CN112521493A

CN112521493A - Anti-foot-and-mouth disease O-type virus monoclonal antibody and application thereof

Info

Publication number: CN112521493A
Application number: CN201910881627.5A
Authority: CN
Inventors: 田克恭; 燕贺
Original assignee: Luoyang Pu Tai Biotechnology Co ltd
Current assignee: Luoyang Pu Tai Biotechnology Co ltd
Priority date: 2019-09-18
Filing date: 2019-09-18
Publication date: 2021-03-19
Anticipated expiration: 2039-09-18
Also published as: CN112521493B

Abstract

The invention provides monoclonal antibodies 6G6 and 7D7, which can be specifically combined with different genetic topology type O-type foot-and-mouth disease viruses, and the monoclonal antibodies can be used for carrying out high-sensitivity detection on the different genetic topology type O-type foot-and-mouth disease viruses by using ELISA detection kits prepared from 6G6 and 7D 7.

Description

Anti-foot-and-mouth disease O-type virus monoclonal antibody and application thereof

Technical Field

The invention relates to a monoclonal antibody capable of reacting with a foot-and-mouth disease O-type virus, a variable region sequence of the monoclonal antibody, a hybridoma cell strain secreting the monoclonal antibody, a kit prepared by using the monoclonal antibody and application, belonging to the field of antibody-containing medical preparations and immunoassay methods.

Background

Foot-and-mouth disease (FMD) is an acute and highly contagious disease caused by foot-and-mouth disease virus, even-hoof animals are most susceptible to the disease, the outbreak and the epidemic of the disease bring huge economic losses to the global breeding industry, and the animal health organization in the world classifies the disease as a type of infectious disease. Foot and mouth disease viruses are divided into 7 serotypes, namely A type, O type, Asia I type, C type, SAT1 type, SAT2 type and SAT3 type, wherein the O type foot and mouth disease virus is most prevalent. According to genetic classification, three kinds of genetic topological type (Topo type) O type foot-and-mouth disease viruses mainly prevail in China at present and respectively belong to CATHOY type (Chinese type), ME-SA type (middle east-south subtype) and SEA type (southeast subtype). At present, the prevention and control of the foot-and-mouth disease are mainly based on inactivated vaccine immunization, and the monitoring of the antibody level after the vaccine immunization is an effective means for controlling the vaccine immunization quality, so that the effect of preventing the disease is achieved.

A common reagent kit for detecting the antibody level in the market is a liquid phase blocking ELISA reagent kit, and the reagent kit adopts a polyclonal antibody which has the problems of large batch-to-batch difference and poor stability. The monoclonal antibody has single biological activity, is easy to produce and standardize, the O-type foot-and-mouth disease is popular in China and belongs to different genetic topotypes, but a solid-phase blocking ELISA kit with better detection capability aiming at different genetic topotypes of antibodies is not available all the time, so that the screening of a proper monoclonal antibody has important significance for the diagnosis and prevention of the disease.

Disclosure of Invention

In order to solve the defects of the prior art, the invention provides a monoclonal antibody reacting to O-type foot-and-mouth disease virus, and the monoclonal antibody is combined with the same sites of different genetic topology type O-type foot-and-mouth disease viruses; a monoclonal antibody reacting with different genetic topological type O type foot-and-mouth disease viruses simultaneously; and a kit containing the monoclonal antibody and application thereof.

The invention relates to a variable region sequence of a monoclonal antibody 6G6 which reacts to O-type foot-and-mouth disease viruses and is combined with the same sites of different genetic topological O-type foot-and-mouth disease viruses, wherein the variable region of a heavy chain of the monoclonal antibody 6G6 is a sequence shown in SEQ.ID No.1 or a degenerate sequence code thereof or a conservative variant obtained by conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications; the variable region of the monoclonal antibody 6G6 light chain is a conservative variant obtained by coding the sequence shown in SEQ ID No.2 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications.

The variable region sequence of 6G6 of the invention can specifically combine with different genetic topology type O type foot-and-mouth disease viruses and can detect the different genetic topology type O type foot-and-mouth disease viruses in a broad spectrum way.

The invention relates to an antibody or an antibody fragment which reacts to O-type foot-and-mouth disease viruses and is combined with the same sites of the O-type foot-and-mouth disease viruses with different genetic topologies, wherein the heavy chain variable region of the antibody or the antibody fragment is a sequence shown in SEQ.ID No.1 or a degenerate sequence code thereof or a conservative variant obtained by adding, deleting, replacing or modifying conservative mutation of one or more amino acids; and the variable region of the light chain of the antibody or the antibody fragment is a conservative variant obtained by the sequence shown in SEQ ID No.2 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications.

As an embodiment of the present invention, the antibody is a monoclonal antibody, a genetically engineered antibody; wherein the genetically engineered antibody comprises a single chain antibody, a chimeric monoclonal antibody, a reshaped monoclonal antibody, or a fragment of the antibody; the antibodies or fragments of the antibodies still retain the ability to specifically bind to different genetic topotype type O foot and mouth disease viruses.

As a preferred embodiment of the present invention, the antibody is monoclonal antibody 6G6, wherein the heavy chain variable region of monoclonal antibody 6G6 is a conservative variant obtained by coding the sequence shown in seq.id No.1 or a degenerate sequence thereof or conservative mutation thereof by one or more amino acid additions, deletions, substitutions or modifications; the variable region of the monoclonal antibody 6G6 light chain is a conservative variant obtained by coding the sequence shown in SEQ ID No.2 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications.

The invention also relates to hybridoma cell 6G6 strain secreting said monoclonal antibody 6G 6.

The invention also relates to a variable region sequence of the monoclonal antibody 7D2 which can be simultaneously and specifically combined with different genetic topology type O type foot-and-mouth disease viruses, wherein the variable region of the heavy chain of the monoclonal antibody 7D2 is a sequence shown in SEQ.ID No.3 or a degenerate sequence code thereof; the variable region of the monoclonal antibody 7D2 light chain is encoded by the sequence shown in SEQ ID No.4 or a degenerate sequence thereof.

The variable region sequence of the 7D2 can be specifically combined with different genetic topological type O type foot-and-mouth disease viruses, and can be used for detecting the different genetic topological type O type foot-and-mouth disease viruses in a broad spectrum manner.

The invention also relates to an antibody or an antibody fragment which can be simultaneously and specifically combined with different genetic topological type O-type foot-and-mouth disease viruses, wherein the variable region of the heavy chain of the antibody or the antibody fragment is a sequence shown in SEQ ID No.3 or a coding sequence of a degenerate sequence thereof or a conservative variant thereof obtained by conservative mutation of one or more amino acids through addition, deletion, substitution or modification; and the variable region of the light chain of the antibody or the antibody fragment is a conservative variant obtained by the sequence shown in SEQ ID No.4 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications.

As a preferred embodiment of the present invention, the antibody is monoclonal antibody 7D2, wherein the heavy chain variable region of monoclonal antibody 7D2 is encoded by the sequence shown in SEQ ID No.3 or a degenerate sequence thereof; the variable region of the monoclonal antibody 7D2 light chain is encoded by the sequence shown in SEQ ID No.4 or a degenerate sequence thereof.

The present invention also relates to hybridoma cell strain 7D2, which secretes monoclonal antibody 7D2 as claimed in claim 7.

The invention also relates to an ELISA detection kit, wherein the kit comprises an effective amount of coating antibody, the coating antibody is the monoclonal antibody 6G6, the O-type foot-and-mouth disease virus antigen and the labeled antibody, the labeled antibody is the monoclonal antibody 7D2, and the detection reagent for antigen-antibody reaction detection, wherein the effective amount of the coating antibody is the monoclonal antibody 6G6, the effective amount of the O-type foot-and-mouth disease virus antigen and the effective amount of the labeled antibody are the monoclonal antibody 7D2, and the detection reagent for antigen-antibody reaction.

The ELISA detection kit can carry out high-sensitivity detection on different genetic topology type O type foot-and-mouth disease viruses.

As an embodiment of the invention, said monoclonal antibody 6G6 is coated on a support medium, and said foot-and-mouth disease virus type O antigen is bound to said monoclonal antibody 6G 6; preferably, the support medium is a microtiter plate.

In one embodiment of the invention, the content of the monoclonal antibody 6G6 is 1-8 mug/ml, the content of the O-type foot-and-mouth disease virus antigen is 50-400 mug/ml, and the content of the monoclonal antibody 7D2 is 3-10 mug/ml; preferably, the O type foot-and-mouth disease virus antigen content is 200 mug/ml.

As an embodiment of the invention, the ELISA detection kit further comprises a sample diluent, a washing solution, a developing solution, a stop solution, a positive pig serum control and a negative pig serum control.

As an embodiment of the present invention, the sample diluent is a phosphate buffer containing skim milk, preferably a phosphate buffer containing 10% (W/V) skim milk; the washing solution is phosphate buffer solution containing Tween-20, preferably phosphate buffer solution containing 0.05% (V/V) Tween-20; the color development liquid comprises color development liquid A and color development liquid B, wherein the color development liquid A contains 20mg of TMB and 10ml of absolute ethyl alcohol, and ddH is used₂O is added to 100ml to fix the volume; the color developing solution B contains 2.1g of citric acid and anhydrous Na₂HPO₄2.82g, 0.75% (V/V) Urea hydrogen peroxide 0.64ml, in ddH₂O is added to 100ml to fix the volume; the stop solution is 2mol/L H₂SO₄(ii) a The positive control is hyperimmune serum of the O-type foot-and-mouth disease virus antigen, and the negative control is pig serum without foot-and-mouth disease antibodies.

The pH value of the phosphate buffer solution is 7.4, and the volume formula of 1L of the phosphate buffer solution is as follows: NaCl 8.0g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄ 0.24g。

The term "phosphate buffer" refers to a solution containing phosphoric acid or a salt thereof and adjusted to a desired pH, and is one of the most widely used buffers in biochemical studies. Typically, phosphate buffers are prepared from phosphoric acid or phosphates (including but not limited to sodium and potassium salts). Some phosphates are known in the art, such as sodium and potassium dihydrogen phosphate, disodium and dipotassium hydrogen phosphate, sodium and potassium phosphate. Phosphate salts are known to exist as hydrates of salts. The buffered pH ranges widely, for example, from about 4 to about 10, preferably from about 5 to about 9, more preferably from about 6 to about 8, and most preferably about 7.4, due to secondary dissociation of the buffer. Further preferably, the phosphate buffer is a phosphate buffer containing sodium chloride and potassium chloride.

Detailed Description

Hereinafter, embodiments of the present invention will be described.

The term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical, except for the possible presence of a small number of possible spontaneous mutations. Thus, the modifier "monoclonal" indicates that the antibody is not a mixture of discrete antibodies in nature. Preferably, the monoclonal antibodies include monovalent or single chain antibodies, diabodies, chimeric antibodies, porcine-derived antibodies, as well as derivatives, functional equivalents and homologues of the above, and also antibody fragments and any polypeptides containing an antigen binding domain. An antibody is any specific binding member which encompasses a binding domain with the desired specificity, and thus, this term encompasses antibody fragments, derivatives, porcine-derived antibodies, and functional equivalents and homologues of antibodies homologous thereto, as well as any polypeptide, whether natural or synthetically produced, which contains an antigen-binding domain. Examples of antibodies are immunoglobulin subtypes (e.g., IgG, IgE, IgM, IgD and IgA) and subclasses thereof; or a fragment comprising an antigen binding domain such as Fab, scFv, Fv, dAb, Fd; and diabodies (diabodies). Chimeric molecules comprising an antigen binding domain fused to another polypeptide or an equivalent are also included. Cloning and expression of chimeric antibodies is described in ep.a.0120694 and ep.a.0125023. Antibodies can be modified in a number of ways and recombinant DNA techniques can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody. Such techniques may involve the introduction of DNA encoding the immunoglobulin variable regions or Complementarity Determining Regions (CDRs) of antibodies into the constant regions or constant region plus framework regions of different immunoglobulins, see ep.a.184187, GB2188638A or ep.a.239400. Genetic mutations or other changes may also be made to the hybridoma cells or other cells that produce the antibody, which may or may not alter the binding specificity of the produced antibody. "monoclonal antibodies" useful in the present invention can also be produced by the hybridoma method, since the DNA sequence encoding the murine antibody of the present invention can be obtained by conventional means well known to those skilled in the art, such as artificial synthesis of the amino acid sequence according to the present disclosure or by PCR amplification, and thus can also be obtained by recombinant DNA methods, and the sequence can be ligated into an appropriate expression vector by various methods well known in the art. Finally, the transformed host cell is cultured under conditions suitable for the expression of the antibody of the present invention, and then purified by a person skilled in the art using a conventional separation and purification means well known to those skilled in the art to obtain the monoclonal antibody of the present invention. Antibodies comprise polypeptide chain geometries linked together by disulfide bridges, with the two polypeptide backbones, termed light and heavy, constituting all major structural classes (isoforms) of antibodies. Both the heavy and light chains can be further divided into subregions known as variable and constant regions. The heavy chain comprises a single variable region and three different constant regions, and the light chain comprises a single variable region (different from the variable region of the heavy chain) and a single constant region (different from the constant region of the heavy chain). The variable regions of the heavy and light chains are responsible for the binding specificity of the antibody.

The term "heavy chain variable region" refers to a polypeptide of 110 to 125 amino acids in length whose amino acid sequence corresponds to the amino acid sequence of the heavy chain of a monoclonal antibody of the invention starting from the N-terminal amino acid of the heavy chain. Similarly, the term "light chain variable region" refers to a polypeptide of 95 to 115 amino acids in length whose amino acid sequence corresponds to the light chain amino acid sequence of the monoclonal antibody of the invention starting from the N-terminal amino acid of the light chain. It is obvious to those skilled in the art that, based on the amino acid sequences of the heavy chain variable region and the light chain variable region of the monoclonal antibody specifically disclosed in the present invention, modifications such as addition, deletion, substitution and the like of one or more amino acids can be performed by conventional genetic engineering and protein engineering methods to obtain conservative variants, while still maintaining specific binding with type O foot-and-mouth disease. The monoclonal antibodies of the invention also include active fragments or conservative variants thereof.

The term "conservative variant" refers to a variant that substantially retains the characteristics of its parent, such as basic immunological biological, structural, regulatory, or biochemical characteristics. Generally, the amino acid sequence of a conservative variant of a polypeptide differs from the parent polypeptide, but the difference is limited so that the sequence with the parent polypeptide is very similar to the conservative variant overall and is identical in many regions. The difference in amino acid sequence between the conservative variant and the parent polypeptide can be, for example: substitutions, additions, and deletions of one or more amino acid residues and any combination thereof. The amino acid residue that is substituted or inserted may or may not be encoded by the genetic code. A conservative variant of a polypeptide may occur naturally, or it may be a non-naturally occurring variant. Non-naturally occurring conservative variants of the polypeptide may be generated by mutagenesis techniques or by direct synthesis.

The term "effective amount" when understood as "detecting an effective amount" refers to an amount that enables a positive sample to be detected with high sensitivity and a negative sample to be distinguished.

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.

The chemical reagents used in the examples of the present invention are all analytical reagents and purchased from the national pharmaceutical group. The experimental methods are conventional methods unless specified otherwise; the biomaterial is commercially available unless otherwise specified.

The antigen used in the embodiment of the invention is foot-and-mouth disease O type virus-like particles disclosed in Chinese patent CN 105566449A.

EXAMPLE 1 obtaining hybridoma cells

1. Immunization of mice

Foot-and-mouth disease O-type virus-like particles are used as immunogen to immunize 5-6 week-old Balb/c mice in a mode of back subcutaneous multipoint injection, and the injection amount of each injection is 200 mu g. The first immunization uses a Freund complete adjuvant, then the boosting immunization uses a Freund incomplete adjuvant, the immunization time interval is two weeks each time, the serum of the mouse is collected 1 week after the three immunizations, the antibody titer (blue animal research liquid phase blocking ELISA kit) is detected, when the antibody titer is not less than 1:1440, the impact immunization is carried out, namely the tail vein is injected with the antigen, three days after the impact immunization, the spleen of the mouse is taken and fused with myeloma cells.

2. Cell fusion

Taking a mouse spleen under an aseptic condition, placing the mouse spleen on an aseptic 200-mesh screen for grinding to obtain a spleen cell suspension, mixing the spleen cell suspension with myeloma cells according to a ratio of 10:1, adding 1mL of PEG 1500 within 1 minute at 37 ℃, adding 1mL of DMEM culture solution preheated at 37 ℃ after acting for 1 minute to stop reaction, adding the PEG 1500 within 1 minute, acting for 1 minute, adding the DMEM culture solution preheated at 37 ℃ for 5 times in all (the fusion tube needs to be shaken in the whole fusion process), supplementing the preheated DMEM culture solution to 20mL, and centrifuging at 800rpm for 7 minutes after acting for 15 minutes at 37 ℃; discarding the supernatant, and resuspending with fetal bovine serum culture medium (adding HAT selection medium); adding the cells into a 96-well plate, wherein each well contains 50 mu L of the cells; place the plates at 37 ℃ 5% CO₂Culturing in an incubator. Half-changes were made on day 3 and day 6 with 20% fetal bovine serum culture medium (plus HAT selection medium) and supernatants were removed to detect specific antibodies when hybridoma cells were observed to grow to about 1/5% bottom of the wells.

3. Screening for hybridoma cells

According to the specification of a liquid phase blocking ELISA detection kit for O-type antibodies of the foot-and-mouth disease virus (FMDV) ground by Lankant, the hybridoma cells detected to be positive are counted by using 20% fetal bovine serum culture solution (added with HT selective medium, Sigma), the cells are adjusted to be 3-5 cells/ml, 100 mu l of diluted cells are dropwise added into each hole, then 100 mu l of HT selective medium is added into each hole, and the mixture is placed at 37 ℃ and 5% CO₂An incubator; half-change of 20% fetal bovine serum culture medium (supplemented with HT selection medium, Sigma) was performed on days 3 and 6, and the formation of cell clones was observed to detect antibody activity in time. After subcloning to obtain positive monoclonal cells, they were subcultured on an expansion scale at 2X 10⁶Cell/branch in liquidStoring in nitrogen. Finally, ten hybridoma cells are screened and named as hybridoma cell 1, hybridoma cell 2, hybridoma cell 3, hybridoma cell 4, hybridoma cell 5, hybridoma cell 6, hybridoma cell 7, hybridoma cell 8, hybridoma cell 9 and hybridoma cell 10.

EXAMPLE 2 preparation and characterization of monoclonal antibodies

1. Preparation of monoclonal antibody ascites

0.5mL of sterile liquid paraffin is used for sensitizing 7-8 weeks old Balb/c mice, and 0.5mL (about 3.0 multiplied by 10) is injected into the abdominal cavity within 10-18 days⁶Individual cells/mL). Gently Rou the abdomen of the mouse every day after injection, and extract ascites when the abdomen of the mouse has significantly risen laterally. Centrifuging ascites at 10000rpm for 10min, collecting supernatant, packaging, and storing at-70 deg.C.

2. Hybridoma cell ascites titer determination

According to the specification of a liquid phase blocking ELISA detection kit for the O-type antibody of the foot-and-mouth disease virus of the Lanzhou animal, the antibody titers of the prepared monoclonal antibody 1, monoclonal antibody 2, monoclonal antibody 3, monoclonal antibody 4, monoclonal antibody 5, monoclonal antibody 6, monoclonal antibody 7, monoclonal antibody 8, monoclonal antibody 9 and monoclonal antibody 10 are respectively determined to be 1:16384, 1:11520, 1:32760, 1:16384, 1:5760, 1:11520, 1:16384, 1:11520 and 1: 5760. The monoclonal antibody 1, the monoclonal antibody 2, the monoclonal antibody 3, the monoclonal antibody 4, the monoclonal antibody 5, the monoclonal antibody 6, the monoclonal antibody 7, the monoclonal antibody 8, the monoclonal antibody 9 and the monoclonal antibody 10 are proved to have good reactivity with viruses, and can be used for developing diagnostic reagents.

EXAMPLE 3 purification and assay of monoclonal antibodies

The purification of the ascites of the hybridoma mouse is carried out by adopting affinity chromatography, and the specific operation is as follows: thawing frozen ascites sample, centrifuging at 10000rpm and 4 deg.C for 10min, sucking clear liquid, and loading with 3 column volumes of loading buffer (Binding buffer formula: 20mM Na)₂HPO₄0.15M NaCl, pH8.0), purifying with Protein G affinity column chromatography, eluting with glycine buffer solution of pH 2.50.1M, and washingThe stripped antibody was immediately adjusted to neutral pH with a neutralization buffer (1M Tris-HCl, pH8.5), and analyzed on SDS-PAGE gel and assayed for protein content, showing that: the purities of the purified monoclonal antibodies 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 are all more than 90%, the protein contents are respectively 3.3mg/ml, 3.5mg/ml, 3.6mg/ml, 3.2mg/ml, 3.3mg/ml, 3.5mg/ml, 3.3mg/ml, 3.2mg/ml, 3.8mg/ml and 3.0mg/ml, and the requirements of clinical application can be met.

EXAMPLE 4 labeling and paired detection of monoclonal antibodies

1. Horse Radish Peroxidase (HRP) labeling of monoclonal antibodies

The horseradish peroxidase is labeled by a sodium periodate method, and the specific operation is as follows: weighing 2mg of HRP and dissolving in 0.5ml of triple distilled water; adding 0.5ml of newly prepared 0.06mol/L NaIO into the supernatant₄The solution is kept in the dark at 4 ℃ for 30 min; adding 0.5ml of 160mmol/L ethylene glycol into the supernatant, and reacting at room temperature for 30 min; adding 2mg of aftosa O-type monoclonal antibody into the supernatant, mixing uniformly, filling into a treated dialysis bag, dialyzing in 1000ml of 0.05mmol/L sodium carbonate buffer solution, and standing at 4 ℃ overnight; absorbing the dialysate into a 10ml centrifuge tube, adding 0.25ml of newly prepared 5g/L NaBH 4 solution, uniformly mixing, and then placing at 4 ℃ for 2 h; adding saturated ammonium sulfate solution with the same volume, acting at 4 deg.C for 30min, centrifuging at 4 deg.C for 25min at 3000r/min, and removing supernatant; dissolving the precipitate in 1.5ml 0.02mol/L PBS pH7.4, sucking into dialysis bag, dialyzing in 0.02mol/L PBS pH7.4, and standing at 4 deg.C overnight; sucking the dialysate into a microfuge tube, centrifuging at 4 deg.C at 10000r/min for 30min, sucking out the supernatant, adding equal amount of glycerol, mixing, and storing at-20 deg.C.

2. Paired antibody Activity assay

The positive serum immunized by the antigen of the southeast subtype O-type foot-and-mouth disease virus-like particle is selected, different collocation modes are detected, and the result is shown in table 1.

TABLE 1 monoclonal antibody collocation and detection of activity on southeast subtype O-type foot-and-mouth disease

Note: + positive, -negative

The results show that the coated monoclonal antibody 1/the labeled monoclonal antibodies 2, 3, 6 and 7 are negative in detection; the coated monoclonal antibody 2/the marked monoclonal antibody 1, 3, 4, 5, 8 and 9 are negative in detection; the detection of the coating monoclonal antibody 3/the marked monoclonal antibody 2, 4, 6, 7, 9 and 10 is negative; the detection of the coating monoclonal antibody 4/the marked monoclonal antibody 2, 3, 5, 7, 8 and 10 is negative; the detection of the coating monoclonal antibody 5/the marked monoclonal antibodies 1, 6, 8 and 9 is negative; the coated monoclonal antibody 6/the labeled monoclonal antibody 2, 3, 5, 7 and 8 are negative in detection; the detection of the coating monoclonal antibody 7/the marked monoclonal antibody 2, 4, 5, 6, 9 and 10 is negative; the coated monoclonal antibody 8/the marked monoclonal antibody 1, 3, 4, 9 and 10 are negative in detection; the detection of the coating monoclonal antibody 9/the marked monoclonal antibodies 1, 3, 6, 7 and 10 is negative; the detection of the coating monoclonal antibody 10/the marked monoclonal antibodies 1, 4, 5 and 8 is negative; all other matches were positive.

Selecting Chinese type O foot-and-mouth disease virus-like particle antigen immune positive serum, detecting the positive detection matching mode, and displaying the result that only the coated monoclonal antibody 1/marked monoclonal antibody 10, the coated monoclonal antibody 1/marked monoclonal antibody 1 and the coated monoclonal antibody 10/marked monoclonal antibody 10 in the multi-pair combination are detected to be positive.

3. Nonspecific detection of paired antibodies

And (3) selecting positive serum immunized by the A type foot-and-mouth disease virus-like particle antigen, detecting the monoclonal antibody collocation modes (monoclonal antibody 1/labeled monoclonal antibody 10, coated monoclonal antibody 1/labeled monoclonal antibody 1 and coated monoclonal antibody 10/labeled monoclonal antibody 10) for simultaneously detecting southeast subtype O type foot-and-mouth disease positive and Chinese type O type foot-and-mouth disease positive, and obtaining results shown in table 2.

TABLE 2 monoclonal antibody collocation and nonspecific detection

Note: + positive, -negative

The results show that, the monoclonal antibody 1 coating and the monoclonal antibody 1 labeling, the monoclonal antibody 10 coating and the monoclonal antibody 10 labeling, both of the two combinations show non-specific reaction, and the other two combinations do not show non-specific reaction. The technical scheme that the monoclonal antibody 1 coating and the monoclonal antibody 1 labeling and the monoclonal antibody 10 coating and the monoclonal antibody 10 labeling can not be used simultaneously is explained, but the monoclonal antibody 1 coating and the monoclonal antibody 10 labeling can be used.

The results of paired activity detection and nonspecific detection show that the various paired combinations can simultaneously detect the southeast subtype O-type foot-and-mouth disease antibody and the Chinese O-type foot-and-mouth disease antibody, namely the enveloped monoclonal antibody 1 and the labeled monoclonal antibody 10, and the other combinations can not achieve the purpose of comprehensively detecting different genetic topology type O-type foot-and-mouth disease antibodies.

The two monoclonal antibodies 1 and 10 which are finally screened are named as 6G6 and 7D2 respectively.

EXAMPLE 5 identification of monoclonal antibodies

1. Subtype identification of monoclonal antibodies

The subtype of the 2-strain monoclonal antibody Ig was identified according to the instructions of the mouse monoclonal antibody subtype identification kit (purchased from Proteitech corporation), and the identification results are shown in Table 3. Monoclonal antibodies 6G6 and 7D2 both had heavy chains of IgG1 and both had light chains of Kappa.

TABLE 3 subtype identification results of monoclonal antibodies

2. Variable region sequencing of monoclonal antibodies

mRNA of hybridoma cells 6G6 and 7D2 was extracted, reverse transcribed into cDNA, amplified by high fidelity PCR using variable region universal primers, and the PCR products were subjected to DNA sequencing. DNA sequencing results: the DNA sequence of the gene for coding the heavy chain variable region of the monoclonal antibody 6G6 is shown as SEQ ID No: 1, the light chain variable region coding gene DNA sequence is shown as SEQ ID No: 2, the DNA sequence of the gene of the monoclonal antibody 7D2, the heavy chain variable region coding gene thereof is shown as SEQ ID No: 3, the light chain variable region coding gene DNA sequence is shown as SEQ ID No: 4, respectively.

Example 6 preparation of ELISA test kit

A solid phase competition ELISA method is established by adopting an O-type monoclonal antibody 6G6, O-type foot-and-mouth disease virus-like particle protein and an HRP-marked O-type foot-and-mouth disease monoclonal antibody 7D2, and the optimal working concentration of the coated monoclonal antibody 6G6 is determined to be 0.08 mu G/well (50 mu l per well), the optimal working concentration of the O-type virus-like particle protein is determined to be 10 mu G/well (50 mu l per well), the optimal working concentration of the enzyme-labeled monoclonal antibody is 1:1000 dilution (50 mu l per well, containing 0.2 mu G/well), and the optimal dilution multiple of serum is 1: 30.

coating: slightly shaking and uniformly mixing the foot-and-mouth disease O type coated antibody 6G6 at a concentration of 0.08 mu G/hole, and coating overnight at the temperature of 2-8 ℃;

and (3) sealing: discarding the liquid in the plate, washing for 3-5 times, beating to dry, adding 100 mu l of 1.5% BSA into each hole, slightly shaking and uniformly mixing, sealing the plate with a sealing plate membrane, and then incubating for 2h at 37 ℃;

adding an antigen: discarding liquid in the plate, washing for 3-5 times, beating to dry, adding 50 mu l of foot-and-mouth disease O-type protein diluted to a working concentration into each hole, and incubating for 1h at 37 ℃ after sealing the plate by a sealing plate membrane;

sample adding: the plate contents were discarded, washed 3-5 times, patted dry, and 50. mu.l of negative control (replicate 2 wells), 50. mu.l of positive control ((replicate 2 wells)), and 50. mu.l of 1: slightly oscillating and uniformly mixing 30-fold diluted serum to be detected, and incubating for 1h at 37 ℃ after sealing a plate by a sealing plate membrane;

adding an enzyme-labeled antibody: adding 50 mul of aftosa O type enzyme-labeled antibody 7D2(1:1000) with the concentration of 4 mug/ml into each well without washing, gently shaking and uniformly mixing, and incubating for 1h at 37 ℃ after sealing with a sealing plate membrane;

color development: discarding liquid in the plate, washing for 3-5 times, patting to dry, adding 50 μ l of chromogenic solution A and chromogenic solution B into each hole, and incubating for 10-15 min at 37 ℃ in a dark place;

and (4) terminating: according to the order of addition of the substrate, 50. mu.l of a stop solution (1M concentrated sulfuric acid) was added to each well to terminate the reaction, and the OD value was read at a wavelength of 450nm using a microplate reader.

And (4) judging a result:

blocking rate PI (%) ═ 1- (test serum OD/negative control mean OD) × 100

The blocking rate is positive when the blocking rate is more than or equal to 50 percent, and the blocking rate is negative when the blocking rate is less than 50 percent

The OD value of the negative control is not less than 1 and the blocking rate of the positive control is more than 50 percent under the condition of test establishment

Example 7 identification of ELISA kits

1. Identification of specificity

Three serial kits were used to detect 10 negative sera to foot and mouth disease and to evaluate their specificity. The results are shown in Table 4.

TABLE 4 result of ELISA kit for detecting negative serum

And detecting the positive serum of the foot-and-mouth disease A-type antibody, the positive serum of the hog cholera virus antibody, the positive serum of the porcine reproductive and respiratory syndrome virus antibody, the positive serum of the porcine Sernica valley virus antibody and the positive serum of the escherichia coli antibody respectively, and setting the southeast subtype O-type foot-and-mouth disease positive serum, the Chinese type O-type foot-and-mouth disease positive serum and the foot-and-mouth disease negative serum as controls. The results are shown in Table 5.

TABLE 5 ELISA kit specificity test results

Three continuous batch number kits detect that 10 negative serums are all negative, and prove that the specificity of the three continuous batch number kits meets the requirement; the three continuous batch kits detect other swine disease viruses, and except positive swine serum control, the rest are negative; the ELISA kit has no cross reaction with other antiviral agents, and the kit has good specificity.

2. Determination of sensitivity

The southeast subtype O-type foot-and-mouth disease positive serum is subjected to 2-fold gradient dilution, the solid-phase blocking kit prepared by the invention is used for determination, the sensitivity of the kit to the positive control serum is determined, and the result is shown in table 6.

TABLE 6 sensitivity test on titer diluted southeast subtype positive control sera

The result shows that the kit detects the positive serum diluted by titer, and the detection titer reaches 1: 256.

The Chinese type O foot-and-mouth disease positive serum is subjected to 2-fold gradient dilution, the solid-phase blocking kit prepared by the invention is used for determination, the sensitivity of the kit to the positive control serum is determined, and the result is shown in Table 7.

TABLE 7 sensitivity test on titer diluted China-type positive control sera

Dilution factor	Blocking Rate (%)	Determination of results
			1:8	95.66％	+
1:16	91.57％	+
			1:32	89.31％	+
1:64	79.29％	+
			1:128	63.23％	+
1:256	52.32％	+
			1:512	21.26％	-
Positive control	91.45％	+
			Negative control	0	-

The result shows that the kit detects the positive serum diluted by titer, and the detection titer reaches 1: 256. The solid-phase blocking ELISA kit has high sensitivity.

3. Repeatability test

Using enzyme labeling plates of 3 different batches, carrying out batch-to-batch repeated tests on one plate, carrying out batch-to-batch repeated tests on different plates, measuring OD450 values, and calculating the coefficient of variation. The coefficient of variation is less than 10%, which shows that the repeatability and stability of the kit are good. The results show that the detection results of the ELISA plates of 3 different batches are consistent, the variation coefficients are less than 5%, and the repeatability of the kit is good.

Coefficient of variation (C · V) calculation formula:

C.V (%) - (OD450 standard deviation/OD 450 average). times.100%

EXAMPLE 8 use of the kit comparative experiment

The southeast subtype O-type foot-and-mouth disease PCR method is identified as 186 parts of positive serum, and three batches of the solid phase blocking kit prepared by the invention and three batches of the commercialized liquid phase blocking kit are respectively used for comparison detection. The results are shown in Table 8.

TABLE 8 kit for southeast Asia subtype detection and comparison experiment results

The results show that the three batches of the solid-phase blocking kit can detect positive by 100 percent; the detection results of three batches of the liquid phase blocking kit are different, wherein the detection results are 95.7 percent at the lowest and 98.3 percent at the highest, and the detection results are different among the batches.

The PCR method of Chinese type O foot-and-mouth disease is identified as 194 parts of positive serum, and the three batches of the solid phase blocking kit prepared by the invention and the three batches of the commercialized liquid phase blocking kit are respectively used for comparison and detection. The results are shown in Table 9.

TABLE 9 test results of Chinese type detection and comparison with kit

The results show that the three batches of the solid-phase blocking kit can detect positive by 100 percent; the detection results of three batches of the liquid phase blocking kit are different, wherein the detection results are 90.2 percent at the lowest and 94.3 percent at the highest, and the detection is missed.

The above shows that the solid-phase blocking kit has better detection capability for different genetic topological antibodies, and can also better solve the problems of larger batch-to-batch difference and poor stability of the current liquid-phase blocking ELISA kit.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.

SEQUENCE LISTING

<110> Luoyang Putai Biotech Ltd

<120> foot-and-mouth disease resistant O-type virus monoclonal antibody and application thereof

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 378

<212> DNA

<213> hybridoma cell (hybridoma cell)

<400> 1

gaggtccagc tgcaacagtc tggacctgag ctggtgaagc ctggggcttc agtgaagata 60

tcctgcaaga cttctggata cacattcact gaatacacca tgcactgggt gaagcagagc 120

catggaaaga gccttgagtg gattggaggt attaatccta acaatggtgt tcctagttac 180

aaccagaagt tcaagggcaa ggccacattg actgtagaca agtcctccag cacaccctac 240

atggacctcc gcagcctgac atctgaggat tctgcagtct attactgtgt aagaaaagga 300

tactataggt acgacgtaga gacgtcgggg tttgctatgg actactgggg tcaaggaacc 360

tcagtcaccg tctcctca 378

<210> 2

<211> 342

<212> DNA

<213> hybridoma cell (hybridoma cell)

<400> 2

gacattgtga tgtcacagtc tccatcctcc ctagctgtgt cagttggaga gaaggttact 60

gtgagctgca ggtccagtca gagcctttta tatagtagca atcaaaagaa ctacttggcc 120

tggtaccagc agaaaccagg gcagtctcct aaactgctga tttactgggc atccactagg 180

gaatctgggg tccctgatcg cttcacaggc agtggatctg ggacagattt cactctcacc 240

atcagcagtg tgaaggctga agacctggca gtttattact gtcagcaata ttatagctat 300

ccgtacacgt tcggaggggg gaccaagctg gaaataaaac gg 342

<210> 3

<211> 354

<212> DNA

<213> hybridoma cell (hybridoma cell)

<400> 3

caggtccaac tgcagcaacc tgggtctgag ctggtgaggc ctggagcttc agtgaagctg 60

tcctgcaagg cttctggcta cacattcacc agctactgga tgcactgggt gaagcagagg 120

cctggacaag gccttgagtg gattggaaat atttatcctg atagtggtag ttttaattac 180

gatgagaaat tcaagagcaa ggccacactg actgtagaca catcctccag cacagcctac 240

atgcagctca gcagcctgac atctgaggac tctgcggtct attattgtac aagagtccac 300

tataggcacg ggtttgctta ctggggccaa gggactctgg tcactgtctc tgca 354

<210> 4

<211> 321

<212> DNA

<213> hybridoma cell (hybridoma cell)

<400> 4

gacattgtga tgacccagtc tcacaaattc atgtccacat cagtaggaga cagggtcagc 60

atcacctgca aggccagtca ggatgtgagt actgctgtag tctggtatca agagaaacca 120

ggacaatctc ctaaactact aatttactcg gcatccttcc ggtactctgg agtccctgat 180

cgcttcactg gcagtggatc tgggacggat ttcactttca ccatcagcag tgttcaggct 240

gaagacctgg cagtttatta ctgtcagcaa cattatacta ctccgctcac gttcggtgct 300

gggaccaagc tggagctgaa a 321

Claims

1. A variable region sequence of a monoclonal antibody 6G6 which reacts to O-type foot-and-mouth disease virus and is combined with the same site of different genetic topologic type O-type foot-and-mouth disease virus, wherein, the heavy chain variable region of the monoclonal antibody 6G6 is a sequence shown in SEQ.ID No.1 or a degenerate sequence code thereof or a conservative variant obtained by conservative mutation of one or more amino acids through addition, deletion, substitution or modification; the variable region of the monoclonal antibody 6G6 light chain is a conservative variant obtained by coding the sequence shown in SEQ ID No.2 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications.

2. An antibody or antibody fragment which reacts to O-type foot-and-mouth disease virus and is combined with the same site of different genetic topology type O-type foot-and-mouth disease virus, wherein the heavy chain variable region of the antibody or antibody fragment is a sequence shown in SEQ.ID No.1 or a degenerate sequence code thereof or a conservative variant obtained by adding, deleting, replacing or modifying conservative mutation of one or more amino acids; and the variable region of the light chain of the antibody or the antibody fragment is a conservative variant obtained by the sequence shown in SEQ ID No.2 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications; preferably, the antibody is a monoclonal antibody, a genetically engineered antibody; wherein the genetically engineered antibody comprises a single chain antibody, a chimeric monoclonal antibody, a reshaped monoclonal antibody, or a fragment of the antibody; said antibody or fragment of said antibody still retains the ability to specifically bind to a different genetic topology type O foot and mouth disease virus; preferably, the antibody is monoclonal antibody 6G6, wherein, the heavy chain variable region of monoclonal antibody 6G6 is a conservative variant obtained by coding the sequence shown in SEQ ID No.1 or a degenerate sequence thereof or conservative mutation thereof through one or more amino acid additions, deletions, substitutions or modifications; the variable region of the monoclonal antibody 6G6 light chain is a conservative variant obtained by coding the sequence shown in SEQ ID No.2 or a degenerate sequence thereof or conservative mutation of the sequence through one or more amino acid additions, deletions, substitutions or modifications.

3. A hybridoma cell strain 6G6 that secretes monoclonal antibody 6G6 of claim 3.

4. The variable region sequence of monoclonal antibody 7D2 capable of simultaneously and specifically binding different genetic topological type O foot-and-mouth disease viruses, wherein the variable region of the heavy chain of monoclonal antibody 7D2 is the sequence shown in SEQ ID No.3 or the code of degenerate sequence thereof; the variable region of the monoclonal antibody 7D2 light chain is encoded by the sequence shown in SEQ ID No.4 or a degenerate sequence thereof.

5. An antibody or antibody fragment which can simultaneously and specifically bind different genetic topological type O foot-and-mouth disease viruses, wherein the heavy chain variable region of the antibody or antibody fragment is a sequence shown in SEQ.ID No.3 or a degenerate sequence code thereof or a conservative variant thereof obtained by adding, deleting, replacing or modifying conservative mutation by one or more amino acids; and the variable region of the light chain of the antibody or the antibody fragment is a conservative variant obtained by the sequence shown in SEQ ID No.4 or the coding of the degenerate sequence thereof or conservative mutation of the conservative variant through one or more amino acid additions, deletions, substitutions or modifications; preferably, the antibody is a monoclonal antibody, a genetically engineered antibody; wherein the genetically engineered antibody comprises a single chain antibody, a chimeric monoclonal antibody, a reshaped monoclonal antibody, or a fragment of the antibody; said antibody or fragment of said antibody still retains the ability to specifically bind to a different genetic topology type O foot and mouth disease virus; preferably, the antibody is monoclonal antibody 7D2, wherein the heavy chain variable region of monoclonal antibody 7D2 is encoded by the sequence shown in SEQ ID No.3 or a degenerate sequence thereof; the variable region of the monoclonal antibody 7D2 light chain is encoded by the sequence shown in SEQ ID No.4 or a degenerate sequence thereof.

6. Hybridoma cell strain 7D2, secreting the monoclonal antibody 7D2 of claim 5.

7. An ELISA detection kit, wherein the kit comprises an effective amount of coating antibody, the coating antibody is the monoclonal antibody 6G6 of claim 3, an effective amount of O-type foot-and-mouth disease virus antigen, an effective amount of labeled antibody, the labeled antibody is the monoclonal antibody 7D2 of claim 7, and a detection reagent for detecting antigen-antibody reaction.

8. The ELISA test kit of claim 7, wherein said monoclonal antibody 6G6 is coated on a support medium, and said foot-and-mouth disease virus type O antigen binds to said monoclonal antibody 6G 6; preferably, the support medium is a microtiter plate.

9. The ELISA detection kit of claim 7, wherein the monoclonal antibody 6G6 is 1-8 μ G/ml, the foot-and-mouth disease virus O antigen is 50-400 μ G/ml, and the monoclonal antibody 7D2 is 3-10 μ G/ml; preferably, the O type foot-and-mouth disease virus antigen content is 200 mug/ml.

10. The ELISA detection kit of claim 7, wherein the ELISA detection kit further comprises a sample diluent, a washing solution, a developing solution, a stop solution, a positive pig serum control and a negative pig serum control.

11. The ELISA test kit of claim 10, wherein theThe sample diluent is phosphate buffer solution containing skim milk, the pH value of the phosphate buffer solution is 7.4, and the formula of 1L volume of the phosphate buffer solution is as follows: NaCl 8.0g, KCl 0.2g, Na₂HPO₄·12H₂O 2.9g、KH₂PO₄0.24g, preferably phosphate buffer containing 10% (W/V) skim milk; the washing solution is phosphate buffer solution containing Tween-20, preferably phosphate buffer solution containing 0.05% (V/V) Tween-20; the developing solution comprises developing solution A and developing solution B, wherein the developing solution A contains 20mg of TMB and 10ml of absolute ethyl alcohol, and ddH2O is used for fixing the volume to 100 ml; the color developing solution B contains 2.1g of citric acid and anhydrous Na₂HPO₄2.82g, 0.75% (V/V) Urea hydrogen peroxide 0.64ml, in ddH₂O is added to 100ml to fix the volume; the stop solution is 2mol/L H₂SO₄(ii) a The positive control is hyperimmune serum of the O-type foot-and-mouth disease virus antigen, and the negative control is pig serum without foot-and-mouth disease antibodies.