CN114805589A - Monoclonal antibody capable of simultaneously recognizing antibodies of cattle, goats and sheep - Google Patents

Monoclonal antibody capable of simultaneously recognizing antibodies of cattle, goats and sheep Download PDF

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Publication number
CN114805589A
CN114805589A CN202210668484.1A CN202210668484A CN114805589A CN 114805589 A CN114805589 A CN 114805589A CN 202210668484 A CN202210668484 A CN 202210668484A CN 114805589 A CN114805589 A CN 114805589A
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monoclonal antibody
sheep
amino acid
acid sequence
cattle
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CN114805589B (en
Inventor
张春鸽
王格
赵巧辉
柴丹丹
徐延伟
李桂林
付光宇
杨增利
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Zhengzhou Immuno Biotech Co Ltd
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Zhengzhou Immuno Biotech Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/42—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins
    • C07K16/4208—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig
    • C07K16/4241—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against immunoglobulins against an idiotypic determinant on Ig against anti-human or anti-animal Ig
    • 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
    • G01N33/56911—Bacteria
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • 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
    • 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
    • G01N2333/08—RNA viruses
    • G01N2333/085—Picornaviridae, e.g. coxsackie virus, echovirus, enterovirus
    • G01N2333/09—Foot-and-mouth disease virus
    • 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
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/23—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Brucella (G)
    • 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
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Abstract

The invention relates to the technical field of in-vitro diagnosis, in particular to a monoclonal antibody capable of simultaneously recognizing antibodies of cattle, goats and sheep. The invention provides a monoclonal antibody, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence shown as SEQ ID No. 1. The monoclonal antibody can identify bovine serum, goat serum, sheep serum and antibodies thereof; can also be used as a secondary antibody for goat and sheep polyclonal antibody development and detection; can also be used as a secondary antibody of a bovine and ovine immune antibody detection kit.

Description

Monoclonal antibody capable of simultaneously recognizing antibodies of cattle, goats and sheep
Technical Field
The invention relates to the technical field of in vitro diagnosis, in particular to a monoclonal antibody capable of simultaneously identifying antibodies of cattle, goats and sheep.
Background
Immunoglobulins are globulins with antibody activity or chemical structure similar to antibodies, which are mainly present in blood and certain secretions. Immunoglobulins generally have antibody activity and specifically bind to the corresponding antigenic determinant, but are themselves antigenic substances for other species of animals. Immunoglobulins are produced in different genetic backgrounds and also in different antigenicities, and they can generate immune responses in xenogeneic, allogeneic and the same body, producing corresponding antibodies (anti-antibodies). Human immunoglobulins can be classified into five classes, IgG, IgA, IgM, IgD and IgE, depending on the antigenic specificity of the heavy chain constant region of the immunoglobulin. Wherein IgG is immunoglobulin with the highest content in serum and accounts for 75-80% of the total amount of the serum. The content of immunoglobulin in normal human is relatively constant. Only in certain disease cases can the immunoglobulin side undergo qualitative or quantitative changes.
The IgG species heterogeneity is derived from the 1/2 (about 105 amino acid residues) located near the C-terminus of the L chain and the 3/4 or 4/5 region (about 119 amino acids to the C-terminus) of the H chain, which are located near the C-terminus of the immunoglobulin constant region (C region). The composition and arrangement of amino acids in this region are relatively constant in both the Ig isotype L chain and the H chain of the same species in animals of the same species. Because the presence of such species specificity results in interspecies heterogeneity of immunoglobulins, which causes immune responses between species.
When the in vivo IgG antibody concentration is measured by ELISA, a secondary antibody is usually used.
The advantage of polyclonal antibody lies in that the site coverage rate of human IgG protein is high, and theoretically, the detection process basically has no possibility of missed detection. Meanwhile, the sensitivity of the kit produced by oneself can be improved by utilizing the high affinity of the multi-antibody.
Along with the advantages of the application of the polyclonal antibody, the development and research of the polyclonal antibody are more in recent years, and when the polyclonal antibody is developed, a mouse-anti-sheep monoclonal antibody is often used as a secondary antibody for screening.
The sheep monoclonal antibody has the advantage of strong affinity, the development and research of the sheep monoclonal antibody are more recently, and the mouse anti-sheep monoclonal antibody is often used as a secondary antibody in screening when the sheep monoclonal antibody is developed.
The animal is usually used for disease preventive vaccination, and the affected antibody detection is carried out. The indirect detection kit can use secondary antibodies of mouse-resistant sheep, mouse-resistant cattle and the like. If a secondary antibody can simultaneously react with monoclonal antibodies of goats, sheep and cattle, a kit can be developed, and the kit is universal for the cows and the sheep.
Disclosure of Invention
In view of this, the present invention provides a monoclonal antibody capable of simultaneously recognizing antibodies of cattle, goats and sheep, and the monoclonal antibody of the present invention is suitable for preparation of a reagent and/or a kit for cattle and sheep.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a monoclonal antibody, wherein the heavy chain variable region of the monoclonal antibody is as follows:
(I) has an amino acid sequence shown as SEQ ID No. 1; and/or
Its light chain variable region:
(II) has an amino acid sequence shown as SEQ ID No. 2; and/or
Its heavy chain variable region and/or light chain variable region:
(III) has an amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in the (I) or (II), and the function of the amino acid sequence is the same as or similar to that of the amino acid sequence shown in the (I) or (II); and/or
(IV) an amino acid sequence having at least 80% homology with any of the amino acid sequences shown in (I) to (III);
the number of the plurality is 2 to 30.
The invention also provides application of the monoclonal antibody in preparing a reagent and/or a kit for recognizing the antibody and/or the serum of the cattle, the goat and/or the sheep.
In some embodiments of the invention, the bovine, goat and/or sheep antibodies are derived from body fluids or exudates.
The invention also provides application of the monoclonal antibody in preparation of a universal immunological detection reagent and/or a universal immunological detection kit for cattle and sheep.
The invention also provides application of the monoclonal antibody in preparation of a reagent and/or a kit for detecting cattle and sheep diseases.
In some embodiments of the present invention, the cattle and sheep diseases in the above-mentioned application include foot-and-mouth disease and/or brucellosis.
In some embodiments of the present invention, the diseases of cattle and sheep further include bacterial diseases, viral diseases and parasitic diseases of cattle and sheep; the bacterial diseases include clostridial disease, pasteurellosis, salmonellosis, listeriosis, tetanus, and streptococcosis; the virus diseases comprise cattle plague, cattle infectious diarrhea, cattle infectious rhinotracheitis, cattle leukemia, cattle epidemic fever, cattle coronavirus diseases, bluetongue disease, rift valley fever, Peste des petits ruminants diseases, cattle and sheep pox, and cattle sarcoidosis; the parasitic diseases include pyrosis, helminthiasis, cattle and sheep nasal fly disease, taeniasis, ascariasis, and enterobiasis.
The invention also provides a preparation method of the monoclonal antibody, which comprises the following steps:
immunizing a mouse by adopting bovine IgG as immunogen;
fusing splenocytes of the mice obtained in the step (a) with myeloma cells to obtain hybridoma cells;
screening and purifying the hybridoma cells obtained in the step (b);
step (d) obtaining said monoclonal antibody from a culture of said hybridoma cells of step (c);
the mice include BALB/c mice; and/or
The myeloma cells include myeloma cell NS 1.
In some embodiments of the present invention, the conditions for screening in the above preparation method include: the monoclonal antibodies secreted by the hybridoma cells can specifically bind to bovine, goat and/or sheep antibodies.
The invention also provides a reagent which comprises the monoclonal antibody and acceptable auxiliary materials and/or auxiliary agents.
The invention also provides a kit which comprises the monoclonal antibody or the reagent and acceptable auxiliary materials and/or auxiliary agents.
The invention also provides a device coated with the monoclonal antibody or the reagent, and acceptable components.
The invention relates to a mouse monoclonal antibody for identifying bovine serum, goat serum, sheep serum and antibodies thereof, which is characterized in that: the mouse monoclonal antibody is named as a second antibody 4C-1, and has a heavy chain variable region with an amino acid sequence shown in SEQ ID No.1 and a light chain variable region with an amino acid sequence shown in SEQ ID No. 2.
The mouse monoclonal antibody for identifying the bovine serum, the goat serum, the sheep serum and the antibody thereof is obtained by taking bovine IgG as immunogen to immunize a BALB/c mouse, then taking spleen and myeloma cell NS1 for fusion, and screening and purifying.
The invention has the following effects:
the monoclonal antibody has the advantage of universality, and the murine monoclonal antibody can identify bovine serum, goat serum, sheep serum and antibodies thereof. Can be used as a universal secondary antibody for goat and sheep polyclonal antibody and monoclonal antibody development and detection. Can be used as a general secondary antibody of a bovine and ovine immune antibody detection kit, and contains foot-and-mouth disease, brucellosis and the like.
1. The result of detecting the titer of the mouse monoclonal antibody shows that the titer of the monoclonal antibody prepared by the invention is all up to 1/256000 by adopting a bovine IgG, a goat IgG and a sheep IgG coated enzyme immune plate to detect, and the titer of the monoclonal antibody is higher;
2. the ELISA detection results of the monoclonal antibody and the bovine, goat and sheep antibodies show that the murine anti-goat secondary antibody has weak reactivity to the sheep antibody IgG and the bovine serum IgG; the mouse anti-sheep secondary antibody has weak reactivity to goat serum IgG and bovine serum IgG; the monoclonal antibody prepared by the invention has strong reactivity to sheep antibody IgG, goat serum IgG and bovine serum IgG.
Detailed Description
The invention discloses a monoclonal antibody capable of simultaneously recognizing antibodies of cattle, goats and sheep, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
Unless otherwise specified, the reagents used in the present invention are conventional, and the apparatus and method used are also conventional in the art. In the detection reagent and the detection kit provided by the invention, the used raw materials and reagents can be purchased from the market.
The invention is further illustrated by the following examples:
example 1: preparation of murine monoclonal antibody
1. Immunization of mice
Fully emulsifying bovine IgG by Freund complete adjuvant, and immunizing female BALB/c mice of 5 weeks in abdominal cavity, wherein the priming dose is 150 mu g per mouse; after the first immunization, the second and third immunizations were performed at intervals of 21 days and 42 days, respectively, and the immunization doses were 70. mu.g/mouse. After the third immunization, blood was collected from the tail about 10 days, and the serum titer was measured by an indirect method using a 96-well plate coated with bovine IgG.
2. Hybridoma cell preparation
Selective indirect method for detecting serum titer greater than 10 4 The mice were boosted intraperitoneally at an immunization dose of 100. mu.g/mouse. The spleen of the mouse 3 days after the booster immunization was fused with a mouse myeloma cell NS1 at a ratio of 10:1, and the fused cells were cultured in a HAT-containing DMEM medium (Gibco).
And (3) about 6-7 days after fusion, detecting the content of specific antibodies in cell culture supernatant by using a 96-well plate coated with bovine IgG, goat IgG and sheep IgG, and performing 3 rounds of subcloning by using a limiting dilution method by using positive wells with reaction OD values not lower than 0.5 in the three 96-well plates respectively to finally obtain the hybridoma cell strain 4C-1 capable of stably secreting anti-bovine IgG.
3. Purification of murine monoclonal antibodies
Injecting the obtained mouse hybridoma cells capable of stably secreting the monoclonal antibody into the abdominal cavity of a mouse, collecting ascites, and purifying by SPA to obtain the anti-mouse monoclonal antibody with the purity of more than 90%.
4. Potency assay for murine monoclonal antibodies
Bovine IgG, goat IgG, and sheep IgG were diluted to 1 μ g/mL with 0.05mmol/L, pH ═ 9.6 CB buffer, and 50 μ L was added to each well of a 96-well enzyme-immune plate (Corning), and the plate was coated overnight at 4 ℃. The next day was washed 3 times with PBST and then blocked with 1% Casein at 100. mu.L/well for 2 hours at 37 ℃. The purified monoclonal antibody 4C-1 (at 5mg/mL in each case) was diluted at a ratio of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, … … in CB buffer 0.05mmol/L, pH ═ 9.6. The diluted antibodies were added to an enzyme-immune plate coated with bovine IgG, goat IgG, and sheep IgG, and 0.05mmol/L, pH ═ 9.6 CB buffer was added to the negative control wells, at 50 μ L/well. After 30 minutes of reaction at 37 ℃, the plates were washed 5 times with PBST, patted dry, and then reacted for 30 minutes at 37 ℃ with HRP-goat anti-mouse IgG (SIGMA) diluted at 1:2000, 100. mu.L/well. The plate was washed 5 times with PBST, patted dry, then the ordinary enzyme-free substrate was added, 100. mu.L/well was left for 10 minutes at room temperature in the dark, 50. mu.L of sulfuric acid at 0.1mol/L was added to terminate the reaction, and the absorbance at 450nm was measured. The results of the measurements are given in table 1 below:
TABLE 1 test results
Figure BDA0003693897700000051
As can be seen from Table 1, the OD value of the negative control is 0.01 on average, and the titer of the monoclonal antibody reaches above 1/256000 and shows higher titer when the bovine IgG, the goat IgG and the sheep IgG are detected by the enzyme-coated immunoplates.
Example 2: ELISA detection results of monoclonal antibody and bovine, goat and sheep antibodies
1. Magnetic particle coating
And (3) washing 30 mu L of the uniformly mixed stock solution of the magnetic particles with 300 mu L of PBS buffer solution for 5 times, activating the magnetic particles with 10% glutaraldehyde for 1 hour, and then washing the activated magnetic particles with PBS buffer solution with the pH value of 7-8 for 2 times. The bovine, goat and sheep antibodies were added to the beads at a rate of 0.3. mu.g/human and coated at 4 ℃ for 2 hours. Finally blocking with blocking solution containing BSA for 2 hours.
2. HRP-labeled mouse monoclonal antibody 4C-1
Monoclonal antibody 4C-1 was labeled with HRP at a mass ratio of 1.5: 1. The HRP-labeled monoclonal antibody 4C-1 was diluted to 1:4000 with a diluent.
3. Detection of magnetic particle chemiluminescence by direct methods. The results of the measurements are shown in Table 2 below:
TABLE 2 test results
Figure BDA0003693897700000061
And (4) analyzing results:
1. the detection result of the luminescence value of the mouse-anti-goat secondary antibody shows that the mouse-anti-goat secondary antibody reacts with goat IgG, the detection luminescence value is more than 100000, the detection luminescence value is lower than 5000, the mouse-anti-goat secondary antibody only reacts with the goat IgG, and the mouse-anti-goat secondary antibody can be regarded as not reacting with the goat IgG and the cow IgG;
2. the detection result of the luminescence value of the mouse anti-sheep secondary antibody shows that the mouse anti-sheep secondary antibody reacts with sheep IgG, the detection luminescence value is more than 100000, the detection luminescence value is lower than 5000, the mouse anti-sheep secondary antibody only reacts with the sheep IgG, and the mouse anti-sheep secondary antibody can be regarded as not reacting with the sheep IgG and the cattle IgG;
3. the secondary antibody 4C-1 screened by the invention reacts with goat IgG, sheep IgG and cattle IgG, the detected luminescence values are all above 100000, and the secondary antibody 4C-1 is proved to react with the goat IgG, the sheep IgG and the cattle IgG.
The mouse monoclonal antibody obtained by the invention is named as a second antibody 4C-1, and the amino acid sequences of a heavy chain variable region and a light chain variable region are as follows:
amino acid sequence of heavy chain variable region (SEQ ID No. 1):
QVQLKQSGPELVKPGASVKMSCKASGYTFTSYVIHWVKQKPGQGLEWIGYINPYNDNTEYNEKFKGKATLTSDKSSSTAYMELSSLTSEDSAVYYCARREGGYYVFTYWGQGTLVTVSA
amino acid sequence of light chain variable region (SEQ ID No. 2):
EMVLTQSPALMAASPGEKVTITCSVSSSISSSNFHWYQQKSETSPKPWIYGTSNLASGVPVRFSGSGSGTSYSLTISSMEAEDAATYYCQQWSSYPLTFGGGTKLEIK
the foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
<110> Zhengzhou Yimeino Biotechnology Ltd
<120> monoclonal antibody capable of simultaneously recognizing bovine, goat and sheep antibodies
<130> MP21028305
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 119
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Gln Val Gln Leu Lys Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr
20 25 30
Val Ile His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile
35 40 45
Gly Tyr Ile Asn Pro Tyr Asn Asp Asn Thr Glu Tyr Asn Glu Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Ser Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Arg Glu Gly Gly Tyr Tyr Val Phe Thr Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ala
115
<210> 2
<211> 108
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Glu Met Val Leu Thr Gln Ser Pro Ala Leu Met Ala Ala Ser Pro Gly
1 5 10 15
Glu Lys Val Thr Ile Thr Cys Ser Val Ser Ser Ser Ile Ser Ser Ser
20 25 30
Asn Phe His Trp Tyr Gln Gln Lys Ser Glu Thr Ser Pro Lys Pro Trp
35 40 45
Ile Tyr Gly Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser
50 55 60
Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Met Glu
65 70 75 80
Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Tyr Pro
85 90 95
Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
100 105

Claims (10)

1. A monoclonal antibody characterized by having a heavy chain variable region:
(I) has an amino acid sequence shown as SEQ ID No. 1; and/or
Its light chain variable region:
(II) has an amino acid sequence shown as SEQ ID No. 2; and/or
Its heavy chain variable region and/or light chain variable region:
(III) has an amino acid sequence obtained by substituting, deleting or adding one or more residues in the amino acid sequence shown in the (I) or (II), and the function of the amino acid sequence is the same as or similar to that of the amino acid sequence shown in the (I) or (II); and/or
(IV) an amino acid sequence having at least 80% homology with any of the amino acid sequences shown in (I) to (III);
the number of the plurality is 2 to 30.
2. Use of a monoclonal antibody according to claim 1 for the preparation of a reagent and/or kit for the recognition of bovine, goat and/or sheep antibodies and/or serum.
3. The use of the monoclonal antibody of claim 1 in the preparation of a universal immunological detection reagent and/or kit for cattle and sheep.
4. The use of the monoclonal antibody of claim 1 in the preparation of a reagent and/or a kit for detecting cattle and sheep diseases.
5. The use of claim 4, wherein the bovine or ovine disease comprises foot and mouth disease and/or brucellosis.
6. The method of producing a monoclonal antibody according to claim 1, comprising the steps of:
immunizing a mouse by adopting bovine IgG as immunogen;
fusing splenocytes of the mice obtained in the step (a) with myeloma cells to obtain hybridoma cells;
screening and purifying the hybridoma cells obtained in the step (b);
step (d) obtaining said monoclonal antibody from a culture of said hybridoma cells of step (c);
the mice include BALB/c mice; and/or
The myeloma cells include myeloma cell NS 1.
7. The method of claim 6, wherein the screening conditions comprise: the monoclonal antibodies secreted by the hybridoma cells can specifically bind to bovine, goat and/or sheep antibodies.
8. Reagent, characterized in that it comprises the monoclonal antibody according to claim 1 and acceptable adjuvants and/or adjuvants.
9. Kit comprising the monoclonal antibody according to claim 1 or the reagent according to claim 8, together with acceptable adjuvants and/or adjuvants.
10. Device, characterized in that it is coated with the monoclonal antibody according to claim 1 or the reagent according to claim 8, and acceptable means.
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