CN118027181A - Hexon protein antibodies and uses thereof - Google Patents
Hexon protein antibodies and uses thereof Download PDFInfo
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- CN118027181A CN118027181A CN202311774540.0A CN202311774540A CN118027181A CN 118027181 A CN118027181 A CN 118027181A CN 202311774540 A CN202311774540 A CN 202311774540A CN 118027181 A CN118027181 A CN 118027181A
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/081—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from DNA viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
- C07K2317/565—Complementarity determining region [CDR]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/01—DNA viruses
- G01N2333/075—Adenoviridae
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Biomedical Technology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Physics & Mathematics (AREA)
- Organic Chemistry (AREA)
- Pathology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Food Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biophysics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to a novel anti-adenovirus (AdV) hexon protein monoclonal antibody, a kit comprising the antibody and related applications thereof. The anti-adenovirus hexon protein monoclonal antibody can specifically bind with hexon proteins of various adenoviruses, shows high sensitivity and high specificity for adenoviruses, can be used for detecting various adenoviruses, greatly improves the detection rate of adenoviruses, reduces the detection cost, and provides a feasible scheme for rapid detection of adenoviruses of respiratory tracts and digestive tracts.
Description
The invention patent application is a divisional application of the invention patent application with the application number of CN202310709351.9 and the invention name of 'anti-adenovirus monoclonal antibody and application' which are filed on the 14 th month 6 of 2023.
Technical Field
The invention relates to the field of biological medicine, in particular to an anti-adenovirus (AdV) hexon protein monoclonal antibody, a kit comprising the antibody and related applications.
Background
Adenovirus particles are in a capsule-free spherical structure with a diameter of 70-90 nm, and each virus particle contains 36kb of linear double-stranded DNA. The adenovirus capsid is in icosahedral symmetry and consists of 252 capsomers with diameters of 8-10 nm, wherein the icosahedral apex capsomers are 12 pentons (penton), and 240 non-apex capsomers are arranged outside the pentons, which are called hexons (Hexon). Hexon carries major genus and subgenera specific antigenic determinants and minor species specific antigenic determinants and can serve as criteria for diagnosis of different serotypes.
Adenovirus is widely distributed in nature, 7 groups of A-G adenovirus capable of infecting human are available, 55 different serotypes are currently known, the most common pathotypes are types 1-8, and the 55 adenovirus is a novel virus recombinantly produced by human type 11 adenovirus and 14 adenovirus.
Adenovirus is mainly transmitted through air droplets, and most adenovirus types can also be transmitted through the digestive tract. Various diseases can be caused after adenovirus infects human beings, such as adenovirus acute upper respiratory tract infection, adenovirus pneumonia, fulminant conjunctivitis, acute hemorrhagic cystitis, infant gastroenteritis, etc. Adenovirus pneumonia accounts for about 10% of childhood pneumonia, and is common to infants from 6 months to 2 years old, and the illness is serious, and is one of the important causes of death and disability of infant pneumonia at present, wherein types 3 and 7 are the main types of infant adenovirus pneumonia, and type 55 is the most common type causing adult adenovirus community pneumonia.
At present, the diagnosis of adenovirus infection mainly comprises the following steps: virus isolation and serological identification, virus nucleic acid detection and antigen detection. The traditional virus separation and serotype method is a gold standard for diagnosing adenovirus, but is not suitable for clinical early diagnosis, and has high sensitivity for detecting virus nucleic acid, high diagnosis cost and strict experimental condition. Aiming at antigen detection of adenovirus capsid hexon, nasopharyngeal swab, sputum or feces are taken as specimens, and the antigen can be detected with high efficiency within 3-5 days of onset.
The region of China is wide, and adenovirus respiratory tract infection can occur all the year round. The types of adenovirus infection present a complex and differentiated epidemic profile in time and space. Adenovirus dominant strains in different areas are different in types and time, and are common in winter and spring in north of China, and more common in spring and summer in south of China. Dominant epidemic strains in northern, northwest and southern areas belong to the HAdV-B group and are mainly type 3 and type 7; while dominant epidemic strains in eastern region belong to the HAdV-C group, mainly type 1 and type 2; meanwhile, in recent years, the novel adenovirus 55 type infection is locally generated in the south and the north of China. Enteroadenoviruses, mainly type 40 and type 41 of subgroup F, are second only to rotaviruses, causing the second most cause of diarrhea in children.
Differentiation of adenovirus types in regions and time brings inconvenience to rapid diagnosis of adenovirus infection. At present, one antibody can only detect adenovirus of one type, so that a plurality of antibodies are needed to improve the antibody detection rate when detecting throat swab samples or fecal samples, the detection cost is improved, and certain false negative probability still exists.
Therefore, there is a need for an adenovirus detection product and method that can detect multiple types simultaneously, with high specificity and high sensitivity.
Disclosure of Invention
As previously mentioned, there is a need in the art for an anti-adenovirus antibody that has high specificity and high sensitivity and is suitable for use with multiple types of adenoviruses.
Considering that the Hexon proteins of different types of adenoviruses have high conservation (78% -95%), the type-to-tissue specific epitope is positioned in the conservation areas and possibly has certain antigenicity, the inventor takes the Hexon protein as a target site for diagnosing adenovirus infection antigen, uses the 3-type adenovirus (HAdV 3) Hexon protein (Hexon) (the amino acid sequence is SEQ ID NO: 62) to immunize a mouse, takes spleen cells of the mouse to fuse with myeloma cells, screens out hybridoma cell strains which are specifically combined with the Hexon proteins of various types of adenoviruses by an ELISA method, constructs a recombinant antibody, and obtains the recombinant anti-adenovirus antibody of the Hexon protein of the targeted adenovirus. Thus, the present invention has been achieved.
Accordingly, in a first aspect, the present invention provides an anti-adenovirus monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein,
A) The heavy chain variable region comprises a heavy chain complementarity determining region V H CDR1、VH CDR2 and a V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 1-3, respectively, and the light chain variable region comprises a light chain complementarity determining region V L CDR1、VL CDR2 and a V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 4-6, respectively;
b) The heavy chain variable region comprises a heavy chain complementarity determining regions V H CDR1、VH CDR2 and V H CDR3, the amino acid sequences of which are shown by SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 8, respectively, and the light chain variable region comprises a light chain complementarity determining regions V L CDR1、VL CDR2 and V L CDR3, the amino acid sequences of which are shown by SEQ ID NO. 9, SEQ ID NO. 5 and SEQ ID NO. 10, respectively;
c) The heavy chain variable region comprises a heavy chain complementarity determining regions V H CDR1、VH CDR2 and V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 8, respectively, and the light chain variable region comprises a light chain complementarity determining regions V L CDR1、VL CDR2 and V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 9, SEQ ID NO. 11 and SEQ ID NO. 6, respectively;
d) The heavy chain variable region comprises a heavy chain complementarity determining region V H CDR1、VH CDR2 and a V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 12-14, respectively, and the light chain variable region comprises a light chain complementarity determining region V L CDR1、VL CDR2 and a V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 15-17, respectively; or alternatively
E) The heavy chain variable region comprises the heavy chain complementarity determining regions V H CDR1、VH CDR2 and V H CDR3, shown in SEQ ID NOS: 18-20, respectively, and the light chain variable region comprises the light chain complementarity determining regions V L CDR1、VL CDR2 and V L CDR3, shown in SEQ ID NOS: 15, 16 and 21, respectively.
In a second aspect, there is provided a nucleic acid molecule encoding an anti-adenovirus monoclonal antibody according to the first aspect.
In a third aspect, there is provided a vector comprising the nucleic acid molecule of the second aspect.
In a fourth aspect, there is provided an expression cell comprising a nucleic acid molecule according to the second aspect or a vector according to the third aspect.
In a fifth aspect, there is provided a method of detecting an adenovirus for non-diagnostic or diagnostic purposes comprising the step of using an anti-adenovirus monoclonal antibody according to the first aspect.
In a sixth aspect, there is provided the use of an anti-adenovirus monoclonal antibody according to the first aspect in the preparation of a reagent for detection of adenovirus.
In a seventh aspect, there is provided a kit for detecting an adenovirus comprising an anti-adenovirus monoclonal antibody according to the first aspect and instructions for use.
In summary, the invention provides a novel anti-adenovirus hexon protein monoclonal antibody which can specifically bind to hexon proteins of various types of adenoviruses including type 1, type 2, type 3, type 4, type 7, type 40, type 41 and type 55, thereby being used for detecting various types of adenoviruses, showing high sensitivity and high specificity for adenoviruses, being capable of realizing more sensitive detection such as picogram concentration, greatly improving the detection rate of adenoviruses, reducing the detection cost and providing a feasible scheme for rapid detection of adenoviruses of respiratory tracts and digestive tracts.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows the results of the binding reaction of monoclonal antibody A18 of the present invention with hexon proteins of eight types of adenoviruses and with the Np protein of the novel coronavirus (control).
FIG. 2 shows the results of the binding reaction of monoclonal recombinant antibody A18 of the present invention with hexon proteins of eight types of adenoviruses and with the Np protein of the novel coronavirus (control).
FIG. 3 shows graphs of the binding reaction of monoclonal antibodies A54, A15, A5, A7, A13, A1, A27 and A8 prepared according to one embodiment of the invention with hexon proteins of eight types of adenoviruses and with novel coronavirus Np proteins (controls).
Detailed Description
Hereinafter, the present invention will be described in detail with reference to the accompanying drawings. It is to be understood that the following description is intended to illustrate the invention by way of example only, and is not intended to limit the scope of the invention as defined by the appended claims. And, it is understood by those skilled in the art that modifications may be made to the technical scheme of the present invention without departing from the spirit and gist of the present invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter described herein belongs. Before describing the present invention in detail, the following definitions are provided to better understand the present invention.
Where a range of values is provided, such as a range of concentrations, a range of percentages, or a range of ratios, it is to be understood that each intervening value, to the tenth of the unit of the lower limit, between the upper and lower limit of the range, and any other stated or intervening value in that stated range, is encompassed within the subject matter unless the context clearly dictates otherwise. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and such embodiments are also included in the subject matter, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the subject matter.
In the context of the present invention, many embodiments use the expression "comprising", "including" or "consisting essentially/mainly of … …". The terms "comprises," "comprising," or "consists essentially of … …" are generally understood to be open ended terms that include not only the individual elements, components, assemblies, method steps, etc., specifically listed thereafter, but also other elements, components, assemblies, method steps. In addition, the expression "comprising," "including," or "consisting essentially of … …" is also to be understood in this document as a closed-form expression, in certain instances, to mean that only the elements, components, assemblies, method steps specifically listed thereafter are included, and that no other elements, components, assemblies, method steps are included. At this time, the expression is equivalent to the expression "consisting of … …".
For a better understanding of the present teachings and without limiting the scope of the present teachings, all numbers expressing quantities, percentages or proportions used in the specification and claims, and other numerical values, are to be understood as being modified in all instances by the term "about" unless otherwise indicated. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
As used herein, the term "antibody" refers to an immunoglobulin molecule that is typically composed of two pairs of polypeptide chains, each pair having one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as kappa and lambda light chains. Heavy chains can be classified as μ, δ, γ, α or ε, and the isotypes of antibodies can be defined accordingly as IgM, igD, igG, igA and IgE, respectively. Within the light and heavy chains, the variable and constant regions are linked by a "J" region (hinge region) of about 12 or more amino acids, and the heavy chain also comprises a "D" region of about 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H) and a heavy chain constant region (C H). The heavy chain constant region consists of 3 domains (C H1、CH2 and C H3). Each light chain consists of a light chain variable region (V L) and a light chain constant region (C L). The light chain constant region consists of one domain C L. The constant region of an antibody may mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (C1 q). The V H and V L regions can also be subdivided into regions of high denaturation, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). For each heavy or light chain, its variable region comprises three CDRs, CDR1, CDR2 and CDR3, respectively. Thus, each of V H and V L is defined by the following sequence: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 consist of 3 CDRs and 4 FRs arranged from amino-terminus to carboxy-terminus. The variable regions (V H and V L) of each heavy/light chain pair form antigen binding sites, respectively.
The rules of allocation of amino acids to regions or domains are defined in a number of documents ：Kabat Sequences of Proteins of Immunological Interest(National Institutes of Health,Bethesda M.d.(1987and 1991));Chothia&Lesk J.Mol.Biol.1987;196:901-917;Chothia et al.,Nature 1989;342:878-883;Ehrenmann,Francois,Quentin Kaas,and Marie-PauleLefranc."IMGT/3Dstructure-DB and IMGT/DomainGapAlign:a database and a tool for immunoglobulins or antibodies,T cell receptors,MHC,IgSF and MhcSF."Nucleic acids research 2009;38(suppl_1):D301-D307.
The exact boundaries of CDRs have been defined differently from system to system, and the Kabat system provides not only a clear residue numbering system applicable to any variable region of an antibody, but also precise residue boundaries defining 3 CDRs, referred to as Kabat CDRs; chothia found that some subfractions within the CDRs of the Kabat system, which are termed Chothia CDRs with boundaries overlapping the Kabat CDRs, have almost identical peptide backbone conformations, despite great diversity at the amino acid sequence level. The overlapping boundaries are again described by Padlan and MacCallum, and CDR boundary definitions may not strictly adhere to the above system, such as the AbM definition. In this context, the CDRs may be defined according to any of these systems, although the preferred embodiment uses the antibody numbering system of Chothia et al.
As used herein, the term "mab" or "monoclonal antibody" refers to an antibody or a fragment of an antibody from a population of highly homologous antibody molecules, i.e., a population of identical antibody molecules except for natural mutations that may occur spontaneously. The antibody molecule may be an immunoglobulin, whether it be a natural immunoglobulin or an immunoglobulin obtained partially or wholly by synthetic means. The antibody molecules may also include all polypeptides or proteins having antibody domains, antibody fragments having antibody domains are molecules such as Fab, scFv, fv, dAb, fd, and bifunctional antibodies. Monoclonal antibodies have a high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, which typically comprise at least 2 or more different antibodies, which typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtainable by the hybridoma technique first reported by Kohler et alG,Milstein C.Continuous cultures of fused cells secreting antibody of predefined specificity[J].nature,1975;256(5517):495), But can also be obtained using recombinant DNA techniques (see, for example, U.S. patent 4,816,567). As used herein, the terms "monoclonal antibody" and "mab" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "polyclonal antibody" have the same meaning and are used interchangeably; the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. And in the present invention, amino acids are generally indicated by single-letter and three-letter abbreviations well known in the art. For example, alanine can be represented by A or Ala.
As used herein, the term "recombinant antibody" refers to an antibody that is expressed by cloning an antibody gene into an expression vector by molecular biological techniques and then transfecting the expression vector into a suitable host cell line. The recombinant antibody-encoding gene may or may not be identical to the naturally derived antibody-encoding gene. For example, the whole coding gene of an antibody obtained by immunizing an animal may be cloned into an expression vector to express, thereby obtaining an antibody identical to the antibody obtained by immunizing an animal, or a gene coding for a variable region (including a heavy chain variable region and a light chain variable region) of an antibody obtained by immunizing an animal may be cloned into an expression vector together with a gene coding for a constant region of an antibody derived from another species (e.g., human), thereby obtaining an antibody comprising a heavy chain and a light chain variable region sequence from one species and a constant region sequence from another species, e.g., an antibody having a mouse heavy chain and a light chain variable region linked to a human constant region. Such antibodies are commonly referred to in the art as "chimeric antibodies".
As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen against which it is directed.
In the context of the present invention, the terms "anti-adenovirus antibody", "anti-adenovirus monoclonal antibody", "anti-adenovirus hexon protein monoclonal antibody" and "hexon protein antibody" are used interchangeably and refer to antibodies which are obtained by immunoscreening and genetic recombination techniques using hexon protein as a target site for diagnosis of an adenovirus infection antigen, and which are capable of specifically binding to hexon protein of an adenovirus to effect diagnosis of an adenovirus infection.
In the present invention, PCR amplification is also performed using the nucleotide sequences encoding the antibodies with the primers. In the primer sequence, some of the sites involve only a single base, such as any of adenine (A), guanine (G), cytosine (C) and thymine (T), while some involve a combination of two, three or four bases, in which case these bases are referred to as degenerate bases to each other, which is determined primarily based on the degeneracy of the codons. Degenerate bases may be represented by letters R, Y, M, K, S, W, H, B, V, D, N, where R represents A/G, Y represents C/T, M represents A/C, K represents G/T, S represents C/G, W represents A/T, H represents A/T/C, B represents G/T/C, V represents G/A/C, D represents G/A/T, and N represents A/T/C/G.
The terms "sequence identity", "identity" or "homology" as used herein have art-recognized meanings and the percent sequence identity between two nucleic acid or polypeptide molecules or regions can be calculated using the disclosed techniques. Sequence identity can be measured along the full length of a polynucleotide or polypeptide or along a region of the molecule (see, e.g., Computational Molecular Biology,Lesk,A.M.,ed.,Oxford University Press,New York,1988;Biocomputing：Informatics and Genome Projects,Smith,D.W.,ed.,Academic Press,New York,1993;Computer Analysis of Sequence Data,Part I,Griffin,A.M.,and Griffin,H.G.,eds.,Humana Press,New Jersey,1994;Sequence Analysis in Molecular Biology,von Heinje,G.,Academic Press,1987;and Sequence Analysis Primer,Gribskov,M.and Devereux,J.,eds.,M Stockton Press,New York,1991)., although there are many methods of measuring identity between two polynucleotides or polypeptides, the term "identity" is well known to the skilled artisan to be suitable for conservative amino acid substitutions in peptides or proteins and can generally be made without altering the biological activity of the resulting molecule.
As described above, the present invention aims to provide an anti-adenovirus monoclonal antibody having high sensitivity, high specificity and against various types of adenoviruses. As described above, the present inventors used Hexon protein as a target site for diagnosis of adenovirus infection antigen, immunized mice with adenovirus type 3 (HAdV 3) Hexon protein (Hexon) (amino acid sequence SEQ ID NO: 62), fused mouse spleen cells with myeloma cells, and screened hybridoma cell lines specifically binding to Hexon proteins of various adenovirus types by ELISA method, designated A54, A15, A5, A1, A27, A8, A18, A7, and A13, respectively.
Accordingly, in a first aspect, the present invention provides an anti-adenovirus monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein,
A) The heavy chain variable region comprises a heavy chain complementarity determining region V H CDR1、VH CDR2 and a V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 1-3, respectively, and the light chain variable region comprises a light chain complementarity determining region V L CDR1、VL CDR2 and a V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 4-6, respectively;
b) The heavy chain variable region comprises a heavy chain complementarity determining regions V H CDR1、VH CDR2 and V H CDR3, the amino acid sequences of which are shown by SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 8, respectively, and the light chain variable region comprises a light chain complementarity determining regions V L CDR1、VL CDR2 and V L CDR3, the amino acid sequences of which are shown by SEQ ID NO. 9, SEQ ID NO. 5 and SEQ ID NO. 10, respectively;
c) The heavy chain variable region comprises a heavy chain complementarity determining regions V H CDR1、VH CDR2 and V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 8, respectively, and the light chain variable region comprises a light chain complementarity determining regions V L CDR1、VL CDR2 and V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 9, SEQ ID NO. 11 and SEQ ID NO. 6, respectively;
d) The heavy chain variable region comprises a heavy chain complementarity determining region V H CDR1、VH CDR2 and a V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 12-14, respectively, and the light chain variable region comprises a light chain complementarity determining region V L CDR1、VL CDR2 and a V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 15-17, respectively; or alternatively
E) The heavy chain variable region comprises the heavy chain complementarity determining regions V H CDR1、VH CDR2 and V H CDR3, shown in SEQ ID NOS: 18-20, respectively, and the light chain variable region comprises the light chain complementarity determining regions V L CDR1、VL CDR2 and V L CDR3, shown in SEQ ID NOS: 15, 16 and 21, respectively.
In a specific embodiment, the antibody is an intact antibody comprising a variable region and a constant region. For the antibodies of the invention, any Framework Region (FR) as well as any constant region may be used. The amino acid sequence of the FR or constant region used in the antibody of the invention may be the amino acid sequence of the original FR or constant region from which it is derived, or may be a different amino acid sequence obtained by substituting, deleting, adding and/or inserting 1 or more amino acids into the amino acid sequence of the original FR or constant region. The structure used to support the CDRs or sets of CDRs of the invention typically belong to an antibody heavy or light chain sequence or a major portion thereof, wherein the CDRs or sets of CDRs are located at positions corresponding to the CDRs or sets of CDRs of naturally occurring V H and V L antibody variable domains encoded by rearranged immunoglobulin genes.
As one example, each Framework Region (FR) may have the following sequence:
HFR1 (heavy chain framework region 1): QLQQSGPGLVAPSQSLSITCTVS A
HFR2 (heavy chain framework region 2): WMHWVMQRPGQGLEWIGVI A
HFR3 (heavy chain framework region 3): TNYNSALMSRLSISKDNSKSQVFLKMN SLQSEDSAVYYCAR A
HFR4 (heavy chain framework region 4): WGQGTTLTVS A
LFR1 (light chain framework region 1): VVMTQTPLTLSVTIGQPASISC A
LFR2 (light chain framework region 2): WYLQKPGQSPKLLIY A
LFR3 (light chain framework region 3): GVPSRFSSSGSGTDFVFKISRVEAEDLG VYYC A
LFR4 (light chain framework region 4): FGGGTKLEI A
In a specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 44 or a sequence identical to SEQ ID NO:44, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 45 or a sequence identical to SEQ ID NO:45 has a sequence that is 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical.
In yet another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 46 or a sequence complementary to the amino acid sequence shown in SEQ ID NO:46, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 47 or a sequence having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence set forth in SEQ ID NO:47 has a sequence that is 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical.
In yet another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 48 or a sequence complementary to the amino acid sequence shown in SEQ ID NO:48, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID No. 49 or a sequence identical to SEQ ID NO:49 has a sequence that is 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical.
In yet another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence set forth in SEQ ID No. 50 or a sequence identical to SEQ ID NO:50, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:51 or a sequence having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence set forth in SEQ ID NO:51 has a sequence that is 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical.
In yet another specific embodiment, the heavy chain variable region of the antibody comprises the amino acid sequence shown in SEQ ID NO. 52 or a sequence identical to SEQ ID NO:52, or a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:53 or a sequence having 80% or more, 85% or more, 90% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identity to the amino acid sequence set forth in SEQ ID NO:53 has a sequence that is 80% or more, 85% or more, 90% or more, or 95% or more, or even 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical.
In a specific embodiment, the antibody further comprises a constant region sequence, such as, but not limited to, a constant region sequence selected from any one of IgG, lgA, igM, igE and IgD, which is not particularly limited herein, and can be selected by one of skill in the art based on the need.
In yet another specific embodiment, the constant region sequence may be derived from a rat, mouse, rabbit, goat, sheep, horse, dog, cow, pig, chicken, duck, goose, or human, but is not limited thereto.
In a specific embodiment, the constant regions of the antibodies of the invention are derived from mice.
In some embodiments, the constant region sequence is derived from a mouse.
In a specific embodiment, the monoclonal antibody targets the hexon protein of the adenovirus. Hexon proteins are highly conserved (78% -95%) adenoviruses and carry major genus and subgenera specific epitopes and minor species specific epitopes, which can be used as criteria for diagnosis of different serotypes.
In a second aspect, the invention provides a nucleic acid molecule encoding an anti-adenovirus monoclonal antibody according to the first aspect.
It will be apparent to those skilled in the art that the determination of the nucleic acid coding sequence is well within the ability of a protein, such as the amino acid sequence of an anti-adenovirus monoclonal antibody of the invention. In addition, in order to obtain monoclonal antibodies by recombinant means, the nucleic acid molecules may be cloned into a vector and the vector further introduced into an expression cell to express the antibody protein using the expression cell.
In a third aspect, the invention provides a vector comprising a nucleic acid molecule of the second aspect of the invention.
In a preferred embodiment, the vector may be a plasmid vector, such as pEE12, pCAGGS, pTOPO, pcDNA, pTT, pTT, pEFBOS, pBV, pJV and pBJ.
In a specific embodiment, the vector is a pTOPO vector.
In yet another specific embodiment, the vector is a eukaryotic expression vector.
In a preferred embodiment, the pcDNA vector may be a pcDNA3.1 vector.
In a fourth aspect, the present invention provides an expression cell comprising the nucleic acid molecule of the second aspect or the vector of the third aspect.
The expression cells are prepared by introducing the above-described nucleic acid molecules or the above-described vectors into host cells by molecular biological methods well known to those skilled in the art.
As described above, the present inventors used Hexon protein as a target site for diagnosis of adenovirus infection antigen, immunized mice with adenovirus type 3 (HAdV 3) Hexon protein (Hexon), fused spleen cells of the mice with myeloma cells, and screened out hybridoma cell lines specifically binding to Hexon proteins of various adenovirus types by ELISA method, designated A54, A15, A5, A1, A27, A8, A18, A7, A13, respectively. After screening a monoclonal cell line secreting the antibody of interest, heavy and light chain variable region cdnas may be recovered from the cell line by reverse transcriptase-PCR, and appropriate immunoglobulin constant regions (e.g., human constant regions) are selected, and then the heavy and light chain variable region cdnas and the constant region cdnas are transferred into host cells such as COS or CHO cells, thereby obtaining the expression cells expressing the antibody of interest of the present invention. Other antibodies or chimeric molecules may be produced that retain the specificity of the original antibody using monoclonal antibodies and other antibodies and recombinant DNA techniques, which may include introducing DNA encoding the immunoglobulin variable or Complementarity Determining Regions (CDRs) of the antibodies into the constant regions or constant region plus framework regions of different immunoglobulins.
In a specific embodiment, the expression cell may be a mammalian cell, such as a chinese hamster ovary cell, a little hamster kidney cell, a monkey kidney cell, a mouse thymoma cell, a human embryonic kidney cell. In a more specific embodiment, the expression cell may be, for example, a monkey kidney cell transformed with SV40 (COS-7, ATCC CRL 1651), a human embryonic kidney cell (HEK 293 or HEK293 cell subcloned for growth in suspension culture, graham et al, 1977,J.Gen Virol.36:59), baby hamster kidney cell (BHK, ATCC CCL 10), chinese hamster ovary cell/-DHFR 1 (CHO, urlaub et al, 1980, proc.Natl. Acad.sci.usa77:4216; such as DG 44), mouse thymoma cells (NSO), mouse testis support cells (TM 4, mather,1980, biol. Reprod. 23:243-251), monkey kidney cells (CV-1, ATCC CCL 70), african green monkey kidney cells (VERO-76, ATCC CRL-1587), human cervical cancer cells (HELA, ATCC CCL 2), canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3A, ATCC CRL 1442), human lung cells (W138, ATCC CCL 75), human liver cells (HepG 2, HB 8065), mouse mammary tumors (MMT 060562, ATCC CCL 51), TR1 cells (Mather et al, 1982,Annals N.Y.Acad.Sci.383:44-68), MRC5 cells, FS4 cells, etc., but are not limited thereto.
In a fifth aspect, the present invention provides a method of detecting an adenovirus for non-diagnostic or diagnostic purposes comprising the step of using an anti-adenovirus monoclonal antibody according to the first aspect.
In a specific embodiment, the detection of adenovirus is achieved by specific binding of the anti-adenovirus monoclonal antibodies of the invention to adenovirus hexon proteins.
In yet another specific embodiment, the detection is performed by immunochromatography, enzyme-linked antibody (ELISA), chemiluminescence, electrochemiluminescence.
In a preferred embodiment, the detection may be direct, indirect, sandwich and competition.
In a preferred embodiment, the immunochromatography includes, but is not limited to, fluorescent microsphere immunochromatography, colloidal gold immunochromatography, color latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography.
In the present invention, the anti-adenovirus monoclonal antibody can be used as a labeled antibody. For example, the anti-adenovirus monoclonal antibody may be conjugated to magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, and the like.
Without wishing to be bound by theory, the anti-adenovirus monoclonal antibodies of the invention may also be used as coating antibodies. For example, an anti-adenovirus monoclonal antibody may be bound to a solid phase such as a solid support. The solid support used in the detection method of the present invention is not particularly limited, and may be a porous or nonporous material such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, or the like.
For example, when performing a colloidal gold immunochromatographic assay, the anti-adenovirus monoclonal antibodies of the present invention (e.g., a54, A5, etc., but not limited thereto) may be labeled with colloidal gold, and the nitrocellulose membrane (NC membrane) may be coated with the anti-adenovirus monoclonal antibodies of the present invention (e.g., antibodies a15, A7, a13, but not limited thereto) or another anti-adenovirus monoclonal antibody of the prior art, and scored to obtain a detection line (T line). And assembling according to the preparation mode of the immune test strip to obtain the colloidal gold test paper. During detection, an analyte in the positive sample is combined with the colloidal gold labeled anti-adenovirus monoclonal antibody to form a complex, the complex is combined with the coated antibody at a T line to form a sandwich complex, and the colloidal gold is aggregated and precipitated to display red, so that the positive sample is indicated.
It will be appreciated by those skilled in the art that in the context of the present invention, by "another anti-adenovirus monoclonal antibody" is meant an antibody capable of binding to the same antigen, preferably a different epitope of the same antigen, as the anti-adenovirus monoclonal antibody of the present invention, which may be one of the anti-adenovirus monoclonal antibodies of the present invention, or other anti-adenovirus monoclonal antibodies of the prior art, but is not limited thereto. It is noted that the anti-adenovirus monoclonal antibody of the invention, whether used as a coating antibody or a labeling antibody, can be used in combination with another anti-adenovirus monoclonal antibody to detect adenovirus in a highly sensitive and highly specific manner.
In a sixth aspect, the invention provides the use of an anti-adenovirus monoclonal antibody according to the first aspect in the preparation of a reagent for detection of adenovirus.
In a specific embodiment, the detection of adenovirus is achieved by specific binding of the anti-adenovirus monoclonal antibodies of the invention to adenovirus hexon proteins.
In yet another specific embodiment, the detection is performed by immunochromatography, enzyme-linked antibody (ELISA), chemiluminescence, electrochemiluminescence. The immunochromatography may include, but is not limited to, fluorescent microsphere immunochromatography, colloidal gold immunochromatography, color latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, quantum dot immunochromatography, and the like. The detection can be selected by those skilled in the art as necessary, and the present invention is not particularly limited.
In yet another preferred embodiment, the ELISA may be a direct method, an indirect method, a sandwich method, and a competition method.
As described above, the anti-adenovirus monoclonal antibody of the present invention can be used as a labeled antibody or a coated antibody. For example, the anti-adenovirus monoclonal antibody may be used as a labeled antibody by binding to magnetic beads, microspheres, enzymes, fluorescent dyes, biotin, streptavidin, quantum dots, colloidal gold, or the like. For another example, an anti-adenovirus monoclonal antibody may be bound to a solid phase such as a solid support, and used as a coating antibody. The solid support used in the detection method of the present invention is not particularly limited, and may be a porous or nonporous material such as magnetic beads, latex microspheres, fluorescent microspheres, microtiter plates, nitrocellulose membranes, microfluidic chips, or the like.
In the context of the present invention, "another anti-adenovirus monoclonal antibody" may also be used. By "another anti-adenovirus monoclonal antibody" is meant an antibody capable of binding to the same antigen, preferably to a different epitope of the same antigen, as the anti-adenovirus monoclonal antibody of the invention, which may be one of the anti-adenovirus monoclonal antibodies of the invention, or may be another anti-adenovirus monoclonal antibody of the prior art. It is noted that the anti-adenovirus monoclonal antibody of the invention, whether used as a coating antibody or a labeling antibody, when used in combination with another anti-adenovirus monoclonal antibody (of the invention or of the prior art), enables detection of adenovirus in a highly sensitive and highly specific manner when used in immunochromatographic reactions such as colloidal gold-based immunochromatographic reactions.
In a seventh aspect, the invention provides a kit for detecting an adenovirus comprising an anti-adenovirus monoclonal antibody according to the first aspect and instructions for use.
The kit of the invention may be used in point of care testing (POCT) or electrochemical immunoassay systems. Kits according to the invention and any of the exemplary forms thereof may be used in automated and semi-automated systems and are optimized.
Examples
In the following examples, the methods of preparation of the antibodies of the invention and characterization of relevant properties are shown. Unless otherwise indicated, all test procedures used herein were conventional, and all test materials used in the examples described below were purchased from a conventional reagent store, unless otherwise indicated. Unless defined otherwise, 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 belongs.
It should be noted that the terminology used in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The foregoing summary of the invention and the following detailed description are only for the purpose of illustrating the invention and are not intended to limit the invention in any way. The scope of the invention is determined by the appended claims without departing from the spirit and scope of the invention.
Example 1: preparation of anti-adenovirus monoclonal antibody
Antigen coupling and immunization: the purified adenovirus hexon protein (amino acid sequence SEQ ID NO:62：MATPSMMPQWAYMHIAGQDASGYLSPGLVQFARATDTYFS MGNKFRNPTVAPTHDVTTDRSQRLMLRFVPVDREDNTYSYKVRYTLAVGDNRVLDMASTFFDIRGVLDRGPSFKPYSGTAYNSLAPKGAPNTSQWIVTTNGDNAVTTTTNTFGIA SMKGGNITKEGLQIGKDITTTEGEEKPIYADKTYQPEPQVGEESWTDTDGTNEKFGGRALKPATNMKPCYGSFARPTNIKGGQAKNRKVKPTTEGGVETEEPDIDMEFFDGRDAVAGALAPEIVLYTENVNLETPDSHVVYKPETSNNSHANLGQQAMPNRPNYIGFRDNFVGLMYYNSTGNMGVLAGQASQLNAVVDLQDRNTELSYQLLLDSLGDRTRYFSMWNQAVDSYDPDVRIIENHGIEDELPNYCFPLNGIGPGHTYQGIKVKTDDTNGWEKDANVAPANEITIGNNLAMEINIQANLWRSFLYSNVALYLPDVYKYTPPNITLPTNTNTYEYMNGRVVSPSLVDSYINIGARWSLDPMDNVNPFNHHRNAGLRYRSMLLGNGRYVPFHIQVPQKFFAVKNLLLLPGSYTYEWNFRKDVNMVLQSSLGNDLRTDGATISFTSINLYATFFPMAHNTASTLEAMLRNDTNDQSFNDYLSAANMLYPIPANATNIPISIPSRNWAAFRGWSFTRLKTKETPSLGSGFDPYFVYSGSIPYLDGTFYLNHTFKKVAIMFDSSVSWPGNDRLLSPNEFEIKRTVDGEGYNVAQCNMTKDWFLVQMLANYNIGYQGFYIPEGYKDRMYSFFRNFQPMSRQVVDEVNYTDYKAVTLPYQHNNSGFVGYLAPTMRQGEPYPANYPYPLIGTTAVKSVTQKKFLCDRTMWRIPFSSNFMSMGALTDLGQNMLYANSAHALDMTFEVDPMDEPTLLYLLFEVFDVVRVHQPHRGVIEAVYLRTPFSAGNATT) was used as immunogen for immunization of mice, mice were selected 6-8 weeks old, female BALB/c mice, immunized 4 times at an interval of 2 weeks each time at a dose of 100. Mu.g/mouse, the hexon protein was mixed with equal volumes of Freund's complete adjuvant (Sigma-Aldrich Co.) for the first immunization, injected subcutaneously via the back, the hexon protein was mixed with equal volumes of Freund's incomplete adjuvant (Sigma-Aldrich Co.) for the last three times, and injected intraperitoneally, 7 days after the fourth immunization, tail-broken blood was collected from the mice, serum was isolated, the antibody titer level of the immunized mice was examined by an indirect ELISA method to observe immune response effects, mice with serum antibody titers higher than 1:10000 were selected for cell fusion experiments, and the hexon protein without adjuvant was boosted by injection (100. Mu.g/100 days intraperitoneally).
Establishment of hybridoma cells: on the day of fusion, spleens of immunized mice were removed under sterile conditions and the organs were made into single cell suspensions. Mouse myeloma cells (SP 2/0) were taken with the immunized BALB/c mouse spleen cells described above at a ratio of 1:5, and washing the cells twice before fusing with PEG. The cells were washed with pre-warmed PEG1500, gently shaken, and pre-warmed serum-free RPMI-1640 medium, and resuspended in HAT selective medium. The cell suspension was plated at 200. Mu.L/well into 96-well plates and the cells were cultured at 37℃under 5% CO 2. After 4 to 7 days of culture, the culture was changed to HT medium, and when the fused cells grew to 1/10-1/5 of the bottom area of the wells of the 96-well plate, the supernatant was taken for antibody detection.
Screening of positive hybridoma cells: hexao-protein was diluted with coating buffer (0.05 mol/L, pH9.6, PBS) to a final concentration of 1. Mu.g/ml, added to a 96-well plate at 100. Mu.L/well, and coated overnight at 4 ℃; discarding the coating liquid, washing 3 times by using Phosphate Buffer Solution (PBST), and beating to dry; blocking with 2% BSA in PBST, 150. Mu.L/well, incubating at 37℃for 2h, washing with PBST 3 times, and drying by pipetting; fusion cell supernatants, 1:1000 diluted immune mouse positive serum (as positive control) and 1:1000 diluted mouse negative serum (as negative control) were added to the corresponding wells at 100 μl/well, incubated for 1h at 37 ℃, washed 3 times with PBST, and patted dry; adding 1:4000 dilution of horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (purchased from Sigma), 100 μl/well, incubated for 1h at 37 ℃, washed 3 times with PBST, and patted dry; adding tetramethyl benzidine (3, 3', 5' -Tetramethylbenzidine, TMB) substrate, and developing at room temperature and in dark place for 10min at 100 μl/hole; the reaction was terminated by adding 50. Mu.L of 2mol/L sulfuric acid per well.
OD 450nm values of all wells in the microplate were measured at 450nm wavelength of the microplate reader. When OD 450nm of negative serum is less than or equal to 0.1, the absorbance OD 450nm of the measured well is more than 2.1 times of that of OD 450nm of the negative well, and the measured well is taken as a judgment standard. Positive hybridoma cells were screened for further cloning.
Cloning of positive cell lines: after counting positive cell wells secreting antibodies, the samples were diluted to 100 cells/10 mL medium, and the diluted cell suspension was plated at 100 μl/well to 96 well cell culture plates and incubated in a 5% co 2 cell incubator at 37 ℃. After 6-7 days, the formation of cloned cells was observed under a microscope, single gram Long Sheng long holes were marked, cell supernatants were taken out, ELISA detection (same as the fusion detection described above) was performed, and positive monoclonal cells were selected. Limiting dilution is carried out on positive hole cells, ELISA values are measured 5-6 days after limiting dilution, and monoclonal holes with high OD450nm positive values obtained by ELISA detection are selected for limiting dilution until the ELISA measurement 96-well plate whole plate results are positive. And selecting monoclonal fixed strains with high positive values. Finally, nine cell lines which stably secrete anti-hexon antibodies are obtained and named hybridoma cell lines A54, A15, A5, A1, A27, A8, A18, A7 and A13.
Preparation and purification of cell-on-list antibody: the hybridoma cell lines were cultured in 10cm dishes with RPMI-1640 containing 15% serum, centrifuged at 800rpm for 5min when expanded to about 4X 10 7 cells/dish, the supernatant was discarded and the cells were transferred to 2L flasks and serum-free medium was added to give a cell density of about 3X 10 5 cells/ml, and the flasks were cultured. After further culturing for 1-2 weeks, when the cell death rate reached 80% -90% (at this time, the cell density was approximately 1X 10 6-2×106 cells/ml), the cell suspension was collected, centrifuged at 6000rpm for 20min at high speed, and the supernatant was collected and purified by Protein A immunochromatography.
Monoclonal antibodies prepared from hybridoma cells were also designated as a54, a15, A5, A1, a27, A8, a18, A7, a13 antibodies, respectively.
The concentrations of the monoclonal antibodies are all larger than 4mg/mL through identification by a micro-spectrophotometer. And measuring the concentration of the purified monoclonal antibody, sub-packaging, and storing at 4-8 ℃.
The monoclonal antibodies described above were identified by SDS-PAGE electrophoresis, the antibodies having an antibody heavy chain band of about 51KD and an antibody light chain band of about 26 KD.
And (3) purity detection: the monoclonal antibody is analyzed by size exclusion chromatography (SEC-HPLC), and under the condition that all components in the sample to be detected are ensured to have peaks, the purity percentage of the main peak is calculated by using a peak area normalization method, and the purity is more than 98%.
Example 2: indirect ELISA method for detecting antibody titer
The hexon protein was diluted to a concentration of 1. Mu.g/ml with 0.05mol/L carbonate buffer pH 9.6, added to a 96-well ELISA plate at 100. Mu.L/well, coated overnight at 4℃and washed 3 times with PBST on an automatic plate washer, and patted dry. Blocking with 2% BSA in PBST, 150. Mu.L/well, incubation at 37℃for 2h, washing 3 times with PBST, and drying. The nine anti-adenovirus hexon protein monoclonal antibodies obtained in example 1 were each subjected to gradient dilution with PBS buffer at pH7.4, 0.02M, and the initial concentration of the antibodies was 5. Mu.g/ml, followed by three-fold gradient dilution in order to obtain a series of monoclonal antibody samples of different concentrations. The diluted monoclonal antibody sample was incubated at 37℃for 1h at 100. Mu.L/Kong Jiaru to the above ELISA plate, washed 3 times and dried by pipetting. Adding 1:5000 dilutions of horseradish peroxidase (HRP) -labeled goat anti-mouse IgG (ex sigma), 100. Mu.L/well, incubated for 1h at 37℃and washed 3 times with PBST and patted dry. TMB substrate was added at 100. Mu.L/well and developed for 10min at room temperature in the dark. The reaction was terminated by adding 2mol/L sulfuric acid at 50. Mu.L/well. OD 450nm values were measured by a microplate reader and the results are shown in fig. 1 and 3. FIG. 1 shows the results of the binding reaction of monoclonal antibody A18 to hexon proteins of eight types of adenoviruses (Adv) and to the novel coronavirus Np protein (as a control). As can be seen from FIG. 1, the monoclonal antibody A18 can be combined with hexon proteins of eight types of adenoviruses, and is not combined with Np proteins of the novel coronaviruses, so that the antibody specificity is good. In addition to antibody a18, other eight monoclonal antibodies were also able to bind to hexon proteins of eight types of adenovirus, but not to the new coronavirus Np protein (as shown in fig. 3).
In addition, the above ELISA results were further analyzed by software to obtain EC 50 values for each monoclonal antibody and hexon protein of eight types of adenovirus, and EC 50 values are shown in Table 1 below.
Table 1: EC 50 (nM) values for hexon proteins of nine monoclonal antibodies and eight types of adenovirus
| Monoclonal antibodies | 1 Type | 2 Type | 3 Type | 4-Type | 7 Type | 40 Type | 41 Type | 55 Type |
| A54 | 0.490 | 0.365 | 0.328 | 0.455 | 0.306 | 0.610 | 0.635 | 0.285 |
| A15 | 0.356 | 0.420 | 0.296 | 0.472 | 0.330 | 0.636 | 0.613 | 0.340 |
| A5 | 0.473 | 0.477 | 0.312 | 0.353 | 0.296 | 0.509 | 0.549 | 0.349 |
| A7 | 0.427 | 0.422 | 0.424 | 0.547 | 0.520 | 0.308 | 0.320 | 0.474 |
| A13 | 0.349 | 0.332 | 0.471 | 0.605 | 0.429 | 0.307 | 0.289 | 0.521 |
| A1 | 0.486 | 0.365 | 0.278 | 0.534 | 0.328 | 0.616 | 0.608 | 0.308 |
| A27 | 0.335 | 0.366 | 0.305 | 0.446 | 0.311 | 0.605 | 0.610 | 0.282 |
| A18 | 0.313 | 0.303 | 0.275 | 0.353 | 0.500 | 0.302 | 0.310 | 0.341 |
| A8 | 0.336 | 0.338 | 0.537 | 0.303 | 0.511 | 0.645 | 0.622 | 0.610 |
As can be seen from the data presented in Table 1, nine clones all had nM levels of affinity for the hexon protein of each type of adenovirus, with A54, A27 having the highest affinity for the hexon protein of adenovirus type 55, A15, A1, A18 having the highest affinity for the hexon protein of adenovirus type 3, and A5 having the highest affinity for the hexon protein of adenovirus type 7.
Example 3: cloning and sequencing of antibody variable region sequences
Total RNA was isolated from the nine hybridoma cell lines obtained in example 1, cDNA was prepared by reverse transcription to clone immunoglobulin sequences from the hybridoma cell lines, and the variable region sequences of the hybridoma cell lines were determined as follows.
Extraction of RNA: the total RNA extraction of the hybridoma cell line was performed by referring to the instructions of the total RNA M5 extraction kit (purchased from Beijing polymeric Biotech Co., ltd.);
Reverse transcription of RNA into cDNA: reverse transcription of total RNA extracted in the previous step is performed with reference to M5 FIRST STRAND CDNA SYNTHESIS KIT polymeric America (purchased from Beijing polymeric Biotechnology Co., ltd.) to obtain cDNA, and frozen at-20deg.C for use;
c. PCR amplification and recovery of variable region sequences: amplifying immunoglobulin heavy chain (IgH) cDNA by PCR using the cDNA obtained in the above step as a template and using the universal heavy chain primers Mu Ig VH 5'-A and Mu IgG VH 3' -2; similarly, the PCR products were recovered by PCR amplification of immunoglobulin light chain (IgK) cDNA using light chain primers Mu Ig kappa VL 5'-a and Mu Ig kappa VL 3' -1; the PCR reaction used a thermostable pfu dna polymerase throughout.
D. Cloning and sequencing of variable region sequences: according to the specification of cloning vector pTOPO-Blunt Cloning kit (purchased from Beijing polymeric Biotechnology Co., ltd.), the heavy chain and light chain variable region genes were respectively linked to pTOPO vector, E.coli DH 5. Alpha. Was transformed, positive clones were picked up, and submitted to Beijing Rui Boxing biotechnology Co., ltd for sequencing.
The heavy chain variable region gene sequence and the light chain variable region gene sequence of the antibody obtained by sequencing the hybridoma cell line were analyzed, and the complementarity determining region sequences of the heavy chain and the light chain are shown in the following table 2.
Table 2: heavy chain complementarity determining region and heavy chain variable region and light chain complementarity determining region sequence and light chain variable region of nine monoclonal antibodies
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Example 4: preparation and purification of recombinant antibodies
Recombinant antibodies are constructed, cell lines for stably expressing the antibodies are prepared through eukaryotic expression, and the cell lines are cultured and purified on a large scale.
The heavy and light chain variable region genes (V H and V L) of the murine monoclonal antibodies were amplified by PCR using cDNA obtained by reverse transcription as a template and sequenced. For the V L and V H genes of the nine antibodies shown in Table 2, a recombinant antibody eukaryotic expression vector was constructed by molecular cloning. The heavy chain and light chain gene expression plasmid pCDNA3.1 of the antibody is electrically transduced into CHO host cells, the cells are added into a pressure screening culture medium (50 mu M MSX) after being electrically transduced, and after 20d culture, the supernatant is taken for ELISA detection (horseradish peroxidase (HRP) is used for marking sheep anti-mouse IgG as secondary antibody for screening, and the method is the same as above) to screen out recombinant antibody cell strains with stable expression.
And (3) carrying out large-scale cell culture on the screened stable transgenic cell strain by adopting a cell roller bottle culture technology, and preparing a recombinant antibody. Cells were inoculated in (0.2-0.3). Times.10 6 cells/ml with medium (Vega CHO) in roller bottles, 1L roller bottles contained 300ml of medium (Vega CHO), the number of inoculated bottles was determined according to production requirements, and the roller bottles inoculated with cells were placed in a cell transfer machine and cultured in a cell culture tank. The culture conditions were 900 rpm, the temperature was 37℃and the carbon dioxide was 5%. After 7-9 days of culture, observing under a sampling microscope, and centrifuging to collect samples when the cell activity rate is less than 50%. And carrying out affinity purification on the sample by using a protein A affinity chromatographic column to obtain the recombinant monoclonal antibody.
Each recombinant antibody was tested for its ability to bind to hexon proteins of eight types of adenovirus according to the method described in example 2. FIG. 2 shows the binding curves of recombinant antibody A18 to hexon proteins of eight types of adenoviruses. From the figure, it can be seen that the recombinant antibody A18 also has strong binding ability to hexon proteins of eight types of adenovirus, but does not bind to the control (novel coronavirus Np protein), and has good antibody specificity. In addition to antibody a18, the other eight monoclonal antibodies gave similar results with the hexon protein of the eight types of adenovirus, but not with the Np protein of the new coronavirus (results not shown).
Further, the above ELISA results were analyzed by software to obtain EC 50 (nM) values of nine recombinant antibodies, as shown in Table 3 below, respectively.
Table 3: EC 50 (nM) values of recombinant antibodies and hexon proteins of eight types of adenoviruses
From the data presented in Table 3, it can be seen that nine recombinant antibodies have affinities on the order of nM for the hexon protein of each type of adenovirus, and that the affinities are substantially similar to the monoclonal antibodies from which they were derived.
Example 5: double-sandwich ELISA method for verifying recombinant antibody sensitivity
In this example, the following steps were performed using one recombinant antibody (first antibody) of the present invention as a labeled antibody and the other recombinant antibody (second antibody) of the present invention as a coated antibody:
a) The primary antibody was labeled with horseradish peroxidase (HRP) to give a primary antibody-HRP conjugate.
B) The sensitivity of the primary antibody-HRP was verified using a double antibody sandwich ELISA according to the following procedure:
coating: diluting the coated antibody/secondary antibody with 0.05mol/L carbonate buffer solution with pH of 9.6 to make the concentration of the coated antibody/secondary antibody be 1 mug/mL, adding the coated antibody/secondary antibody into a 96-well ELISA plate at 100 mug/well, coating the ELISA plate at 4 ℃ overnight, washing the ELISA plate with PBST for 3 times on an automatic plate washer, and performing beating drying;
closing: adding 120 mu L of blocking solution (PBST containing 2% BSA) into each hole, blocking at 37 ℃ for 2 hours, and spin-drying and airing for later use;
sample adding: respectively adding 50 mu L of a sample to be detected, a positive control and a negative control, and incubating at 37 ℃ for 60min;
adding enzyme: adding the primary antibody-HRP conjugate into enzyme diluent according to a certain concentration, and uniformly mixing. 50 μl was added to each well and incubated at 37deg.C for 30min;
washing: beating the liquid in the pore plate, and washing the plate for 5 times;
color development: adding 100 mu L of a chromogenic agent TMB substrate into each hole, gently shaking and uniformly mixing, and developing at room temperature in a dark place for 30min;
And (3) terminating: adding 2mol/L sulfuric acid into each 50 mu L/hole to terminate the reaction, and gently shaking and uniformly mixing;
reading: OD values were read on an ELISA reader at 450nm,630 nm;
The recombinant hexon protein was diluted several gradients from 100ng/mL in a double ratio, and then different concentrations of hexon protein were detected using the recombinant antibodies of the invention, while the same concentration (of the novel coronavirus Np protein) was diluted as a control. The detection value of the Np protein of the new coronavirus is taken as a standard, and the lowest concentration of hexon protein with the detection value being more than or equal to 2.1 times of the detection value of the Np protein of the same new coronavirus is taken as the sensitivity of the method for detecting the antigen. The results of detection of the nine recombinant monoclonal antibodies are shown in table 4 below.
Table 4: results of the recombinant monoclonal antibodies of the invention for detecting recombinant hexon proteins at different concentrations
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As can be seen from the results of Table 4, even when the concentration of the recombinant adenovirus hexon protein was diluted to 10pg/mL (i.e., 0.01 nm/mL), the ELISA detection result obtained from the antibody of the present invention was about 3-5 times that of the corresponding concentration of the novel coronavirus Np (as a control), and thus the minimum concentration of the recombinant hexon protein of the antibody of the present invention for detecting eight types of adenovirus could be 10pg/mL according to the above-mentioned judgment criteria. This result also demonstrates that the recombinant monoclonal antibodies of the invention have good affinity and extremely high sensitivity.
Example 6: colloidal gold immunochromatographic assay
In this example, a colloidal gold immunochromatographic test was performed using one recombinant antibody (first antibody) of the present invention as a labeled antibody and another recombinant antibody (second antibody) of the present invention.
Preparing colloidal gold: 200ml of ultrapure water was added to the Erlenmeyer flask, heated to boiling, 1ml of 2% chloroauric acid (Sigma-Aldrich Co., ltd., cat# 16961-25-4) solution was added, 1ml of 2% trisodium citrate (Sigma-Aldrich Co., ltd., cat# 6132-04-3) aqueous solution was added immediately after boiling, stirring was continued for 10 minutes, and natural cooling was performed for use.
Labeling colloidal gold conjugates: adding 140 μl of 0.2: 0.2M K 2CO3 into 10ml of the colloid Jin Fangru beaker, adjusting pH to 7.4, and stirring for 10 seconds; 100 μg of primary antibody was added and stirring continued for 5 minutes; 0.1ml of 10% BSA was added and stirring was continued for 5 minutes; 12000g was centrifuged for 10 minutes, the supernatant was discarded, and the precipitate was fixed to 1ml with colloidal Jin Xishi (10mM PB,150mM NaCl,0.2%BSA,0.1%TritonX-100,3% sucrosi, 0.01% procrin300) as an anti-adenovirus hexon protein antibody colloidal gold complex.
Preparing a colloidal gold pad: the colloidal gold composite is diluted by 10 times of colloid Jin Xishi liquid respectively, then soaked in glass fiber (Shanghai gold standard company) and freeze-dried, thus obtaining the gold standard pad.
Nitrocellulose membrane (NC membrane) coating: the second antibody was diluted to 1.2mg/ml to prepare a detection line working solution, which was streaked onto a corresponding position of a nitrocellulose membrane (Millipore Co., ltd., product No. HF 135002) with a spot film reader, and dried at 50℃for 1 hour for use.
Assembling a colloidal gold immunochromatography test reagent strip: and assembling the gold-labeled pad, the nitrocellulose membrane coated with the antibody, the absorbent paper, the polyester plate and the sample pad into a colloidal gold immunoassay reagent strip.
Sensitivity detection: samples of hexon protein antigens of eight types of adenovirus were tested at different concentrations. Specifically, 80. Mu.l of the sample to be tested was dropped onto the sample pad, left at room temperature for 10-15 minutes, and the result was judged. The activity of antigen binding to antibodies in the sample can be indicated by the shade of the color of the displayed band. And comparing the color of the T line color developed by the colloidal gold test paper with a standard color card, selecting the closest color, and marking the activity of the product by the grade number of the color number corresponding to the color. The results are shown in Table 5 below.
Table 5: detection results of recombinant monoclonal antibodies of the invention on hexon proteins of eight types of adenoviruses
* A1/A2, wherein A1 represents a first antibody and A2 represents a second antibody.
From the above results, it was found that when the recombinant monoclonal antibody of the present invention was assembled into a colloidal gold chromatographic reagent, the minimum detection limit of hexon protein for eight types of adenovirus could be 100pg (C8).
And (3) specificity detection: the test was performed on 500 nasopharyngeal swab samples from healthy people using the test strips prepared with the recombinant monoclonal antibodies of the invention. The result shows that the reagent strip prepared by the recombinant monoclonal antibody has no false positive result and the specificity is 100%.
Claims (10)
1. An anti-adenovirus monoclonal antibody comprising a heavy chain variable region and a light chain variable region, wherein,
A) The heavy chain variable region comprises a heavy chain complementarity determining region V HCDR1、VH CDR2 and a V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 1-3, respectively, and the light chain variable region comprises a light chain complementarity determining region V L CDR1、VL CDR2 and a V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 4-6, respectively;
b) The heavy chain variable region comprises a heavy chain complementarity determining region V H CDR1、VH CDR2 and a heavy chain V H CDR3, the amino acid sequences of which are shown as SEQ ID NO. 1, SEQ ID NO. 7 and SEQ ID NO. 8, respectively, and the light chain variable region comprises a light chain complementarity determining region V L CDR1、VL CDR2 and a light chain V L CDR3, the amino acid sequences of which are shown as SEQ ID NO. 9, SEQ ID NO. 5 and SEQ ID NO. 10, respectively; or alternatively
C) The heavy chain variable region comprises a heavy chain complementarity determining regions V H CDR1、VH CDR2 and V H CDR3, the amino acid sequences of which are shown by SEQ ID NO:1, SEQ ID NO:7 and SEQ ID NO:8, respectively, and the light chain variable region comprises a light chain complementarity determining regions V L CDR1、VL CDR2 and V L CDR3, the amino acid sequences of which are shown by SEQ ID NO:9, SEQ ID NO:11 and SEQ ID NO:6, respectively.
2. The anti-adenovirus monoclonal antibody according to claim 1, wherein,
A) The amino acid sequence of the heavy chain variable region is shown as SEQ ID NO. 44 or a sequence similar to SEQ ID NO:44, and the light chain variable region has a sequence with 80% or more, 85% or more, 90% or more, or 95% or more identity to the amino acid sequence shown in SEQ ID No. 45 or to the sequence shown in SEQ ID NO:45 has a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity;
b) The amino acid sequence of the heavy chain variable region is a sequence shown as SEQ ID NO. 46 or a sequence similar to SEQ ID NO:46, and the light chain variable region has a sequence with 80% or more, 85% or more, 90% or more, or 95% or more identity to the sequence shown in SEQ ID No. 47 or to the sequence shown in SEQ ID NO:47 has a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity; or alternatively
C) The amino acid sequence of the heavy chain variable region is a sequence shown as SEQ ID NO. 48 or a sequence similar to SEQ ID NO:48, and the amino acid sequence of the light chain variable region is the sequence shown in SEQ ID NO. 49 or a sequence identical to SEQ ID NO:49, has a sequence having 80% or more, 85% or more, 90% or more, or 95% or more identity.
3. A nucleic acid molecule encoding the anti-adenovirus monoclonal antibody of claim 1 or 2.
4. A vector comprising the nucleic acid molecule of claim 3; preferably, the vector is a plasmid vector, for example, any one of pEE12, pCAGGS, pTOPO, pcDNA such as pCDNA3.1, pTT3, pEFBOS, pBV, pJV and pBJ.
5. An expression cell comprising the nucleic acid molecule of claim 3 or the vector of claim 4, preferably the expression cell is a mammalian cell, e.g. selected from chinese hamster ovary cells, little hamster kidney cells, monkey kidney cells, mouse thymoma cells, human embryonic kidney cells.
6. A method of detecting adenovirus for non-diagnostic purposes comprising the step of using an anti-adenovirus monoclonal antibody according to claim 1 or 2.
7. The method of claim 6, wherein the detecting is performed by immunochromatography, enzyme-labeled antibody (ELISA), chemiluminescence, electrochemiluminescence; preferably, the immunochromatography includes fluorescent microsphere immunochromatography, colloidal gold immunochromatography, color latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography; ELISA such as direct method, indirect method, sandwich method and competition method.
8. Use of an anti-adenovirus monoclonal antibody according to claim 1 or 2 in the preparation of a reagent for detection of adenovirus.
9. The use according to claim 8, wherein the detection is performed by immunochromatography, enzyme-linked antibody (ELISA), chemiluminescence, electrochemiluminescence; preferably, the immunochromatography includes fluorescent microsphere immunochromatography, colloidal gold immunochromatography, color latex microsphere-based immunochromatography, time-resolved fluorescent microsphere immunochromatography, magnetic microsphere immunochromatography, and quantum dot immunochromatography; ELISA such as direct method, indirect method, sandwich method and competition method.
10. A kit for detecting an adenovirus comprising the anti-adenovirus monoclonal antibody of claim 1 or 2 and instructions for use.
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| US4487829A (en) * | 1982-03-23 | 1984-12-11 | Massachusetts Institute Of Technology | Production and use of monoclonal antibodies against adenoviruses |
| JP2000072800A (en) * | 1998-08-26 | 2000-03-07 | Toagosei Co Ltd | Antiadenovirus 37-type monoclonal antibody and its use |
| CN103242433B (en) * | 2012-02-14 | 2016-12-14 | 中国医学科学院病原生物学研究所 | A kind of adenovirus non-structural protein immunogen, its antibody and application |
| CN104086650B (en) * | 2014-06-16 | 2017-01-04 | 广州锐达生物科技有限公司 | Neutralizing monoclonal antibody of people 7 type adenovirus and its preparation method and application |
| CL2016000164A1 (en) * | 2016-01-21 | 2016-07-29 | Pontificia Universidad Católica De Chile | Monoclonal antibodies specific for the human adenovirus piii antigen (adv), produced and secreted by cellular hybridomas, useful for the detection and diagnosis of adv caused infection. |
| CN108918869B (en) * | 2018-06-15 | 2021-12-28 | 新兴县国研科技有限公司 | Application of fiber2 protein and recombinant protein thereof in detecting serum type 4 avian adenovirus antibody |
| CN109142724B (en) * | 2018-08-24 | 2021-08-03 | 北京市动物疫病预防控制中心 | Blocking ELISA kit for detecting avian adenovirus group I type 4 antibody and application thereof |
| CN109957009B (en) * | 2019-03-28 | 2021-03-19 | 中国人民解放军军事科学院军事医学研究院 | Anti-human 7-type adenovirus antibody 2-1H and application thereof |
| CN109897104B (en) * | 2019-03-28 | 2020-12-22 | 中国人民解放军军事科学院军事医学研究院 | Human adenovirus 7 monoclonal antibody 3-3E and application thereof |
| JP7291659B2 (en) * | 2020-04-16 | 2023-06-15 | デンカ株式会社 | Adenovirus immunoassay method and immunoassay device |
| JP7511377B2 (en) * | 2020-04-16 | 2024-07-05 | デンカ株式会社 | Method and instrument for immunoassay of adenovirus |
| CN113683685B (en) * | 2021-09-15 | 2024-03-26 | 中国农业科学院上海兽医研究所(中国动物卫生与流行病学中心上海分中心) | anti-I group 4 type avian adenovirus monoclonal antibody, hybridoma cell strain and application of detection kit |
| CN114605526B (en) * | 2022-03-28 | 2023-06-30 | 内蒙古农业大学 | Single-domain heavy chain antibody for adenovirus vector and application thereof |
| CN116023474A (en) * | 2023-02-03 | 2023-04-28 | 中国人民解放军军事科学院军事医学研究院 | Anti-adenovirus monoclonal antibody or antigen binding fragment thereof, and nucleic acid molecule and application thereof |
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