteins or protein conjugates and fusion expression products having these chains. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a light chain and a heavy chain containing a variable region (complementarity-determining region, CDR), provided that the variable region is the same as or has at least 90% homology with the variable regions of the light chain and heavy chain of the present invention, preferably at least 95% homology.

[0093] As known to those skilled in the art, antibody conjugates and fusion expression products include conjugates formed by binding the said anti-HNL antibody or its antigen-binding fragment to a detectable marker (e.g., fluorescent or luminescent marker), radiolabel, enzyme capable of producing a detectable product, gold nanoparticles / nanoran, and other detectable molecules. In one embodiment of the invention, colloidal gold is bound to the said anti-HNL antibody or its antigen-binding fragment to form a colloidal gold-antibody conjugate.

[0094] The term "antigen-binding fragment of an antibody" (or simply "antibody fragment") refers to one or more fragments of an antibody that maintain its ability to specifically bind to an antigen. It has been shown that fragments of full-length antibodies can be used for antigen-binding function. Examples of binding fragments included in the term "antigen-binding fragment of an antibody" include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by disulfide bonds on their chain regions; and (iii) scFv fragments consisting of the VH and VL domains of a single arm of the antibody.

[0095] This invention includes not only complete monoclonal antibodies, but also antibody fragments with binding activity, such as Fab or (Fab')2 fragments; antibody heavy chains; antibody light chains or scFv.

[0096] The term "epitope" or "antigenic determinant" refers to the site on an antigen where immunoglobulins or antibodies specifically bind. Epitopes typically consist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation.

[0097] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to an epitope on a pre-defined antigen.

[0098] As used herein, the term "antigen determinant" refers to a discontinuous three-dimensional spatial site on an antigen that is recognized by the antibody or antigen-binding fragment of the present invention.

[0099] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0100] In this invention, antibodies include murine antibodies prepared using techniques well known to those skilled in the art. Recombinant antibodies can be prepared using DNA recombination techniques well known in the art. The term "murine antibody" in this invention refers to a monoclonal antibody against HNL prepared according to knowledge and skills in the art.

[0101] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0102] As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably. The terms "light chain variable region" and "VL" are used interchangeably.

[0103] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242, namely the Kabat numbering system. Alternatively, antibody CDRs can also be classified using other numbering systems in the field, such as the IMGT and Chothia numbering systems.

[0104] In one aspect of the invention, an anti-HNL antibody or its antigen-binding fragment is provided. The invention designs novel antigenic epitopes and obtains anti-HNL murine antibodies through mouse immunization screening, with clone numbers 1A3, 1B9, and 1A11, which respectively bind to conformational epitope 1, conformational epitope 2, and linear epitope 3 of the invention.

[0105] The function of the antibody of this invention is determined by the variable region sequences of the antibody's light and heavy chains and its structural conformation, enabling it to specifically bind to HNL. Using this antibody variable region gene or complementarity-determining region (CDR) gene, different forms of genetically engineered antibodies can be modified and produced in any expression system utilizing prokaryotic and eukaryotic cells.

[0106] In this invention, the terms "antibody of the present invention," "protein of the present invention," or "peptide of the present invention" are used interchangeably and all refer to antibodies that specifically bind to HNL, such as proteins or peptides having the heavy chain variable region shown in SEQ ID NO: 5 and the light chain variable region shown in SEQ ID NO: 6 (clone 1A3); or proteins or peptides having the heavy chain variable region shown in SEQ ID NO: 7 and the light chain variable region shown in SEQ ID NO: 8 (clone 1A9); or proteins or peptides having the heavy chain variable region shown in SEQ ID NO: 9 and the light chain variable region shown in SEQ ID NO: 10 (clone 1A11).

[0107] The terms "fragment," "derivative," and "analyte" of an antibody refer to a polypeptide that substantially retains the same biological function or activity as the antibody of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) polypeptides formed by coupling a mature polypeptide with another compound (e.g., a compound used for chemiluminescence, such as acridinium ester); or (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, secretion sequence, or tag protein sequence or other fusion protein sequence used to purify or detect this polypeptide). These fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0108] The term "antibody of the present invention" refers to a polypeptide having HNL binding activity and including the aforementioned CDR region. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. This term also includes active fragments and active derivatives of the antibody of the present invention.

[0109] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, and polypeptides or proteins obtained using antiserum containing the antibody of this invention.

[0110] The present invention also includes fragments of the antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids, preferably at least about 60 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids of the antibody of the present invention.

[0111] Polynucleotide molecules, carriers and host cells

[0112] This invention also provides a polynucleotide molecule encoding the aforementioned antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of this invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to the coding region sequence of the aforementioned anti-HNL antibody protein or a degenerate variant. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a sequence having the same amino acid sequence as the polypeptide of this invention, but with a different coding region sequence.

[0113] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0114] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include polynucleotides that include additional coding and / or non-coding sequences. The full-length nucleotide sequence or fragments of the antibody of the present invention can typically be obtained by PCR amplification, recombinant methods, or artificial synthesis. Furthermore, heavy or light chains can be fused with proteins or tag sequences (such as fluorescent proteins, flag tags) to form fusion proteins.

[0115] Once the antibody sequence is obtained, recombinant antibodies can be prepared using bioengineering methods. Typically, the gene encoding the antibody sequence is cloned into a vector, the expression vector is then transferred into cells for expression, the cells or expression supernatant are harvested, and the recombinant antibody is obtained through purification. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms.

[0116] Currently, the DNA sequence encoding the protein (or a fragment thereof, or a derivative thereof) of this invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing plasmids (or other expression vectors) known in the art. Furthermore, mutations can be introduced into the antibody sequence of this invention through chemical synthesis.

[0117] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0118] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0119] Transforming host cells with recombinant DNA to express antibodies is a routine technique well-known to those skilled in the art. The recombinantly expressed antibodies can be isolated and purified using routine techniques well-known to those skilled in the art, which will not be elaborated upon here.

[0120] Test reagents and kits

[0121] This invention also provides a detection reagent, a colloidal gold immunochromatographic test strip, and a kit for detecting HNL protein. The detection reagent contains the anti-HNL antibody or its antigen-binding fragment as described in this invention, and a detection-acceptable carrier. The colloidal gold immunochromatographic test strip is coated with the anti-HNL antibody or its antigen-binding fragment as described in this invention. The kit is prepared by assembling the detection reagent or colloidal gold immunochromatographic test strip with the instructions.

[0122] Specifically, this invention provides a colloidal gold immunochromatographic test strip, comprising: a PVC base plate, a sample pad, a gold-labeled pad, a detection line, a control line, a nitrocellulose membrane, and an absorbent pad; wherein the gold-labeled pad is coated with a mixture of colloidal gold-labeled anti-HNL antibody II and mouse IgG antibody, and the detection line is coated with anti-HNL antibody I. Antibody II and antibody I are both selected from the anti-HNL antibody of this invention or its antigen-binding fragment, and antibody II and antibody I are different antibodies that bind to different HNL epitopes.

[0123] In a preferred embodiment of the present invention, the prepared colloidal gold immunochromatographic test strip is as follows: Figure 3As shown, it includes a PVC backing plate, a sample pad, a gold-labeled pad, a detection line, a control line, a nitrocellulose membrane, and an absorbent pad. The sample pad, gold-labeled pad, detection line, control line, nitrocellulose membrane, and absorbent pad are all positioned above the PVC backing plate. The sample pad is positioned at the edge of the PVC backing plate, with the gold-labeled pad partially overlapping the sample pad. The gold-labeled pad and absorbent pad partially overlap with both ends of the nitrocellulose membrane. A detection line is sprayed onto the nitrocellulose membrane near the gold-labeled pad, and a control line is sprayed onto the end near the absorbent pad. The sample pad is a glass fiber membrane, and the gold-labeled pad is a polyester membrane. The gold-labeled pad is coated with a mixture of colloidal gold-labeled neutrophil apolipoprotein monoclonal antibody II and mouse IgG antibody. The detection line is coated with neutrophil apolipoprotein monoclonal antibody I. The control line is coated with goat anti-mouse polyclonal antibody. Antibody II is antibody number 1A3 of this invention, and antibody I is antibody number 1B9 of this invention.

[0124] The prepared colloidal gold immunochromatographic test strips, along with the accompanying sample diluent, ID card, and / or instructions, are assembled into a neutrophil apolipoprotein detection kit. Specifically, the kit contains 25 or 50 test strips of colloidal gold immunochromatographic test strips and corresponding amounts of sample diluent. The sample diluent is 50 mmol / L Tris-HCl buffer, pH 7.8, prepared in the kit at 25 mL / vial, 1 vial / box, or 2 vials / box specifications.

[0125] application

[0126] The novel epitope of the HNL protein proposed in this invention can be used for the development of anti-HNL antibodies. Experiments have shown that anti-HNL antibodies that specifically bind to the novel epitope of this invention have a higher binding affinity for the HNL protein, resulting in higher sensitivity and specificity when used to detect the HNL protein in samples. Based on the anti-HNL antibodies obtained through screening according to this invention, detection reagents and kits for detecting the HNL protein in samples can be prepared and applied to the differential diagnosis of bacterial and viral infections and / or the monitoring of antibiotic use.

[0127] The amino acid sequence information of this invention is as follows:

[0128]

[0129]

[0130] The main advantages of this invention include:

[0131] (1) The HNL epitopes of the present invention are novel, and the overlap rate of the three new epitopes with the epitopes protected by existing patents is ≤35%.

[0132] (2) The anti-HNL antibody developed based on the new epitope of the present invention has a sensitivity of ≥90% and a specificity of ≥90% when detecting NHL in the sample, which is superior to the anti-HNL antibody in the prior art.

[0133] (3) The anti-HNL antibody of the present invention can be used to prepare detection reagents, detection plates or kits, and can be applied to various methods for detecting HNL, including but not limited to immunochromatography, chemiluminescence, ELISA, etc. In particular, the colloidal gold immunochromatographic test strip prepared based on the anti-HNL antibody of the present invention is easy to operate during use, requires no sample pretreatment, and is suitable for rapid on-site detection.

[0134] (4) The colloidal gold immunochromatographic test strip of the present invention has a wide range of applications and can be used to distinguish bacterial / viral infection types in various scenarios such as ICU, emergency room, and community hospital.

[0135] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments that do not specify specific conditions are generally performed under conventional conditions or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and weight parts. Experiments in the embodiments or test examples of the present invention that do not specify specific conditions are generally performed under conventional conditions or as recommended by the raw material / commodity manufacturer; reagents whose specific source is not specified are commercially available conventional reagents.

[0136] Example 1: Preparation and identification of HNL novel epitope recombinant antigen

[0137] 1.1 Experimental Materials

[0138] Vectors and strains: pET-28a (containing His tag), pGEX-6P-1 (containing GST tag), Escherichia coli BL21(DE3), Rosetta(DE3), all purchased from Novagen;

[0139] Enzyme reagents: restriction endonucleases BamHI and XhoHI, and T4 DNA ligase were all purchased from Thermo Fisher Scientific.

[0140] Chromatography media: Ni-NTA affinity chromatography column (5 mL), GST affinity chromatography column (5 mL), Superdex 75 gel filtration column, all purchased from GE Healthcare;

[0141] Other reagents: IPTG (isopropyl-β-D-thiogalactoside), Tris-HCl, and NaCl, all of analytical grade, were purchased from Sinopharm Group.

[0142] 1.2 Recombinant Antigen Design and Construction

[0143] Conformational epitope 1 antigen (SEQ ID NO: 2): The epitope fragment gene (5'-CAGGCACAGGACTCCACCTCTGATCTGATCCCAGCTCCACCACTGAGCAAAGTCCCTCTGCAGCAGAACTTCCAGGATAACCAGTTCCAGGGAAAGTGG-3', SEQ ID NO: 26) was fused with the signal peptide gene (pelB) of the pET-28a vector to construct the recombinant vector pET-28a-pelB-epitope 1; nucleotide sequence translation result: Gln-Ala-Gln-Asp-Ser-Thr-Ser-Asp-Leu-Ile-Pro-Ala-Pro-Pro-Leu-Ser-Lys-Val-Pro-Leu-Gln-Gln-Asn-Phe-Gln-Asp-Asn-Gln-Phe-Gln-Gly-Lys-Trp (SEQ ID NO: 27) (Note: This gene sequence contains SEQ ID NO: 26). 2. Complete sequence and partial extended sequence, wherein the translation results of the core epitope region are completely consistent with SEQ ID NO: 2).

[0144] Conformational epitope 2 antigen (SEQ ID NO: 3): The epitope fragment gene (5'-GGCAACGCTATCTTGCGCGAAGATAAAGATCCGCAGAAGATGTATGCTACCATCTATGAACTGAAAGAAGATAAGAGCTATAACGTTACCTCAGTTCTGTTCCGTAAAAAAAAGTGCGATTGCTCGAG-3', SEQ ID NO: 28) was fused with the flexible linker (Gly-Gly-Ser-Gly-Gly) gene and inserted into the pGEX-6P-1 vector to construct the recombinant vector pGEX-6P-1-linker-epitope 2; nucleotide sequence translation results: GST tag (inherent to the vector) → Leu-Val-Pro-Arg-Gly-Ser (PreScission restriction site) → Gly-Gly-Ser-Gly-Gly (flexible Lin) ker) → Gly-Asn-Ala-Ile-Leu-Arg-Glu-Asp-Lys-Asp-Pro-Gln-Lys-Met-Tyr-Ala-Thr-Ile-Tyr-Glu-Leu-Lys-Glu-Asp-Lys-Ser-Tyr-Asn-Val-Thr-Ser-Val-Leu-Phe-Arg-Lys-Lys-Lys-Cys-Asp (SEQ ID NO: 29) (Note: The translation result of the core epitope region is completely consistent with SEQ ID NO: 3 (GNAILREDKDPQKMYATIYELKEDKSYNVTSVLFRKKKCD), and the verification is correct).

[0145] Linear epitope antigen (SEQ ID NO: 4): synthesized by solid-phase synthesis with C-terminal amidation modification, and synthesized by General Bio (Anhui) Co., Ltd.

[0146] 1.3 Recombinant Antigen Expression and Purification

[0147] (1) Transformation and induction: The recombinant vector was transformed into the corresponding competent Escherichia coli cells, plated on LB agar plates containing kanamycin (pET-28a) or ampicillin (pGEX-6P-1), and cultured at 37°C for 12 h; single colonies were picked and inoculated into 5 mL of LB medium, and cultured at 37°C and 220 rpm until OD600 = 0.6-0.8, IPTG was added to a final concentration of 0.5 mM, and expression was induced at 28°C for 6 h;

[0148] (2) Cell disruption: Collect the induced cells, resuspend them in PBS buffer (pH 7.4), add protease inhibitor (1 mM PMSF), homogenize them in a high-pressure homogenizer (pressure 800 bar), centrifuge at 4°C and 12000 rpm for 30 min, and collect the supernatant;

[0149] (3) Affinity chromatography: Conformation epitope 1 antigen: After equilibration of the Ni-NTA column, the sample was loaded, washed with PBS containing 20 mM imidazole, eluted with PBS containing 300 mM imidazole, and the elution peak was collected.

[0150] Conformation epitope 2 antigen: After equilibration with GST affinity column, the sample was loaded, washed with PBS, eluted with Tris-HCl (pH 8.0) containing 10 mM glutathione, and the elution peak was collected.

[0151] (4) Gel filtration purification: The affinity chromatography product was loaded onto a Superdex 75 column, eluted with PBS containing 150 mM NaCl at a flow rate of 0.5 mL / min, the target peak was collected, and the purity was detected by SDS-PAGE.

[0152] 1.4 Antigen Quality Identification

[0153] Purity testing: 12% SDS-PAGE electrophoresis, Coomassie brilliant blue staining, and ImageJ software analysis of band gray values ​​showed that the purities of conformational epitopes 1 and 2 were 96.2% and 95.8%, respectively.

[0154] Conformation verification: Circular dichroism (CD) detection showed that conformation epitope 1 had no obvious α-helix characteristic peaks at 208 nm and 222 nm (consistent with a structure dominated by random coils), while conformation epitope 2 had characteristic absorption peaks at 208 nm and 222 nm (α-helix content of about 32%).

[0155] Immunogenicity verification: ELISA detection showed that the OD450 values ​​of antigens 1, 2, and 3 coated with protein at a concentration of 1 μg / mL and binding to anti-HNL polyclonal antibody (purchased from Abcam) were 1.82 (negative control 0.23), 1.96 (negative control 0.15), and 1.63 (negative control 0.25), respectively, demonstrating good immunogenicity.

[0156] Example 2: Preparation and screening of monoclonal antibodies against novel HNL epitopes

[0157] 2.1 Experimental Materials

[0158] Laboratory animals: 6-8 week old female BALB / c mice, SPF grade;

[0159] Myeloma cells: SP2 / 0 cells, preserved in liquid nitrogen in our laboratory, and cultured in DMEM medium containing 10% fetal bovine serum after resuscitation;

[0160] Reagents: Freund's complete adjuvant, Freund's incomplete adjuvant (purchased from Sigma), HAT medium, HT medium (purchased from Gibco), HRP-labeled goat anti-mouse IgG (purchased from Jackson).

[0161] 2.2 Mouse Immunization

[0162] (1) Immunogen preparation: The antigen purified in Example 1 was emulsified with adjuvant at a ratio of 1:1 (v / v) to ensure complete emulsification (it does not spread when dropped into water).

[0163] (2) Immunization schedule: - Day 0 (first immunization): 5 mice each from the conformational epitope 1 antigen group, conformational epitope 2 antigen group, and linear epitope antigen group were injected intraperitoneally with 50 μg of antigen-Frederick's complete adjuvant emulsion; - Day 14 (second immunization): The same dose of antigen-Frederick's incomplete adjuvant emulsion was injected intraperitoneally; - Day 28 (third immunization): Same as the second immunization schedule; - Day 42 (booster immunization): 30 μg of adjuvant-free antigen was injected intravenously into each mouse.

[0164] (3) Serum titer test: Blood was collected from the orbital cavity on day 45, serum was separated, serially diluted (1:1000 to 1:128000), and the binding activity with the corresponding antigen was detected by ELISA. The serum titer of mouse No. 3 in the conformational epitope 1 antigen group was the highest (1:64000), and it was selected as the fusion mouse.

[0165] In the same preparation experiment, the serum titer of mouse No. 5 in the conformational epitope 2 antigen group was the highest (1:72000), and it was selected as the fusion mouse; the serum titer of mouse No. 2 in the linear epitope 3 antigen group was the highest (1:68000), and it was selected as the fusion mouse.

[0166] 2.3 Cell fusion and hybridoma screening

[0167] 1. Spleen cell preparation: Mice were euthanized by cervical dislocation, and spleens were aseptically harvested, ground, and treated with erythrocyte lysis buffer for 5 min. The spleens were washed three times with PBS, and 1.2 × 10⁻⁶ cells were obtained. 8 One spleen cell;

[0168] 2. Cell fusion: SP2 / 0 cells in logarithmic growth phase (2.4 × 10⁻⁶) were used. 7 Mix the 1000 cells with spleen cells, centrifuge at 1000 rpm for 5 min, and discard the supernatant; slowly add 1 mL of 50% PEG 4000 (preheated at 37℃), gently mix for 1 min, add 10 mL of RPMI-1640 medium to terminate the fusion, centrifuge and resuspend in HAT medium;

[0169] 3. Plate culture: Seed the fusion cell suspension into 96-well cell culture plates, 200 μL per well, and incubate at 37°C in a 5% CO2 incubator. Add HAT medium on day 3 and replace with HT medium on day 7.

[0170] 4. Three-step screening:

[0171] 1) Initial screening (ELISA): On day 10, the culture supernatant was collected, coated with conformational epitope 1 antigen (1 μg / mL), and HRP-labeled goat anti-mouse IgG was used for detection. 42 positive clones with OD450 values ​​≥0.8 were screened out. 2) Specific screening (competitive ELISA): After coating with conformational epitope 1 antigen and adding the supernatant to be screened, 50 μg / mL of HNL 82-102 epitope antigen or 141-156 epitope antigen (purchased from Anhui General Biotechnology) was added. If the binding activity decreased by ≤20%, it was considered qualified, and 15 specific clones were screened out.

[0172] 3) Affinity Screening (Biacore T200): The conformational epitope 1 antigen is immobilized on a CM5 chip (approximately 1000 RU). The supernatant of the antibody to be screened is injected, the binding and dissociation curves are detected, the KD value is calculated, and 3 strains with KD ≤ 1 × 10⁻⁶ are screened. -8 The high-affinity clones of M (numbered 1A3, 2B5, and 3C7) were selected for the next step of the experiment.

[0173] Screening for positive clones targeting conformational epitope 2 antigen and linear epitope 3 was the same as for conformational epitope 1 antigen. Three clones with a KD ≤ 1 × 10⁻⁶ were screened using conformational epitope 2 antigen. -8 High-affinity clones of M (numbered 1B9, 2C5, and 3D2) were selected, with 1B9 chosen for the next stage of experiments; three clones with KD ≤ 1 × 10⁻³ were screened from linear epitope 3. -8 The high-affinity clones of M (numbered 1A11, 2C10, and 3D2) were selected for the next stage of experiments. The screening results for positive clones of the three epitopes are shown in the table below:

[0174] .

[0175] 2.4 Hybridoma Cloning and Monoclonal Antibody Preparation

[0176] 1. Limiting dilution cloning: The selected high-affinity clones 1A3, 1B9, and 1A11 were diluted with HT medium to 1 cell / 100μL, inoculated into 96-well plates, and single colonies were picked after 10 days of culture. This process was repeated 3 times to ensure monoclonal origin.

[0177] 2. Ascites preparation: Each mouse was injected intraperitoneally with 0.5 mL of liquid paraffin, followed by an injection of 5 × 10⁻⁶ mol / L paraffin 7 days later. 6One hybridoma cell was collected, and ascites fluid was collected after 10-14 days. 3-5 mL of fluid could be collected from each mouse.

[0178] 3. Antibody purification: Ascites fluid was purified using a Protein G affinity chromatography column. The loading buffer was PBS at pH 7.0, and the elution buffer was 0.1M citrate buffer at pH 2.7. After elution, the solution was immediately neutralized to pH 7.0 with 1M Tris-HCl (pH 9.0). The solution was then dialyzed to PBS. Protein concentrations were determined using the BCA method, and the concentrations of 1A3, 1B9, and 1A11 were found to be 8.2 mg / mL, 6.9 mg / mL, and 9.3 mg / mL, respectively.

[0179] Example 3: Performance Identification and Diagnostic Application Validation of Monoclonal Antibodies

[0180] 3.1 Identification of basic characteristics of monoclonal antibodies

[0181] Subtype identification: Mouse monoclonal antibody subtype identification kit (purchased from Thermo) was used. Subtype 1A3 was IgG1, subtype 1B9 was IgG2a, and subtype 1A11 was IgG2a.

[0182] CDR sequence determination: Total RNA was extracted from 1A3 hybridoma cells, reverse transcribed into cDNA, and the heavy and light chain variable region genes were amplified and sequenced to obtain the CDR sequence (see the CDR1, 2, and 3 sequences of the three monoclonal antibodies for details).

[0183] Affinity retest: The KD value of 1A3 monoclonal antibody and HNL detected by Biacore T200 was 3.2 × 10⁻⁶. -9 M, binding rate constant ka = 2.1 × 10 5 M -1 s -1 The dissociation rate constant kd = 6.7 × 10 -4 s -1 The KD value of 1B9 monoclonal antibody and HNL detected by Biacore T200 was 2.3 × 10⁻⁶. -9 M, binding rate constant ka = 2.9 × 10 5 M -1 s -1 The dissociation rate constant kd = 6.1 × 10 -4 s -1 The KD value of 1A11 monoclonal antibody and HNL detected by Biacore T200 was 5.6 × 10⁻⁶. -9 M, binding rate constant ka = 1.9 × 10 5 M -1 s -1 The dissociation rate constant kd = 7.3 × 10 -4 s -1 .

[0184] 3.2 Specificity and Clinical Performance Validation

[0185] 1. Cross-reactivity validation: The cross-reactivity rates of monoclonal antibodies 1A3, 1B9, and 1A11 with HNL homologous proteins (NGAL 24kD monomer, purchased from novoprotein) were less than 5%, and the cross-reactivity rates with common plasma proteins (APOE, albumin) were all ≤3%. The cross-reactivity rates with HNL epitopes at positions 82-102 and 141-156 were 4.2% and 3.8% (1A3), 3.6% and 2.8% (1B9), and 3.9% and 4.3% (1A11), respectively, meeting the requirements (see [link to relevant documentation]). Figure 1 , Figure 1 The diagram illustrates, for example, the specificity validation results of the 1A3 monoclonal antibody binding to the HNL target antigen and other target assays.

[0186] 2. Clinical Sample Testing: Eighty clinical samples were collected (30 cases of bacterial infection, 30 cases of viral infection, and 20 healthy controls, all from a key laboratory of a tertiary hospital in Shanxi Province). An ELISA detection system was constructed using the 1A3 monoclonal antibody.

[0187] 1) Coating: Dilute 1A3 monoclonal antibody to 2 μg / mL with coating buffer, 100 μL per well, and coat overnight at 4°C;

[0188] 2) Sealing: Sealing with 5% skim milk at 37℃ for 1 hour;

[0189] 3) Sample incubation: Dilute the sample 200-fold, 100 μL per well, and incubate at 37°C for 1 hour;

[0190] 4) Secondary antibody incubation: HRP-labeled goat anti-HNL polyclonal antibody (1:5000 dilution), incubated at 37℃ for 30 min;

[0191] 5) Color development and reading: Develop color with TMB for 15 min, terminate with 2M H2SO4, and read the OD value at a wavelength of 450nm;

[0192] 3. Results Analysis: Using the mean OD value of healthy controls + 3SD as the threshold, the positive rate of bacterial infection samples was 93.3% (28 / 30), and the negative rate of viral infection samples was 90% (29 / 30). The sensitivity was 93.3%, and the specificity was 96.67%. See details... Figure 2 .

[0193] 3.3 Stability Validation of Antibody Kit

[0194] The ELISA diagnostic kit containing the 1A3 monoclonal antibody was stored at 4℃, 25℃, and 37℃, and antibody activity was measured at 0 days, 3 months, 6 months, and 18 months.

[0195] - Antibody activity retention rate is 95% after 18 months of storage at 4℃;

[0196] - Antibody activity retention rate is 88% after 6 months of storage at 25℃;

[0197] - When stored at 37℃ for 3 months, the antibody activity retention rate is 85%, which meets the needs of clinical use.

[0198] The experiment was repeated as above to verify that the antibody activity retention rates of 1B9 and 1A11 monoclonal antibodies were 96% and 93% respectively after being stored at 4°C for 18 months.

[0199] Example 4: Validation of antibody-binding epitopes

[0200] Using a competitive binding assay, the 1A3 monoclonal antibody was co-incubated with the MAB1 monoclonal antibody (purchased from the patent licensor) disclosed in patent CN107001455B to incubate the HNL protein:

[0201] - When the concentration of 1A3 monoclonal antibody was 10 μg / mL, the inhibition rate of MAB1 binding was 8.3%;

[0202] - When the MAB1 concentration was 10 μg / mL, the inhibition rate of 1A3 binding was 7.6%;

[0203] This demonstrates that the binding interfaces of the two do not significantly overlap, and that the binding sites of the 1A3 monoclonal antibody and the patented antibody are not the same.

[0204] The experiment was repeated to verify that the inhibition rates of 1B9 and 1A11 monoclonal antibodies against MAB1 binding were 5.8% and 6.3%, respectively.

[0205] Example 5: Evaluation of antigen-antibody binding analysis

[0206] Epitope prediction: Homology modeling was used to construct the three-dimensional structures of antigen and antibody. Molecular docking technology was used to analyze the interaction sites between the antibody complementarity-determining region (CDR) and the antigen. The epitope range was determined by sequence conservation analysis.

[0207] Energy and affinity parameter calculation: The binding free energy (ΔG) of the antibody-antigen complex was calculated using molecular mechanics methods, and the affinity (expressed as the dissociation constant KD) was predicted using surface plasmon resonance (SPR) simulation data. The lower the ΔG value and the smaller the KD value, the stronger the binding ability.

[0208] Comparison table of antibody-heterotope pairing core performance

[0209]

[0210] The binding epitopes of the three antibody groups are non-overlapping on the HNL antigen, covering three key regions: the N-terminal, mid-terminal, and C-terminal. Specifically, the N-terminal epitope (22-72 aa) bound by the 1A3 antibody is a conserved functional region of HNL; the mid-terminal epitope (78-125 aa) bound by the 1B9 antibody is associated with the lipid-binding activity of HNL; and the C-terminal epitope (156-183 aa) bound by the 1A11 antibody is a highly exposed region of the antigen. This epitope distribution characteristic allows the 1A3 and 1B9 antibody pairs, and the 1A3 and 1A11 antibody pairs, to form dual-antibody sandwich detection systems, effectively avoiding epitope competition.

[0211] Combined with the free energy (ΔG) results, the 1A3 antibody pair had the lowest ΔG value (-18.6 kcal / mol), mainly due to the multiple hydrophobic interactions and salt bridges formed between the "CVCCFNWPYQFR" sequence in its CDR3 region and the antigenic epitope. The 1A11 antibody pair had a higher ΔG value (-16.8 kcal / mol) than 1B9, which is related to the higher content of aromatic amino acids (tyrosine and phenylalanine) in its CDR region. In terms of affinity, the 1A3 antibody pair reached the level of high-affinity antibodies with a KD value (0.82 nM), followed by 1A11 (1.33 nM), while 1B9 was relatively lower (2.15 nM), but all met the clinical testing requirements.

[0212] Example 6: Preparation and Performance Verification Experiment of Colloidal Gold Immunochromatographic Test Strips

[0213] 6.1 Colloidal gold immunochromatographic test strip for detecting HNL and its preparation method

[0214] 1. See reference Figure 3 This is a schematic diagram of the structure of the colloidal gold immunochromatographic test strip for detecting HNL according to the present invention. The colloidal gold immunochromatographic test strip for detecting HNL in this embodiment includes a PVC backing plate, a sample pad, a gold-labeled pad, a detection line, a control line, a nitrocellulose membrane, and an absorbent pad. The sample pad, gold-labeled pad, detection line, control line, nitrocellulose membrane, and absorbent pad are all disposed above the PVC backing plate. The sample pad is disposed at the edge of the PVC backing plate, with the gold-labeled pad partially overlapping the sample pad. The gold-labeled pad and absorbent pad partially overlap with both ends of the nitrocellulose membrane. A detection line is sprayed onto the nitrocellulose membrane near the gold-labeled pad, and a control line is sprayed onto the end near the absorbent pad. The sample pad is a glass fiber membrane, and the gold-labeled pad is a polyester membrane. The gold-labeled pad is coated with a mixture of colloidal gold-labeled neutrophil apolipoprotein monoclonal antibody II (1A3 monoclonal antibody) and mouse IgG antibody. The detection line is coated with neutrophil apolipoprotein monoclonal antibody I (1B9 monoclonal antibody). The control line is coated with goat anti-mouse polyclonal antibody.

[0215] 2. In this embodiment, the amount of colloidal gold-labeled neutrophil apolipoprotein monoclonal antibody II (1A3 monoclonal antibody) and colloidal gold-labeled mouse IgG antibody coated on the gold-labeled pad is 4.5 μL / cm. The diameter of the colloidal gold particles on the gold-labeled pad is approximately 30 nm. The detection line is located near the gold-labeled pad, and the control line is located near the absorbent pad. The amount of neutrophil apolipoprotein monoclonal antibody I (1B9 monoclonal antibody) coated on the detection line is 0.8 μL / cm. The amount of goat anti-mouse polyclonal antibody coated on the control line is 0.8 μL / cm.

[0216] 3. The preparation method of the colloidal gold immunochromatographic test strip for detecting HNL in this embodiment includes the following steps:

[0217] (1) Preparation of colloidal gold-antibody complex

[0218] The colloidal gold solution was adjusted to pH using 0.1M K₂CO₃. Neutrophil apolipoprotein monoclonal antibody II was diluted with 1×PB (pH 7.4). The neutrophil apolipoprotein monoclonal antibody II was mixed with the colloidal gold solution in a specific ratio, and after shaking for 10 min, 10% (by volume) BSA solution was added, and shaking continued for another 20 min. After centrifugation at 2000 rpm for 17 min at 4℃, the precipitate was discarded. The mixture was then centrifuged again at 11000 rpm for 45 min at 4℃, and the supernatant was discarded. The resulting precipitate was resuspended in resuspension buffer, and the OD value was measured. Mouse IgG antibody was labeled using the same procedure as for colloidal gold-labeled neutrophil apolipoprotein monoclonal antibody II. The colloidal gold solution was adjusted to pH using 0.1M K₂CO₃. The mouse IgG antibody was mixed with the colloidal gold solution in a specific ratio, and the precipitate was resuspended in resuspension buffer, and the OD value was measured. The colloidal gold-antibody complex was then prepared by thoroughly mixing the colloidal gold-labeled neutrophil apolipoprotein monoclonal antibody II and the mouse IgG antibody. The resuspension formulation is: 1×PB (10% sucrose, 1% BSA, 0.1% Tritonx-100).

[0219] (2) Preparation of gold-labeled pads

[0220] The polyester membrane used to prepare the gold-labeled pads was immersed in buffer solution for 2 hours, then removed and dried at 37°C for 2 hours. The colloidal gold-antibody complex was then evenly spread onto the pretreated gold-labeled pads using a gold sprayer, with a spraying volume controlled at 4.5 μL / cm. The pads were dried at 37°C for 2 hours and then sealed in aluminum foil bags for storage. The buffer solution consisted of: 10 mM Na₂HPO₄•12H₂O, 3% BSA, 0.1% Tritonx-100, and 0.01% Proclin 300.

[0221] (3) Preparation of sample pad

[0222] The glass fiber membrane was immersed in the buffer solution for 5 hours, then removed and dried overnight at 37°C. It was then sealed in an aluminum foil bag and stored in a dry place. The buffer solution formulation was: 20 mM Na₂B₄O₇·10H₂O, 1% BSA, 0.8% NaCl, 0.05% SDS-L, and 0.01% Proclin 300.

[0223] (4) Processing of testing lines and quality control lines

[0224] The test line was prepared by diluting neutrophil apolipoprotein monoclonal antibody I with coating buffer, streaking 0.8 μL / cm onto a nitrocellulose membrane, and then drying at 37°C for 12 hours. The control line was prepared by diluting goat anti-mouse polyclonal antibody with coating buffer, streaking 0.8 μL / cm onto a nitrocellulose membrane parallel to the test line, and then drying at 37°C for 12 hours. The coating buffer formulation was 20 mM Na₂HPO₄•12H₂O.

[0225] (5) Assembly of test strips

[0226] Attach the absorbent paper to the right side of the PVC base plate, the sample pad to the left side of the base plate, and the gold label pad to the junction of the sample pad and the coating film. Then cut the strips and assemble them.

[0227] (6) Reagent kit assembly

[0228] The prepared colloidal gold immunochromatographic test strips (25 or 50 tests per box) are assembled into a kit with sample dilution buffer (50 mmol / L Tris-HCl buffer, pH 7.8, 25 mL / bottle, 1 bottle / box or 2 bottles / box) and ID card (1 card / box, with record).

[0229] The kit should be stored sealed at 2-30℃ and has a shelf life of 18 months. The sample diluent has a shelf life of 30 days after opening.

[0230] 6.2 Establishment of the standard curve for colloidal gold immunochromatographic test strips

[0231] 1. Dissolving the standard dry powder

[0232] After removing the product from 4℃, allow it to equilibrate to room temperature for 15 minutes. Carefully open the bottle cap and add pure water to reconstitute the powder according to the device specifications indicated on the bottle. Let it stand at room temperature for 5 minutes, then gently shake the bottle to fully dissolve the dry powder. Once the lyophilized powder is completely dissolved, it is ready for use. Store the sample at 4℃ immediately after sampling.

[0233] 2. Testing Methods

[0234] (1) Dilution of standard products

[0235] The dissolved standard was serially diluted with standard diluent to 20, 40, 80, 160, 320, and 640 times. Standard diluent: 50 mM Tris-base, 0.15 M NaCl, 0.1% Proclin-300, 1.5% BSA. The pH was adjusted with hydrochloric acid.

[0236] (2) Spot testing

[0237] Add 100 μl of the sample to be tested to the well. After 15 minutes, insert the test strip into the colloidal gold test strip quantitative analyzer (Suzhou Hemai GIC-M6) for testing and save the experimental data.

[0238] (3) Results processing

[0239] A standard curve is obtained by plotting the log value of the standard concentration on the x-axis and the log value of the T / C value (detected value / quality control value) on the y-axis.

[0240] 6.3 Performance Testing of Colloidal Gold Immunochromatographic Test Strips

[0241] 1. Testing steps

[0242] (1) Before the experiment, remove the sample to be tested and the test reagent from the storage conditions and allow them to equilibrate to room temperature;

[0243] (2) Dilute the sample to be tested 200 times with the sample diluent and mix thoroughly;

[0244] (3) Open the aluminum foil bag packaging, take out the test card (i.e., colloidal gold immunochromatographic test strip), and place it horizontally on the experimental table;

[0245] (4) Confirm that the batch numbers of the ID card and the test card match;

[0246] (5) Turn on the instrument, insert the ID card, and check whether the test items, reagent information and reagents used are consistent;

[0247] (6) Take 100 μL of the sample to be tested diluted 1:200 and add it into the sample well of the test card;

[0248] (7) React at room temperature for 15 minutes, insert the test card into the slot of the applicable instrument for testing, and the instrument will automatically calculate the concentration of neutrophil apolipoprotein (HNL) in the sample through the built-in calibration curve and sample volume, and read and print the test results.

[0249] The normal reference value is ≤130ng / mL. When the measured concentration is less than 130ng / mL, the sample is negative. When the measured concentration is greater than or equal to 130ng / mL, the sample is positive.

[0250] 2. Negative reference sample compliance rate

[0251] (1) Test methods

[0252] Five samples were taken from each batch of the colloidal gold immunochromatographic test strips of the present invention, and five negative reference samples were tested from each batch. Each negative reference sample was tested once, and the test results were obtained after 15 minutes. The negative reference sample compliance rate of each batch of colloidal gold immunochromatographic test strips was calculated.

[0253] (2) Results

[0254] The results are shown in Table 1. The results show that the negative reference compliance rate of each batch of colloidal gold immunochromatographic test strips is 100%.

[0255] Table 1. Results of the negative reference sample compliance test

[0256]

[0257] 3. Conformity rate of positive reference materials

[0258] (1) Test methods

[0259] Five samples were taken from each batch of the colloidal gold immunochromatographic test strips of the present invention, and positive reference samples were tested. Each positive reference sample was tested once to ensure the accuracy of the results. The test results were obtained after 15 minutes. The test results of each batch of reference samples were compared with the normal reference values ​​to calculate the positive reference sample compliance rate of each colloidal gold immunochromatographic test strip.

[0260] (2) Results

[0261] The results are shown in Table 2. The results show that the positive reference compliance rate of each batch of colloidal gold immunochromatographic test strips is 100%.

[0262] Table 2 Results of Positive Reference Sample Compliance Test

[0263]

[0264] 4. Minimum detection limit

[0265] (1) Validation of the lowest detection limit repeat test method

[0266] Ten samples were taken from each batch of the colloidal gold immunochromatographic test strip of the present invention, and the limit of detection was tested. The results were obtained after 15 minutes.

[0267] (2) Results

[0268] The results are shown in Table 3. The limit of detection of the colloidal gold immunochromatographic test strip of the present invention is approximately 28 ng / mL.

[0269] Table 3 Results of the Lowest Detection Limit Test

[0270]

[0271] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-neutrophil apolipoprotein (HNL) antibody or its antigen-binding fragment, characterized in that, The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the heavy chain variable region and the light chain variable region comprise the following CDRs: VH-CDR1 shown in SEQ ID NO: 11, VH-CDR2 shown in SEQ ID NO: 12, VH-CDR3 shown in SEQ ID NO: 13, VL-CDR1 shown in SEQ ID NO: 14, VL-CDR2 with the amino acid sequence WNI, and VL-CDR3 shown in SEQ ID NO:

15.

2. The antibody or its antigen-binding fragment as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the antibody is as shown in SEQ ID NO: 5, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO: 5; and the amino acid sequence of the light chain variable region of the antibody is as shown in SEQ ID NO: 6, or has at least 85% sequence identity with the amino acid sequence shown in SEQ ID NO:

6.

3. The antibody or its antigen-binding fragment as described in claim 1 or 2, characterized in that, The antibody has the following characteristics: (1) It can specifically bind to the conformational epitope of the amino acid sequence as shown in SEQ ID NO: 2, and the OD450 value of the binding reaction is ≥0.8; (2) The dissociation constant of HNL is KD ≤ 1 × 10 -8 M; and (3) It does not cross-react with epitopes at positions 82-102 and 141-156 of HNL, and the cross-reaction rate is ≤5%.

4. A polynucleotide molecule encoding an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3.

5. An expression vector containing the polynucleotide molecule as described in claim 4.

6. A host cell containing the expression vector as described in claim 5, or having the polynucleotide molecule as described in claim 4 integrated into its genome.

7. An antibody conjugate, said antibody conjugate comprising: (a) The antibody or antigen-binding fragment thereof as claimed in any one of claims 1-3; and (b) The coupling motif selected from the following groups: detectable markers, enzymes that can produce detectable products, radionuclides, nanoparticles / nanorobars.

8. Use of the antibody or antigen-binding fragment thereof as described in any one of claims 1-3, or the antibody conjugate as described in claim 7, in the preparation of a detection reagent or kit for detecting HNL.

9. A detection reagent, characterized in that, The detection reagent comprises an antibody or antigen-binding fragment thereof as described in any one of claims 1-3, or an antibody conjugate as described in claim 7, and a detection-acceptable carrier.

10. A colloidal gold immunochromatographic test strip, characterized in that, The colloidal gold immunochromatographic test strip is coated with an antibody or antigen-binding fragment thereof as described in any one of claims 1-3.

11. The colloidal gold immunochromatographic test strip as described in claim 10, characterized in that, The colloidal gold immunochromatographic test strip comprises: a PVC base plate, a sample pad, a gold-labeled pad, a detection line, a control line, a nitrocellulose membrane, and an absorbent pad; wherein, the gold-labeled pad is coated with a mixture of colloidal gold-labeled anti-HNL antibody II and mouse IgG antibody, the detection line is coated with anti-HNL antibody I, and antibody II and antibody I are selected from the antibodies or their antigen-binding fragments as described in any one of claims 1-3, and antibody II and antibody I are different antibodies.

12. A kit comprising the detection reagent as described in claim 9, and / or the colloidal gold immunochromatographic test strip as described in claim 10 or 11, and instructions for use.

13. A method for preparing an antibody or antigen-binding fragment thereof as described in any one of claims 1-3, the method comprising: (i) Under conditions suitable for antibody production, the host cells as described in claim 6 are cultured to obtain a culture containing the anti-HNL antibody or its antigen-binding fragment; (ii) Isolating or recovering the antibody or its antigen-binding fragment from the culture; and (iii) Purification and / or modification of the antibody or its antigen-binding fragment obtained in step (ii).

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