CN117363582B - A hybridoma cell line secreting monoclonal antibodies against the F protein of the peste des petits ruminants virus and its monoclonal antibodies and applications - Google Patents

Abstract

The present application discloses a hybridoma cell strain that secretes anti-PPR F protein monoclonal antibodies and its monoclonal antibodies and applications. The hybridoma cell line in this application is 2D10-F-PPRV, and the deposit number is CGMCC No. 45615. This monoclonal antibody can be used to detect PPRV, with strong specificity and high antibody titer. It is closely related to infection with PPRV Nigeria 75, the peste des petits ruminants virus. The Vero cells of the /1 strain produce specific fluorescence reactions and do not react with normal Vero cells and common sheep viruses such as sheep herpes virus type Ⅰ, bovine viral diarrhea virus type Ⅰ, and bovine viral diarrhea virus type Ⅱ.

Description

A hybridoma cell secreting monoclonal antibodies against the F protein of the peste des petits ruminants virus Strains and their monoclonal antibodies and applications

Technical field

The present application relates to the field of biological detection, and specifically relates to a hybridoma cell line secreting monoclonal antibodies against the F-protein of the PPR virus, its monoclonal antibody, and its application in the detection of PPR virus infection.

Background technique

Peste des petits ruminants (PPR) is a highly contagious viral infectious disease caused by Pestedes petits ruminants virus (PPRV). It mainly infects goats, sheep and other small ruminants, among which goats are highly Susceptible, the incidence rate is as high as 100%, and the fatality rate in severe outbreaks is 100%, causing serious economic losses to the breeding industry. PPRV belongs to the genus Morbillivirus of the family Paramyxoviridae and can be divided into four lineages based on the N gene and F gene. Currently, there is only one serotype.

It has been confirmed that the strains currently existing in China are mainly Series IV. Although the PPRV vaccine strain belongs to Series II, the vaccine strain has a good immune protection effect. By formulating reasonable epidemic prevention measures, the disease is expected to be eliminated from the country.

Contents of the invention

In this field, establishing a rapid, effective, and high-throughput PPRV detection method can provide technical support for the elimination of PPR. Previously, the detection of PPRV pathogens mostly used conventional RT-PCR and qRT-PCR detection methods, which required viral RNA extraction and other operations. The operation was complicated, time-consuming, high-cost, and could not be tested in high-throughput. Monoclonal antibodies play an important role in the diagnosis and prevention of animal viruses. The double-antibody sandwich ELISA detection method or indirect immunofluorescence method established using monoclonal antibodies to detect pathogens plays an important role in the clinical monitoring and laboratory testing of pathogens. At present, there are no monoclonal antibodies and high-throughput detection methods targeting the F protein of the PPRV pathogen in China. Therefore, it is necessary to provide an effective detection method that is simple, fast, highly sensitive and specific to detect the PPRV pathogen in a large number of samples.

The technical problem to be solved by this application is to provide a hybridoma cell strain that secretes anti-PPRV monoclonal antibodies and a preparation method thereof in view of the shortcomings of the existing technology.

The technical problem to be solved by this application is to provide monoclonal antibodies secreted by the above-mentioned hybridoma cell lines and preparation methods thereof.

The final technical problem to be solved in this application is to provide the application of the above-mentioned monoclonal antibodies. Specifically, this application involves the following:

This application provides a hybridoma cell. The deposit number of this hybridoma cell line is CGMCC No. 45615.

In a specific embodiment of the present application, the antigen bound by the hybridoma cells is PPRV Nigeria 75/1 strain F protein. Preferably, the amino acid sequence of the antigenic epitope bound by the hybridoma cells includes the amino acid sequence shown in SEQ ID NO.1 The sequence of the antigen epitope bound by the hybridoma cell is further preferably as shown in SEQ ID NO.1,

Antigen epitope sequence SEQ ID NO.1 is:

ALHQSLMNSQAIESLKTSLEKSNQAIEEIRLANKETILAVQGVQDYINNELVPSVHRMSCELVGHKLSLKLLRYYTEILSIFGPSLRDPIAAEISIQALSYALGGDINKILDKLGYSGGDFLAILESKGIKARVTYVDTRDYFIILSIAYPTLSEIKGVIVHKIEAISYNIGAQEWYTTIPRYVATQGYLISNFDETSCVFTPEGTVCSQNALYPMSP Amino acid sequence of LLQECFRGSTKSCARTLVSGTTSNRFILSKGNLIANCASVLCKCYTTETVINQDPDKLLTVIASDKCPVVEVDGVTIQVGSREYPDSVYLHEIDLGPAISLEKLDVGTNLGNAVTRLENAKELLDASDQILKTVKGVP.

The present application provides a monoclonal antibody produced by a hybridoma cell strain deposited as CGMCC No. 45615.

In a specific embodiment of the present application, the monoclonal antibody is capable of binding to the F protein of PPRV Nigeria 75/1 strain, preferably capable of binding to an antigenic epitope including the sequence shown in SEQ ID NO. 1, preferably capable of binding to an epitope such as SEQ ID NO. The antigenic epitope shown in .1.

In a specific embodiment of the present application, the monoclonal antibody includes the antibody heavy chain complementarity determining region, which includes: CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 has the sequence SEQ ID NO 4: SYGLS The amino acid sequence of CDR-H2 is SEQ ID NO 5: TITWNGGSTYYPDTVKG, and CDR-H3 is the amino acid sequence SEQ ID NO6: ESLLRLPAWFAY.

In a specific embodiment of the present application, the monoclonal antibody includes an antibody light chain complementarity determining region, which includes: CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-L1 has the sequence SEQ ID NO 7: RASKSVSTSGYSYMH The amino acid sequence of CDR-L2 is SEQ ID NO 8: the amino acid sequence of LVSNLES, and CDR-L3 is the amino acid sequence of SEQ ID NO9: QHIRELYT.

In a specific embodiment of the present application, a monoclonal antibody that binds to the F protein of PPRV Nigeria 75/1 strain includes:

a) Antibody heavy chain complementarity determining region, which includes: CDR-H1, CDR-H2, CDR-H3, among which:

CDR-H1 has the sequence SEQ ID NO 4: the amino acid sequence of SYGLS,

CDR-H2 has the amino acid sequence of SEQ ID NO 5: TITWNGGSTYYPDTVKG,

CDR-H3 has the amino acid sequence of SEQ ID NO 6: ESLLRLPAWFAY,

b) Antibody light chain complementarity determining region, which includes: CDR-L1, CDR-L2, CDR-L3, where:

CDR-L1 has the amino acid sequence of SEQ ID NO 7: RASKSVSTSGYSYMH,

CDR-L2 has the sequence SEQ ID NO 8: the amino acid sequence of LVSNLES,

CDR-L3 has the amino acid sequence of SEQ ID NO 9: QHIRELYT.

In a specific embodiment of the present application, the monoclonal antibody includes the antibody heavy chain variable region HCVR as shown in SEQ ID NO 10 or has 90%, 91%, 92%, or 90% similarity with the amino acid sequence shown in SEQ ID NO 10. An antibody heavy chain variable region HCVR that is 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to an antibody light chain variable region LCVR as set forth in SEQ ID NO 11 or as SEQ ID NO The amino acid sequence shown in 11 has an antibody heavy chain variable region LCVR with 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, wherein:

HCVR has the amino acid sequence SEQ ID NO 10: EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYGLSWVRQTPDKRLELVATITWNGGSTYYPDTVKGRFTISRDSAKNILYLQMSSLKSEDTAMYYCARESLLRLPAWFAYWGQGTLVTVSA;

LCVR has the sequence SEQ ID NO 11:

Amino acid sequence of DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELYTFGGGTKLEIK.

In a specific embodiment of the present application, the monoclonal antibody includes a heavy chain as shown in SEQ ID NO 12 or has 90%, 91%, 92%, 93% or 94% of the amino acid sequence shown in SEQ ID NO 12 , 95%, 96%, 97%, 98% or 99% identity of the heavy chain and the light chain as shown in SEQ ID NO 13 or the amino acid sequence shown in SEQ ID NO 13 has 90%, 91%, 92% , a light chain of 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, wherein:

The heavy chain amino acid sequence is SEQ ID NO 12:

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK Amino acid sequence of TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKL;

The light chain amino acid sequence is the amino acid sequence of SEQ ID NO 13: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

A kit for detecting and/or diagnosing PPR virus infection in animals, which includes: the above-mentioned monoclonal antibody or an antibody produced by a hybridoma cell deposited with the deposit number CGMCC No. 45615.

Beneficial effects: Compared with existing technology, this application has the following advantages:

(1) The monoclonal antibody obtained in this application can be used to detect PPRV. It has strong specificity and high antibody titer. It reacts specifically with Vero cells infected with the PPRV Nigeria 75/1 strain of the peste des petits ruminants virus and does not react with normal cells. Vero cells react with common sheep viruses such as sheep herpes virus type I, bovine viral diarrhea virus type I, and bovine viral diarrhea virus type II.

(2) This application is proposed to establish a fast, simple, and accurate detection method, and to provide technical means in the research of immune mechanism, immune function research, establishment of detection methods, etc., and has implications for the prevention and control of PPRV and laboratory research. Significance.

Description of drawings

The accompanying drawings are used for a better understanding of the present application and do not constitute an undue limitation of the present application. in:

Figure 1 shows the identification results of pET28a-PPRV-F recombinant protein.

Figure 2 shows the results of indirect immunofluorescence detection of PPR virus.

Figure 3 shows the Western blot experiment to detect the reaction between monoclonal antibody 2D10-F-PPRV strain and PPRV.

Detailed ways

The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described can be obtained from commercial sources unless otherwise specified.

It should be noted that certain words are used in the description and claims to refer to specific components. Those skilled in the art will understand that skilled persons may use different names to refer to the same component. This description and the claims do not use difference in nouns as a way to distinguish components, but rather use differences in functions of the components as a criterion for distinction. If the words "include" or "including" mentioned throughout the description and claims are open-ended terms, they should be interpreted as "including but not limited to." The following descriptions of the description are preferred embodiments for implementing the present application. However, the descriptions are for the purpose of general principles of the description and are not intended to limit the scope of the present application. The scope of protection of this application shall be determined by the appended claims.

The scientific and technical terms mentioned in this specification have the same meanings as commonly understood by those skilled in the art. If there is any conflict, the definitions in this specification shall prevail.

Generally speaking, the terms used in this specification have the following meanings.

In this specification, the term "monoclonal antibody" means an antibody derived from a population of substantially homologous antibodies, i.e. the individual antibodies constituting said population are identical and/or bind the same epitope, except for possible variants Outside of antibodies (eg, containing naturally occurring mutations or generated during the production of monoclonal antibody preparations), such variants are usually present in trace amounts. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibody preparations have each monoclonal antibody directed against a single determinant on the antigen. Thus, the modifier "monoclonal" indicates the characterization that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be prepared by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and the use of repertoires containing human immunoglobulin loci. or partially transgenic animals, such methods and other exemplary methods of preparing monoclonal antibodies are described herein.

In this specification, the term "hybridoma cell" refers to a cell formed by fusion of myeloma cells and B lymphocytes in the process of preparing monoclonal antibodies, and is generally prepared by culturing tumor cells.

In this specification, the terms "heavy chain" ("CH"), "light chain" ("CL"), "heavy chain constant region", "light chain constant region", "light chain variable region" ("VL") are used. "), "heavy chain variable region" ("VH"), "framework region" ("Framework Region, FR"), and "complementarity determining region" ("CDR") are components of antibodies, which can be Different types of antibodies are formed. For example, conventional IgG antibodies are tetramers composed of two light chains and two heavy chains. Among them, VL and VH are composed of different CDRs and FRs. The CDR contains residues that contact the antigen and determines the antigen specificity of VL and VH. The FR is used to maintain the structure of the variable region and determine the position of the CDR loop. Generally, the heavy chain variable region and the heavy chain constant region form a complete heavy chain, while the light chain variable region and the light chain constant region form a complete light chain.

In this specification, the term "label" refers to a molecule that can indicate the location or amount of the antibody through color, chemical reaction, excitation light, mass spectrometry, etc., after the antibody is bound. Including, for example: alkaline phosphatase, peroxidase, luciferase, luciferin, fluorescent protein, isotope, etc.

In this specification, the terms "polynucleotide" or "nucleic acid" and "nucleic acid molecule" are used interchangeably, including but not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA). ), shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA and/or tRNA.

In this specification, "Enzyme-linked immunosorbent assay (EILSA)" refers to the use of the characteristics of antibody molecules to specifically bind to antigen molecules to combine free impurity proteins with target proteins bound to solid-phase carriers, and use special A detection method for qualitative or quantitative analysis of markers. The principle is: the antigen or antibody can be physically adsorbed on the solid surface and maintain its immune activity; the antigen or antibody can form an enzyme conjugate with the enzyme through covalent bonds while maintaining their respective immune or enzymatic activity; enzyme binding After the substance is combined with the corresponding antigen or antibody, the occurrence of the immune reaction can be determined by adding the color reaction of the substrate. The depth of the color reaction is proportional to the amount of the corresponding antigen or antibody in the specimen. Various types of detection methods can be designed according to the substance to be detected and the conditions for detection. The double-antibody sandwich method is the most commonly used method for detecting antigens. The method is to adsorb the antiserum containing known antibodies to the small holes on the microtiter plate and wash it once; add the antigen to be tested, if the two are specific, binding will occur, and then wash away the excess antibody; add the antigen to be tested and The enzyme-linked antibody that reacts specifically to the detection antigen forms a "sandwich"; when the substrate of the enzyme is added, if a colored enzymatic hydrolysis product is produced, it means that the corresponding antigen is present.

In this specification, "plasmid" refers to DNA molecules other than chromosomes (or nucleoids) in organisms such as bacteria, yeasts, and actinomycetes. They exist in the cytoplasm or nucleus and have the ability to replicate autonomously, allowing them to reproduce in progeny cells. It can also maintain a constant copy number and express the genetic information it carries.

In this specification, the term "vector" refers to a vector through which a polynucleotide sequence (eg, a foreign gene) can be introduced into a host cell to transform the host and promote expression (eg, transcription and translation) of the introduced sequence. Vectors include plasmids, phage vectors, viral vectors, etc. Among them, "viral vectors" are vectors modified from viral genomes, which introduce foreign genes into host cells through viral infection.

This application provides a hybridoma cell line 2D10-F-PPRV. The deposit number of this hybridoma cell line is CGMCC No. 45615.

The present application also provides a monoclonal antibody produced by a hybridoma cell strain deposited as CGMCC No. 45615. The name of the hybridoma cell strain in this application is 2D10-F-PPRV, the classification name is hybridoma cell strain, the deposit number is CGMCC No. 45615, the deposit date: May 17, 2023, the depository unit: China Microbial Culture Collection General Microbiology Center of the Management Committee is located at No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing, Postal Code 100101.

The application provides a monoclonal antibody, characterized in that the antibody includes: a) the antibody heavy chain complementarity determining region, which includes: CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 has the sequence SEQ ID NO 4: The amino acid sequence of SYGLS, CDR-H2 has the amino acid sequence of SEQ ID NO 5: TITWNGGSTYYPDTVKG, CDR-H3 has the amino acid sequence of SEQ ID NO 6: ESLRLPAWFAY; b) Antibody light chain complementarity determining region, which Contains: CDR-L1, CDR-L2, CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 7: RASKSVSTSGYSYMH, CDR-L2 has the amino acid sequence of SEQ ID NO 8: LVSNLES, and CDR-L3 has Sequence SEQ ID NO 9: Amino acid sequence of QHIRELYT.

This application also provides a monoclonal antibody that binds to the F protein of PPRV Nigeria 75/1 strain, which includes: a) the antibody heavy chain complementarity determining region, which includes: CDR-H1, CDR-H2, and CDR-H3, wherein: CDR-H1 has the amino acid sequence of SEQ ID NO4: SYGLS, CDR-H2 has the amino acid sequence of SEQ ID NO 5: TITWNGGSTYYPDTVKG, and CDR-H3 has the amino acid sequence of SEQ ID NO 6: ESLLRLPAWFAY; b) Antibody light chain complementarity The determining region includes: CDR-L1, CDR-L2, and CDR-L3, wherein: CDR-L1 has the amino acid sequence of SEQ ID NO 7: RASKSVSTSGYSYMH, and CDR-L2 has the amino acid sequence of SEQ ID NO 8: LVSNLES, CDR-L3 has the amino acid sequence of SEQ ID NO 9: QHIRELYT.

In a specific embodiment of the present application, the above-mentioned monoclonal antibody binding to the F protein of PPRV Nigeria 75/1 strain includes, but is not limited to, produced by the hybridoma cell line with the deposit number CGMCC No. 45615, or obtained by chemical synthesis.

In a specific embodiment of the present application, the above-mentioned antibody comprises the antibody heavy chain variable region HCVR as shown in SEQ ID NO 10 or has 90%, 91%, 92%, 93%, or 93% similarity with the amino acid sequence shown in SEQ ID NO 10. An antibody heavy chain variable region HCVR that is 94%, 95%, 96%, 97%, 98% or 99% identical to an antibody light chain variable region LCVR as set forth in SEQ ID NO 11 or as set forth in SEQ ID NO 11 An antibody heavy chain variable region LCVR having an amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity, wherein:

HCVR has the sequence SEQ ID NO 10:

Amino acid sequence of EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYGLSWVRQTPDKRLELVATITWNGGSTYYPDTVKGRFTISRDSAKNILYLQMSSLKSEDTAMYYCARESLLRLPAWFAYWGQGTLVTVSA;

LCVR has the sequence SEQ ID NO 11:

Amino acid sequence of DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELYTFGGGTKLEIK.

In a specific embodiment of the present application, the above-mentioned antibody comprises a heavy chain as shown in SEQ ID NO 12 or has an amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, A heavy chain that is 96%, 97%, 98% or 99% identical to a light chain as shown in SEQ ID NO 13 or has an amino acid sequence of 90%, 91%, 92%, 93%, A light chain of 94%, 95%, 96%, 97%, 98% or 99% identity, wherein:

The heavy chain amino acid sequence is SEQ ID NO 12:

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEK Amino acid sequence of TISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKL;

The light chain amino acid sequence is SEQ ID NO 13:

Amino acid sequence of RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

In a specific embodiment of the present application, the method of producing monoclonal antibodies from the hybridoma cell line deposited as CGMCC No. 45615 is a routine method in this field.

In a specific embodiment of the present application, the method for producing monoclonal antibodies from the hybridoma cell line deposited as CGMCC No. 45615 is the mouse ascites method.

This application also provides a method for preparing the above-mentioned monoclonal antibody. In a specific embodiment of this application, sterilized liquid paraffin is injected intraperitoneally into 6-8 week old Balb/c mice; a specific embodiment of this application In, after 7 days, the hybridoma cell line 2D10-F-PPRV is injected into the abdominal cavity of mice; in a specific embodiment of the present application, the ascites of mice with obviously bulging abdomens is collected after 7 days; in a specific embodiment of the present application , centrifuge the ascites of mice for 10 minutes at 4000 rpm, collect the ascites supernatant, aliquot, label, save, and set aside.

This application also provides the application of the monoclonal antibody as described above in the detection of PPR virus infection.

In a specific embodiment of the present application, the hybridoma cell line secreting monoclonal antibodies against the peste des petits ruminants virus (PPRV) is obtained by fusing mouse myeloma cells with mouse spleen cells; in a specific embodiment of the present application, the cells Fusion adopts PEG cell fusion method. In a specific embodiment of the present application, the ratio of mouse myeloma cells (SP2/0) to immunized Balb/c mouse spleen cells is 1:8; in a specific embodiment of the present application, after centrifugation, the supernatant, and then add an appropriate amount of serum-free DMEM medium to resuspend; in a specific embodiment of this application, centrifuge at 1000 rpm and 25°C for 6 minutes and re-spin to wash the cells, discard the supernatant, and tap the bottom of the tube to make the cells loose and even; In a specific embodiment of the application, the evenly dispersed cells are preheated in a 37°C water bath for 1 minute, and then 0.6 mL of preheated PEG1460 is added thereto; in a specific embodiment of the application, the fusion agent is added and then left to stand in the water bath. 90s, add preheated 10 mL DMEM culture medium from slow to fast; in a specific embodiment of this application, mix well and then let it stand in a 37°C incubator for 5 minutes; in a specific implementation of this application, let it stand and then proceed Centrifuge at 500 r/min for 10 minutes and repeat washing twice; in a specific embodiment of this application, DMEM medium containing 15% FBS and HAT is added to resuspend the cells, and 100 μL per well is added to 96-well cells containing feeder cells. Place the culture plate in a constant temperature incubator at 37°C and 5% _CO2 ;

In a specific embodiment of the present application, the antigen of the hybridoma cell line secreting PPRV monoclonal antibodies is the recombinantly expressed PPRV Nigeria 75/1 strain F protein.

In a specific embodiment of the present application, it is preferred that the amino acid sequence of the antigenic epitope bound by the hybridoma cell is shown in SEQ ID NO. 1.

In a specific embodiment of the present application, the preparation method of the recombinantly expressed PPRV Nigeria 75/1 strain F protein is to express the PPRV Nigeria 75/1 strain F protein in an E. coli expression system and purify it.

The present application also provides an anti-PPRV antigen detection kit, which contains a monoclonal antibody produced by the hybridoma cell line deposited as CGMCC No. 45615, which can be used to detect PPRV. In a specific embodiment of the present application, the kit is an in vitro detection kit. In a specific embodiment of the present application, the kit is an immune detection kit, such as an ELISA kit or an immunohistochemistry kit, which contains the aforementioned neutralizing antibody of PPRV or its antigen-binding fragment. In a specific embodiment of the present application, the kit is an in vivo non-invasive diagnostic kit, which contains the aforementioned complex. In a specific embodiment of the present application, the in vivo non-invasive diagnostic kit can be used for imaging methods selected from the following: radioimmunoassay or targeted ultrasound contrast imaging. When used for radioimmunoimaging, the complex is a complex of the aforementioned PPRV neutralizing antibody or its antigen-binding fragment and a radionuclide. When used for targeted ultrasound contrast imaging, the complex is a complex of the aforementioned neutralizing antibody of PPRV or its antigen-binding fragment and an ultrasound contrast agent.

The BL21 (DE3) competent cells used in the examples of this application are competent cells obtained by using Escherichia coli BL21 (DE3) through special processing and can be used for chemical transformation of plasmids.

Western immunoblotting used in the examples of this application is a method of transferring proteins to a membrane and then using antibodies for detection. For known expressed proteins, the corresponding antibody can be used as the primary antibody for detection, and for the expression product of a new gene, the antibody of the fusion part can be detected. Western Blot uses polyacrylamide gel electrophoresis. The detected object is a protein, the probe is an antibody, and a labeled secondary antibody is used for color development. The protein sample separated by PAGE is transferred to a solid-phase carrier. The solid-phase carrier adsorbs the protein in the form of non-covalent bonds and can keep the type of polypeptide separated by electrophoresis and its biological activity unchanged. The protein or peptide on the solid phase carrier is used as the antigen, reacts with the corresponding antibody, and then reacts with the enzyme or isotope-labeled second antibody, and then detects the specific purpose of electrophoretic separation through substrate color development or autoradiography. Protein components of gene expression.

The "recombinantly expressed PPRV Nigeria 75/1 strain F protein" and "recombinant PPRV-F protein" described in this application refer to the protein after expression and are the same protein.

The experimental methods used in the following examples are conventional methods unless otherwise specified.

Materials, reagents, etc. used in the following examples can all be purchased from commercial sources unless otherwise specified.

Example 1 Preparation of recombinant F-His protein

1. PCR amplification of PPRV-F gene

Based on the gene sequence of PPRV Nigeria 75/1 strain (KY628761.1) and the gene sequence of PPRVClone9 strain preserved in our laboratory, the hydrophilicity, antigenicity and functional region of F protein were analyzed. The results showed that the antigenic epitopes of the 132AA-487AA protein sequence are relatively concentrated and can be used as candidate fragments for protein expression. Based on the protein structure analysis, the nucleotide fragment (1071bp) of the above protein region was selected for codon optimization and synthesis. BamHI and XhoI sites were introduced at the 3' and 5' ends, and the F gene fragment was amplified using homologous recombination. The primer sequences are as follows:

F:GtggacagcaaatgggtcgCGGATCCGCACTCCATCAGTCATTGATGAAT (SEQ ID No. 2);

R: CAGTGGTGGTGGTGGTGGTGCTCGAGCAAGGAGTTTTTCTAGGTCTTTATGCG (SEQ ID No. 3); the above primers were synthesized by BGI Bioengineering Co., Ltd. Using the synonymously optimized plasmid of F protein amino acids as a template, use upstream and downstream primers to amplify the target gene with the vector sequence homology arm and enzyme cutting site. According to the instruction manual of KOD PCR enzyme (TOYOBO Company of Japan), a 50 μL reaction system was used. The reaction program was as follows: 94°C for 2 min; 98°C for 10 s, 55°C for 30 s, 68°C for 50 s, and 30 cycles of reaction; 68°C for 7 min. The PCR amplification products were detected by 1% agarose gel electrophoresis. Collect the target band according to the instructions of the gel recovery kit. Double-digest the pET-28a(+) prokaryotic expression vector with BamHI and XhoI (NEB), perform homologous recombination according to the Seamless Cloning Mix homologous recombinase instructions (Beyotime Company), and place it at 50°C for 15 minutes. The homologous recombination products were transformed into TOP10 (Quanshijin Biotechnology Co., Ltd.) competent cells, spread on LB plates containing kanamycin, and cultured at 37°C overnight. A single colony was picked, cultured with shaking in LB medium containing kanamycin, and the plasmid was extracted and identified by PCR and BamHI and XhoI double enzyme digestion. The positive plasmid was sent to BGI Bioengineering Co., Ltd. for sequencing.

The antigenic epitope sequence is as follows:

ALHQSLMNSQAIESLKTSLEKSNQAIEEIRLANKETILAVQGVQDYINNELVPSVHRMSCELVGHKLSLKLLRYYTEILSIFGPSLRDPIAAEISIQALSYALGGDINKILDKLGYSGGDFLAILESKGIKARVTYVDTRDYFIILSIAYPTLSEIKGVIVHKIEAISYNIGAQEWYTTIPRYVATQGYLISNFDETSCVFTPEGTVCSQNALYPMSP LLQECFRGSTKSCARTLVSGTTSNRFILSKGNLIANCASVLCKCYTTETVINQDPDKLLTVIASDKCPVVEVDGVTIQVGSREYPDSVYLHEIDLGPAISLEKLDVGTNLGNAVTRLENAKELLDASDQILKTVKGVP (SEQ ID No. 1).

2. Construction and protein expression of recombinant expression strain BL21(DE3)-F

The recombinant plasmid pET28a-F, whose sequence was determined to be correct, was transformed into E. coli BL21 (DE3) competent cells, and single clones were picked and inoculated into LB medium containing kanamycin, and cultured at 37°C overnight. Take the bacterial solution cultured overnight and inoculate it into a new culture medium. Cultivate it at 37°C until the OD _600nm is about 0.6-0.8. Add IPTG with a final concentration of 1mmol/L and induce expression at 20°C for 12 hours. After centrifugation, the bacteria are lysed by ultrasonic, and the sediment is plated. The clear samples were analyzed by SDS-PAGE. The protein purification method was carried out according to the Ni-NTAAgarose operating instructions of GE Company. The expression supernatant was purified by affinity chromatography on a nickel column and then subjected to SDS-PAGE electrophoresis. There was an obvious target protein expression band around 39kDa (Figure 1A).

3. Western blot identification of recombinant proteins

After SDS-PAGE, the recombinant protein was transferred to a PVDF membrane (Millipore) using the wet transfer method, and blocked with PBST containing 2% skim milk for 2 h; a 1:10000 dilution of His-Tag Mouse mAb monoclonal antibody was added ( proteintech), incubate at room temperature for 2 hours; wash 3 times with PBST; add 1:10000 diluted goat anti-mouse IgG-HRP (Beijing Quanshijin Biotechnology Co., Ltd.), shake gently at room temperature for 1 hour; wash 3 times with PBST, use TMB color development kit (SeraCare) color development. The results showed that a specific band could be detected at 39kDa, proving that the recombinant protein was successfully expressed and purified (Figure 1B). The protein concentration was determined to be 0.81mg/mL by BCA method and stored at -80°C for later use.

Example 2 Screening of hybridoma cell lines with monoclonal antibodies against PPR F protein

1. Immunize Balb/c mice: Inject the purified recombinant F-His protein subcutaneously into multiple points to immunize 6-8 week old female BALB/c mice (Beijing Vitong Lever Company). A total of 3 immunizations, each immunization Three weeks apart, 50 μg of purified recombinant F-His protein and an equal volume of complete Freund's adjuvant (purchased from Sigma) were used for the first immunization, and 100 μg of purified recombinant F-His protein and an equal volume of incomplete Freund's adjuvant (purchased from Sigma) were used for the next two immunizations. Mixed immunization with H1's adjuvant (purchased from Sigma Company); blood was collected 10-14 days after the third immunization, and the immune serum titers of the mice were detected. Mice with ELISA antibody titers >106 were selected and immunized 3 days before cell fusion. Once, 100 μg of purified recombinant F-His protein was injected intraperitoneally.

2. Cell fusion: Cell fusion adopts PEG cell fusion method. Take mouse myeloma cells (SP2/0) and immunized Balb/c mouse spleen cells at a ratio of 1:8 (cell number ratio) and mix thoroughly, centrifuge at 1000 rpm and 25°C for 6 minutes, discard the supernatant, and then add an appropriate amount of Resuspend the serum-free DMEM culture medium, centrifuge at 1000 rpm and 25°C for 6 minutes to wash the cells. Discard the supernatant and tap the bottom of the tube to loosen and evenly distribute the cells. Place it in a 37°C water bath to preheat for 1 minute, then add 0.6 mL to it and place it in a 37°C water bath in advance. Add the fusion agent to the preheated PEG1460 and let it stand in the water bath for 90 seconds. Add the preheated 10 mL DMEM culture medium from slow to fast. After mixing, place it in a 37°C incubator for 5 minutes. Centrifuge at 500 r/min for 10 minutes. Repeat washing 2 more times. Add DMEM medium containing 15% FBS and HAT to resuspend the cells, add 100 μL per well to a 96-well cell culture plate containing feeder cells, and culture in a constant temperature incubator at 37°C and 5% _CO2 . During this period, the cells in the wells were observed. When the cell colony grew to 1/2 of the cell well, the cell culture supernatant was taken for antibody detection.

3. Screening of hybridoma cells: Use 0.05mol/L pH 9.6 carbonate buffer as the coating solution, and 2 μg/μL diluted purified F protein as the coating antigen to coat the enzyme plate, 100 μL/well, 4 °C overnight. Wash 3 times with PBST and pat dry; add the fused cell supernatant, 1:1000 (v/v) diluted Balb/c immune mouse positive serum and 1:1000 (v/v) diluted mouse negative serum to the corresponding In the well, 100 μL/well was incubated at 37°C for 1 h, washed three times with PBST, and patted dry; add horseradish peroxidase (HRP)-labeled goat anti-mouse IgG (purchased from Beijing) diluted at 1:5000 (v/v). Quanshijin Biotechnology Co., Ltd.), 100 μL/well, incubate at 37°C for 1 hour, wash 3 times with PBST, and pat dry. Add substrate TMB (SeraCare), 100 μL/well, and develop color for 10 min at room temperature in the dark; add 50 μL 2mol/L sulfuric acid to each well to terminate the reaction. Use a microplate reader to measure the OD _450nm value of the enzyme plate. P is the OD _450nm value of each detection well, and N is the OD4 _50nm value of the negative serum. When the OD _450nm value of the negative serum is ≤ 0.1 and the OD _450nm value of the positive serum is the same as the OD of the negative serum. The ratio of _450nm is ≥2.1, that is, under the premise that both negative and positive controls are established, the negative and positive detection holes are determined based on the criterion of P/N≥2.1 as a positive hole. Test again after 2 days, and select hybridoma cell lines with positive test results for both times for subcloning.

4. Cloning of hybridoma cells: Stain the selected positive well cell lines with trypan blue, count, dilute it with DMEM medium containing 15% FBS to 100 cells/10mL medium, and suspend the diluted cells. Add the solution to the 96-well plate with feeder cells in advance, 100 μL per well, and culture it in a 37°C, 5% CO ₂ incubator. During the period, observe the cell lines. When they grow to 1/2 of the bottom area of the 96-well plate, proceed as before The established indirect ELISA method enables timely ELISA testing. Record the positive wells of monoclonal cells, and perform the same subcloning more than three times until the supernatants of all cloned cell lines are positive after cloning and the OD _450nm values detected in each well are relatively close. The cloned PPRV F-specific monoclonal antibody hybridoma cell line was expanded and cultured, cryopreserved, and named hybridoma cell line 2D10-F-PPRV.

Example 3 Preparation and Purification of Monoclonal Antibodies

Use the ascites preparation method: Inject sterilized liquid paraffin intraperitoneally into 6- to 8-week-old Balb/c mice (purchased from Beijing Vitong Lever Company), 0.5 mL/mouse, and 7 days later, inject the hybridoma cell line 2D10-F -PPRV was injected into the abdominal cavity of mice, 0.5mL each (10 ⁶ hybridoma cells). After 7 days, collect the ascites from mice with obviously bulging abdomens, centrifuge at 4000 rpm for 10 min, collect the ascites supernatant, aliquot, label and store at -80°C for later use.

Use saturated ammonium sulfate treatment to crudely extract IgG: Add ascites to a 100mL beaker with a rotor, add PBS in the same amount as the ascites volume, place the beaker on ice, and place it on a magnetic stirrer; add 2 dropwise at a slow speed Add saturated ammonium sulfate solution twice the volume of the ascites sample while stirring, and stir at 4°C for more than 4 hours; centrifuge at 10,000g for 10 minutes, discard the supernatant, resuspend the pellet in an equal volume of PBS as the ascites, and resuspend the liquid. into the dialysis bag and seal both ends of the dialysis bag; use PBS to dialyze the IgG sample at 4°C for more than 12 hours. During this period, replace the PBS at least twice to remove residual ammonium sulfate in the sample.

Further purification of crude IgG: Centrifuge the dialyzed crude IgG sample at 10,000g for 10 minutes, collect the supernatant, and use Protein G Resin for purification. Wash the Protein G Resin chromatography column with PBS until the OD280nm of the effluent is less than 0.01. Add the sample to the column, control the flow rate to 5 s/drop, prepare an empty tube in advance to recover the effluent of the sample, add the combined effluent repeatedly to the ProteinG chromatography column, repeat the addition 3 times, and retain the last effluent. At 4°C; use PBS to elute impurity proteins at a flow rate of 5 s/drop until the OD280 nm of the effluent is less than 0.01. Take a certain amount of glycine (such as 10 ml) and add a volume of Tris-HCI so that the pH of the mixture after neutralization is between 7 and 8. Use glycine solution to elute the target protein, and use a sterilized centrifuge tube to collect the eluted target protein. The collected sample is immediately neutralized by adding Tris-HCI with a predetermined neutralization volume ratio to make the pH 7~8, and then flows out Stop collecting samples when the OD280 nm of the solution is <0.1; dialyze the collected samples with PBS at 4°C for more than 12 hours, changing PBS at least twice during this period, and finally collect the purified target antibody and store it at -20°C.

Example 4 Analysis of Antibody Characteristics

1. Monoclonal antibody titer detection

The indirect ELISA method was used to detect the titer of the hybridoma cell culture supernatant and the ascites fluid titer obtained in Example 3. The results are shown in Table 1. The titer of the ascites fluid induced in mice was 1:32000, and the potency of the hybridoma cell culture supernatant was 1:32000. The price is 1:320.

Table 1 Monoclonal antibody titer detection

.

2. Indirect immunofluorescence assay (IFA)

After culturing Vero cells infected with PPRV Nigeria75/1 strain and normal cells for 4 days respectively, discard the supernatant, wash 3 times with PBS, fix the cells with 4% paraformaldehyde (100 μL/well) for 6-8 min, and wash with PBS for 3 times. times; block cells with 100 μL/well 2% BSA, block at room temperature for 2 hours, discard the blocking solution, and wash 3 times with PBS; add 100 μL of hybridoma cell supernatant to each well, and use SP2/0 cell supernatant and pre-immune mouse serum at the same time, after immunization Mouse serum was used as negative and positive control, reacted at 37°C for 1 hour; washed three times with PBS, reacted with FITC-labeled goat anti-mouse IgG (1:200, Sigma Company) at 37°C for 1 hour; washed three times with PBS and patted dry. , observed under a fluorescence microscope.

Indirect immunofluorescence experiments showed that SP2/0 cell supernatant and pre-immune mouse serum had no specific fluorescence reaction with Vero cells infected with PPRV Nigeria75/1 strain and normal cells. After immunization with recombinant PPRV-F protein, mouse serum reacted with PPRV Nigeria75 strain. Vero cells infected with /1 strain reacted to produce specific fluorescence, indicating that both negative and positive controls were established. The monoclonal antibody 2D10-F-PPRV reacted with Vero cells infected with PPR virus Nigeria 75/1 strain, and an obvious specific fluorescence reaction was observed, but did not react with normal Vero cells. The results are shown in Figure 2, where A: Vero cells infected with PPR virus; B: Normal Vero cells.

3. Specificity experiment

Indirect immunofluorescence experiments were used to determine the specificity of sheep herpes virus type Ⅰ (CHpV-1), bovine viral diarrhea virus type Ⅰ (BVDV-1), and bovine viral diarrhea virus type Ⅱ (BVDV-2) stored in our laboratory. The test results showed that the monoclonal antibody 2D10-F-PPRV reacted positively with PPRV but did not react with other viruses, indicating that the method has good specificity. Detailed results are shown in Table 2.

Table 2 Specificity experimental test results

.

4. Western blot detection

Vero cells infected with PPRV Nigeria75/1 strain and normal cells were cultured respectively for 4 days. The supernatant was discarded and washed three times with PBS. The cells were lysed with 100 μL/well lysis solution (Beyotime Biotechnology Co., Ltd.), and then added for protein electrophoresis. Load the sample buffer and boil for 5 minutes to prepare protein samples for electrophoresis. After SDS-PAGE, the proteins were transferred to PVDF membrane (Millipore) using wet transfer method, and blocked with PBST containing 2% skim milk for 2 h; 2D10-F-PPRV monoclonal antibody diluted at 1:2000 was added respectively. , incubate at room temperature for 2 h; wash 3 times with PBST; add 1:10000 diluted goat anti-mouse IgG-HRP (Beijing Quanshijin Biotechnology Co., Ltd.), shake gently at room temperature for 1 h; wash 3 times with PBST, and use TMB color development kit (SeraCare ) color development.

The test results showed that after incubation with 2D10-F-PPRV monoclonal antibody, PPRV-infected Vero cell samples showed obvious bands at 48-63kDa (predicted size is 58kDa), but normal Vero cell samples had no bands (Figure 3). It shows that this monoclonal antibody can be used for laboratory Western blot detection.

5. Neutralizing activity detection

A neutralization test was conducted using fixed virus (the working concentration of PPRV Nigeria75/1 is 100TCID ₅₀ /0.1ml) and dilution of ascites to identify the neutralizing activity of the monoclonal antibody. 2D10-F-PPRV ascites was diluted 1:20, 1:40, 1:80, and 1:160, and the neutralization titer was calculated using the Reed-Muench method.

The results showed that the ascites titer of the 2D10-F-PPRV monoclonal antibody was 1:50.1, indicating that the 2D10-F-PPRV monoclonal antibody had the activity of neutralizing PPRV. Detailed results are shown in Table 3.

Table 3 Neutralizing activity test results

.

In summary, the indirect immunofluorescence detection and neutralization test results show that the monoclonal antibody in this application can react specifically with Vero cells infected with PPRV Nigeria 75/1 strain, but does not react with normal Vero cells, indicating that The monoclonal antibody obtained in this application can react specifically with PPRV and has neutralizing activity, with a neutralizing titer of 1:50.1. It was further found that the monoclonal antibodies obtained in this application did not react with sheep herpes virus, bovine viral diarrhea virus type I, and bovine viral diarrhea virus type II, indicating that the monoclonal antibodies in this application have good specificity.

This application provides a hybridoma cell line that secretes monoclonal antibodies against the peste des petits ruminants virus, as well as ideas and methods for its monoclonal antibodies and applications. There are many specific methods and approaches for realizing this technical solution. The above are only the contents of this application. Preferred embodiment, it should be pointed out that for those of ordinary skill in the art, several improvements and modifications can be made without departing from the principles of the present application, and these improvements and modifications should also be regarded as the protection scope of the present application. . All components not specified in this embodiment can be implemented using existing technologies.

Claims (33)

1. A hybridoma cell, wherein the preservation number of the hybridoma cell strain is CGMCC No.45615.

2. The hybridoma cell according to claim 1, wherein the antigen to which the hybridoma cell binds is PPRV Nigeria 75/1 strain F protein.

3. The hybridoma cell according to claim 2, wherein the amino acid sequence of the epitope bound by the hybridoma cell comprises a sequence as shown in SEQ ID No. 1.

4. Use of a hybridoma cell line according to claims 1-3 for the preparation of a reagent for the detection and/or diagnosis of animals infected with peste des petits ruminants virus.

5. A monoclonal antibody, wherein the monoclonal antibody is produced by a hybridoma cell strain with a preservation number of CGMCC No. 45615.

6. The monoclonal antibody of claim 5, wherein the monoclonal antibody is capable of binding PPRV Nigeria 75/1 strain F protein.

7. The monoclonal antibody of claim 6, wherein the monoclonal antibody is capable of binding an epitope comprising the sequence shown as SEQ ID No. 1.

8. The monoclonal antibody of claims 5-7, wherein the antibody comprises:

a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:

CDR-H1 has the sequence SEQ ID NO 4: the amino acid sequence of SYGLS,

CDR-H2 has the sequence SEQ ID NO 5: the amino acid sequence of TITWNGGSTYYPDTVKG,

CDR-H3 has the sequence SEQ ID NO 6: the amino acid sequence of ESLLRLPAWFAY,

b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:

CDR-L1 has the sequence SEQ ID NO 7: the amino acid sequence of RASKSVSTSGYSYMH,

CDR-L2 has the sequence SEQ ID NO 8: the amino acid sequence of LVSNLES,

CDR-L3 has the sequence SEQ ID NO 9: the amino acid sequence of QHIRELYT.

9. A monoclonal antibody that binds PPRV Nigeria 75/1 strain F protein, wherein the antibody comprises:

a) An antibody heavy chain complementarity determining region comprising: CDR-H1, CDR-H2, CDR-H3, wherein:

the amino acid sequence of CDR-H1 is shown as SEQ ID NO 4: the expression "SYGLS" is used,

the amino acid sequence of CDR-H2 is shown as SEQ ID NO 5: as shown in figure TITWNGGSTYYPDTVKG,

the amino acid sequence of CDR-H3 is shown as SEQ ID NO 6: as shown in figure ESLLRLPAWFAY,

b) An antibody light chain complementarity determining region comprising: CDR-L1, CDR-L2, CDR-L3, wherein:

the amino acid sequence of CDR-L1 is shown as SEQ ID NO 7: as shown in figure RASKSVSTSGYSYMH,

the amino acid sequence of CDR-L2 is shown as SEQ ID NO 8: as shown in the LVSNLES,

the amino acid sequence of CDR-L3 is shown as SEQ ID NO 9: QHIRELYT.

10. The monoclonal antibody of claim 8 or 9, wherein the antibody comprises an antibody heavy chain variable region HCVR as set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR as set forth in SEQ ID NO 11, wherein:

HCVR has the sequence SEQ ID NO 10: EVKLVESGGGLVQPGGSLKLSCAASGFTFSSYGLSWVRQTPDKRLELVATITWNGGSTYYPDTVKGRFTISRDSAKNILYLQMSSLKSEDTAMYYCARESLLRLPAWFAYWGQGTLVTVSA amino acid sequence;

LCVR has the sequence SEQ ID NO 11:

DIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELYTFGGGTKLEIK.

11. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 90% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 90% identity to the amino acid sequence set forth in SEQ ID NO 11.

12. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 91% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 91% identity to the amino acid sequence set forth in SEQ ID NO 11.

13. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 92% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 92% identity to the amino acid sequence set forth in SEQ ID NO 11.

14. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 93% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 93% identity to the amino acid sequence set forth in SEQ ID NO 11.

15. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 94% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 94% identity to the amino acid sequence set forth in SEQ ID NO 11.

16. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 95% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 95% identity to the amino acid sequence set forth in SEQ ID NO 11.

17. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 96% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 96% identity to the amino acid sequence set forth in SEQ ID NO 11.

18. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 97% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 97% identity to the amino acid sequence set forth in SEQ ID NO 11.

19. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 98% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 98% identity to the amino acid sequence set forth in SEQ ID NO 11.

20. The monoclonal antibody of claim 10, wherein the antibody further comprises an antibody heavy chain variable region HCVR having 99% identity to the amino acid sequence set forth in SEQ ID NO 10 and an antibody light chain variable region LCVR having 99% identity to the amino acid sequence set forth in SEQ ID NO 11.

21. The monoclonal antibody of claim 8 or 9, wherein the antibody comprises a heavy chain as set forth in SEQ ID NO 12 and a light chain as set forth in SEQ ID NO 13, wherein:

the heavy chain amino acid sequence is SEQ ID NO 12:

AKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGKL amino acid sequence;

The light chain amino acid sequence is SEQ ID NO 13: RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC.

22. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 90% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 90% identity to the amino acid sequence set forth in SEQ ID NO 13.

23. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 91% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 91% identity to the amino acid sequence set forth in SEQ ID NO 13.

24. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 92% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 92% identity to the amino acid sequence set forth in SEQ ID NO 13.

25. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 93% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 93% identity to the amino acid sequence set forth in SEQ ID NO 13.

26. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 94% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 94% identity to the amino acid sequence set forth in SEQ ID NO 13.

27. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 95% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 95% identity to the amino acid sequence set forth in SEQ ID NO 13.

28. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 96% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 96% identity to the amino acid sequence set forth in SEQ ID NO 13.

29. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 97% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 97% identity to the amino acid sequence set forth in SEQ ID NO 13.

30. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 98% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 98% identity to the amino acid sequence set forth in SEQ ID NO 13.

31. The monoclonal antibody of claim 21, wherein the antibody further comprises a heavy chain having 99% identity to the amino acid sequence set forth in SEQ ID NO 12 and a light chain having 99% identity to the amino acid sequence set forth in SEQ ID NO 13.

32. Use of a monoclonal antibody according to claim 5 or 9 for the preparation of a reagent for detecting and/or diagnosing animals infected with peste des petits ruminants virus.

33. A kit for detecting and/or diagnosing a peste des petits ruminants virus infection in an animal, comprising: the monoclonal antibody of any one of claims 5 to 31 or an antibody produced by the hybridoma of claims 1 to 3.