CN109061166B

CN109061166B - ELISpot detection kit for detecting bovine tuberculosis

Info

Publication number: CN109061166B
Application number: CN201810614743.6A
Authority: CN
Inventors: 焦新安; 陈祥; 李昕; 徐正中; 刘佳莹; 孟闯; 孙林
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2018-06-14
Filing date: 2018-06-14
Publication date: 2021-02-12
Anticipated expiration: 2038-06-14
Also published as: CN109061166A

Abstract

The invention belongs to the technical field of biology, and particularly relates to an ELISpot detection kit for detecting bovine tuberculosis and application thereof in the field of bovine tuberculosis diagnosis. The detection kit comprises a support medium, a capture antibody and a detection antibody, wherein the capture antibody can be prepared from a nucleic acid sequence with a preservation number of CCTCC NO: c2017283, and the detection antibody is a gamma interferon monoclonal antibody different from the capture antibody. The kit of the invention and bovine tuberculosis BOVIGAM^®Compared with the ELISA detection kit and the bovine tuberculosis antibody detection reagent, the kit can realize more sensitive and high-specificity detection of bovine mycobacterium infection, eliminates false positive interference caused by cross reaction, and shows good application prospect.

Description

ELISpot detection kit for detecting bovine tuberculosis

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an ELISpot detection kit for detecting bovine tuberculosis and application thereof in the field of bovine tuberculosis diagnosis.

Background

Bovine tuberculosis is a chronic infectious disease of zoonosis caused by Mycobacterium bovis (Mycobacterium bovis), is characterized by tuberculous granuloma and caseous and calcified necrotic lesions of tissues and organs, and mostly affects lung, breast, intestine, lymph node and the like. Seriously threatens the health condition of cattle, and causes great harm to the animal husbandry production and human health.

The mycobacterium tuberculosis is a facultative intracellular parasitic bacterium, can cause cellular immune response after infecting organisms, further generates a series of cell factors, and plays an important role in the process of resisting infection of the organisms. When Mycobacterium tuberculosis is infected by organisms, specific immunogenic proteins can activate T cell responses, including activation of macrophages and CD4⁺T cells and CD8⁺Toxic T cells, produce several important cytokines, such as gamma interferon (IFN- γ), tumor necrosis factor α (TNF- α), and the like. These cytokines play an important role in the clearance of mycobacterium tuberculosis in the body. Particularly, the secretion of IFN-gamma is an important index of T cell immune response generated by a host, plays an important role in resisting the infection of mycobacterium tuberculosis, and can indirectly reflect the cellular immune condition of an organism after the infection of the mycobacterium tuberculosis by detecting the IFN-gamma secretion condition of antigen specificity.

IFN-gamma is CD4 activated primarily by stimulation with specific antigens⁺Th1 cell, CD8⁺Cytokines produced by T cells and NK cells, etc., have broad antiviral, antitumor activity and immunoregulatory function, and are important components of the body's defense system. The immunological detection of IFN-gamma is an experimental method for qualitative and quantitative analysis of IFN-gamma in a sample, which is established on the basis of a specific anti-IFN-gamma monoclonal antibody (MAb). Various methods have been developed, such as enzyme-linked immunosorbent assays (ELISA), enzyme-linked immunospot assays (ELISpot), Flow Cytometry (FCM) assays, and the like, using various monoclonal antibody labels and detection techniques.

The detection method of the bovine tuberculosis at the present stage mainly comprises the following steps: (1) the bacteriological method has higher reliability and accuracy of diagnosis by culturing pathogens and judging according to biological characteristics of the pathogens; but the mycobacterium bovis grows slowly, the separation rate is low (about 20 percent), biological safety measures are needed, meanwhile, the collection of pathological samples needs to slaughter the cows, and the method is not suitable for quarantine of live animals and seriously influences the popularization of the method; (2) molecular biology techniques, such as established PCR detection methods for specificity of Mycobacterium tuberculosis, but the requirements for experimental conditions are high, and the popularization is inconvenient; (3) the immunological detection method comprises serological detection and cellular immune detection, and the immune response of organisms caused by mycobacteria is mainly cellular immune but not humoral immune, so that the application value of the ELISA method for detecting the antibody is limited. At present, in the quarantine process of bovine tuberculosis in China, bovine tuberculin (hereinafter referred to as PPD) is mainly adopted to carry out simple cervical skin test allergic reaction, but the method has more false positives. In addition, the method has a series of defects of difficult operation process, long time required by the test, more factors interfered by the outside, strong subjectivity of result judgment and the like. (4) The basic principle of the gamma-interferon ELISA detection test and the gamma-interferon release test is the same as that of TST, but the whole operation process is carried out in vitro. Compared with TST, the gamma-interferon ELISA test has higher sensitivity and specificity, more objective and reliable result, no influence of the detection result on the detection result, only one-time contact with animals, and great superiority in detection of wild animals and large animals difficult to be fixed. Meanwhile, the gamma-interferon release test simulates the immune response of a mycobacterium tuberculosis infected organism, and reflects the immune state of a host at the early stage of mycobacterium tuberculosis infection to a certain extent, so the method can be used for detecting the early stage infection of tuberculosis, and particularly has more advantages when the organism has no obvious humoral immune response and pathological changes when the mycobacterium tuberculosis is in a latent infection state. However, the gamma interferon ELISA test is a population level test, and if the cytokine level of a population is increased, ELISA cannot distinguish whether the number of cells secreting cytokines is increased or whether the single secretion level is increased. This distinction is important for studying the cellular immunity of the organisms, but the population-level detection cannot be distinguished, and only the single-cell level detection is adequate.

However, the above detection methods still do not meet the real need in the art for specific detection of M.bovis infection.

Currently, the bovine tuberculin intradermal allergy test is a well-known tuberculosis diagnosis method with relatively high specificity and sensitivity. Because the tuberculin Pure Protein Derivative (PPD) has complex components, a plurality of contained proteins exist in pathogenic mycobacteria and environmental nonpathogenic mycobacteria at the same time, and the conditions of positive tuberculosis infection, immune individuals, environmental mycobacteria infection and the like cannot be distinguished, so that the specificity of tuberculin intradermal allergy is influenced to a certain degree.

Therefore, a kit with higher sensitivity and better specificity needs to be established for detecting bovine tuberculosis.

Disclosure of Invention

In order to overcome the problems in the prior art, the invention aims to provide an ELISpot detection kit for detecting bovine tuberculosis and application thereof in the field of bovine tuberculosis diagnosis.

In order to achieve the above objects and other related objects, the present invention adopts the following technical solutions:

in a first aspect, the present invention provides a monoclonal antibody, which comprises a light chain and a heavy chain, wherein the light chain comprises a CDR region having an amino acid sequence as shown in SEQ ID No. 1-3, and the heavy chain comprises a CDR region having an amino acid sequence as shown in SEQ ID No. 6-8.

In one embodiment, the heavy and light chains are linked by a disulfide bond.

In one embodiment, the monoclonal antibody has the amino acid sequence of SEQ ID No.4 or a conservative variant thereof in the light chain variable region and the amino acid sequence of SEQ ID No.9 or a conservative variant thereof in the heavy chain variable region.

In one embodiment, the light chain variable region encodes a nucleotide sequence of SEQ ID No.14 or a conservative variant thereof, and the heavy chain variable region encodes a nucleotide sequence of SEQ ID No.19 or a conservative variant thereof.

In one embodiment, the monoclonal antibody is murine.

In one embodiment, the monoclonal antibody subclass is IgG2 a.

In one embodiment, the monoclonal antibody is a bovine interferon gamma monoclonal antibody.

In one embodiment, the monoclonal antibody is represented by a sequence with a collection number of CCTCC NO: c2017283, or a subculture cell strain thereof.

The preservation number is CCTCC NO: the hybridoma cell strain of C2017283 has been registered and preserved in China center for type culture Collection (address: Wuhan university, Wuhan city) in 12 months and 7 days of 2017, and the preservation number is CCTCC NO: c2017283, and is classified and named as hybridoma cell strain 3E 9.

In a second aspect of the present invention, there is provided a monoclonal antibody comprising a light chain and a heavy chain, wherein the light chain comprises a CDR region having an amino acid sequence as set forth in SEQ ID No. 21-23, and the heavy chain comprises a CDR region having an amino acid sequence as set forth in SEQ ID No. 26-28.

In one embodiment, the heavy and light chains are linked by a disulfide bond.

In one embodiment, the monoclonal antibody has the amino acid sequence of SEQ ID No.24 or a conservative variant thereof in the light chain variable region and the amino acid sequence of SEQ ID No.29 or a conservative variant thereof in the heavy chain variable region.

In one embodiment, the monoclonal antibody has the light chain variable region encoding nucleotide sequence of SEQ ID No.34 or a conservative variant thereof and the heavy chain variable region encoding nucleotide sequence of SEQ ID No.39 or a conservative variant thereof.

In one embodiment, the monoclonal antibody is murine.

In one embodiment, the monoclonal antibody subclass is IgG₁。

In one embodiment, the monoclonal antibody is represented by a sequence with a collection number of CCTCC NO: c2017282 or a subculture cell strain thereof.

The preservation number is CCTCC NO: the hybridoma cell strain of C2017282 has been registered and preserved in China type culture Collection (address: Wuhan university in Wuhan city) in 12/7 in 2017, and is classified and named as: hybridoma cell line 6F 8.

In a third aspect of the present invention, there is provided a use of the monoclonal antibody of the first aspect and the monoclonal antibody of the second aspect for preparing a detection reagent or a diagnostic reagent for bovine plague.

Further, there is provided the use of the monoclonal antibody of the first aspect and the monoclonal antibody of the second aspect in the preparation of an ELISpot assay kit for bovine gamma interferon.

In a fourth aspect of the present invention, an ELISpot detection kit for bovine gamma interferon is provided, which comprises:

(1) selected from any one of:

1) a support medium and a capture antibody;

2) a support medium coated with a capture antibody;

the capture antibody is a first monoclonal antibody;

(2) detecting the antibody;

the second antibody is a labeled second monoclonal antibody;

in the kit, the capture antibody and the detection antibody can jointly generate antigen-antibody binding action with bovine gamma interferon.

The second monoclonal antibody of the labeled second monoclonal antibody is different from the capture antibody described above.

In one embodiment, the detection antibody is a second monoclonal antibody labeled with biotin or alkaline phosphatase.

Further, the detection antibody is a biotin-labeled second monoclonal antibody.

The method for labeling the second monoclonal antibody with biotin is conventional.

In one embodiment, the capture antibody, i.e. the first monoclonal antibody, is a monoclonal antibody according to the aforementioned first aspect of the invention.

In one embodiment, the detection antibody, i.e. the second monoclonal antibody, is a monoclonal antibody according to the second aspect of the invention.

In the kit, the support medium may be coated with the capture antibody in advance, or only a blank support medium and the capture antibody may be provided, and the capture antibody is coated on the support medium by an operator by using a conventional method before detection.

Wherein, the contact surface of the supporting medium and the reagent can be provided with a microporous filter membrane. Thus, the support medium is a microporous filter membrane sheet. The microporous filter membrane plate can be a microporous filter membrane plate with various common specifications, such as a 96-pore filter membrane plate. More preferably, the support medium is a PVDF-coated membrane micropore culture plate. Wherein, preferably, the PVDF film is arranged on the contact surface of the supporting medium and the reagent.

Further, the kit also comprises one or more of the following reagents:

1) avidin-alkaline phosphatase conjugates;

2) a substrate developing solution;

3) diluting the solution;

4) washing liquid;

5) sealing liquid;

6) negative control;

7) a positive control;

8) a specific stimulant;

9) a non-specific irritant.

The reagents are all general reagents in ELISopt detection, are not limited by specific detection items, so that the reagent kit can be selectively added according to needs, and can be configured by an operator or purchased independently. For the convenience of the operator, it is most preferable to include an avidin-alkaline phosphatase conjugate, a substrate display solution and a washing solution in the kit.

The substrate solution can be a common substrate display solution commonly used in an ELISpot detection kit, such as liquid nitrogen blue tetrazole/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP) substrate display solution.

The washing solution can be a washing solution commonly used in an ELISpot detection kit, such as phosphate buffer solution and the like. Concentrated or unconcentrated washing solutions may be used as desired.

The blocking solution can be a commonly used blocking solution for coating a microfiltration membrane plate, such as skim emulsion, FBS, BSA or casein.

Furthermore, other universal reagents required by ELISpot detection, such as cell culture solution, phosphate buffer solution, phosphate Tween buffer solution and the like, can be selectively included in the kit.

The components of the negative control were identical to those of the sample set being tested, but no stimulus was added.

The components of the positive control are identical to the components of the sample set being tested, but the specific stimulant is replaced by a non-specific stimulant.

Further, the non-specific stimulant is PWM. Specific stimulators are Mb3904-Mb3905(CFP-10 and ESAT-6, i.e., CE protein).

In general, in the kit of the present invention, each reagent is separately stored.

The invention further establishes an ELISpot detection method of the bovine gamma interferon with better specificity and sensitivity based on the BoIFN-gamma ELISpot detection kit, and is used for detecting T lymphocytes secreting the bovine gamma interferon so as to perform the research on the aspects of organism immune state evaluation and disease diagnosis.

The detection method for detecting the bovine peripheral blood mononuclear cell sample by using the kit comprises the following steps:

(1) a capture antibody coated support medium (e.g., a microporous filter plate);

(2) preparing a suspension of bovine peripheral blood mononuclear cells;

(3) cell incubation and detection

1) Taking the support medium (microporous filter plate) coated with the capture antibody, and adding a specific stimulant (such as CE protein), a positive control (such as pokeweed PWM) and a negative control (such as complete 1640 culture medium) into each well respectively;

2) adding PBMC to be detected into each hole, and uniformly mixing the solution in the reaction plate;

3) covering a cover, and incubating at 37 ℃ for 24-48 h;

4) washing each plate well with a wash solution;

5) after washing the plate, adding the diluted avidin-alkaline phosphatase conjugate and incubating at 37 ℃;

6) adding 100 μ L substrate color developing solution into each well, attaching a sealing plate membrane, and developing at 37 deg.C in dark for 5-10 min.

7) Washing with water to terminate the reaction, removing the liquid, and drying overnight at room temperature or in an oven at 37 ℃ for 2-3 hours;

8) counting purple spots in each reaction well using an inverted microscope, each spot representing one bovine interferon gamma-secreting T cell; or using an ELISPOT plate reader for counting, and performing scanning counting and analysis on the experimental result.

In a fifth aspect of the invention, the application of the ELISpot detection kit in the detection of non-diagnostic Mycobacterium tuberculosis infection of cattle is provided. Wherein the non-diagnostic purposes include epidemiological analysis and research, in vitro tissue detection, epitope identification research, and qualitative and quantitative testing of mycobacterium tuberculosis antigen specific bovine IFN-gamma.

The sixth aspect of the invention provides a hybridoma cell strain secreting bovine gamma interferon monoclonal antibodies, wherein the preservation number of the hybridoma cell strain is CCTCC NO: C2017283.

the hybridoma cell strain is registered and preserved in China center for type culture Collection (address: Wuhan university in Wuhan city) in 2017 at 12/7, and the preservation number is CCTCC NO: c2017283, and is classified and named as hybridoma cell strain 3E 9.

The seventh aspect of the invention provides an application of the hybridoma cell strain secreting the bovine gamma interferon monoclonal antibody in preparing a detection reagent or a diagnostic reagent for bovine epidemic diseases.

Furthermore, the hybridoma cell strain secreting the bovine gamma interferon monoclonal antibody can be used for preparing an ELISpot detection kit of the bovine gamma interferon.

The eighth aspect of the invention provides a hybridoma cell strain secreting bovine gamma interferon monoclonal antibodies, wherein the preservation number of the hybridoma cell strain is CCTCC NO: C2017282.

The ninth aspect of the invention provides the use of the hybridoma cell strain secreting the bovine gamma interferon monoclonal antibody in the preparation of a detection reagent or a diagnostic reagent for bovine plague.

The biological material preservation information of the present invention is as follows:

name: hybridoma cell line 3E 9;

the preservation number is as follows: CCTCC NO: c2017283;

the preservation date is as follows: 12 months and 7 days 2017;

the name of the depository: china center for type culture Collection;

the preservation unit is abbreviated as: CCTCC (China center for type communication);

the address of the depository: wuhan city Wuchang Lodoya street Wuhan university's Life sciences college.

Name: hybridoma cell line 6F8

The preservation number is as follows: CCTCC NO: c2017282;

the preservation date is as follows: 12 months and 7 days 2017;

the name of the depository: china center for type culture Collection;

Compared with the prior art, the invention has the following beneficial effects:

1) the monoclonal antibody secreted by the hybridoma cell strain adopted in the kit has the advantages of high titer, good specificity and strong affinity with natural antigen.

2) The Mycobacterium bovis-specific antigens Mb3904, Mb3905 used in the kit of the present invention are encoded by the RD1 gene, and RD1 is present only in Mycobacterium tuberculosis and M.bovis, but is absent in BCG, so that Mb3904 and Mb3905 are more specific as stimulants.

3) The kit based on the invention is based on the detection of cell level, compared with the skin test, the interference of false positive caused by cross reaction is eliminated, and good specificity is displayed;

4) in addition, compared with other commercial ELISA kits based on antibodies, the ELISpot kit based on the invention has higher sensitivity.

5) In addition, compared with skin test and commercial ELISA kits, the ELISpot kit based on the invention has higher coincidence rate.

Drawings

FIG. 1: and detecting the result of the bovine tuberculosis ELISpot detection kit.

FIG. 2: and comparing the ELISpot detection kit based on the non-specific stimulus PWM with the bovine gamma interferon ELISpot detection method, wherein A is a blank control group, B is a negative control group, and C is a positive control group.

FIG. 3: results of comparison of the ELISpot kit with the antibody pair of Mabtech.

FIG. 4: bovine tuberculosis gamma-interferon ELISpot detection kit.

FIG. 5: results of CE protein-based ELISpot detection kit and PPD-based ELISpot detection method, A, C is CE protein stimulated group, and B, D is PPDB stimulated group.

Detailed Description

The ELISpot detection kit for detecting bovine tuberculosis can specifically detect bovine tuberculosis, and the detection sensitivity is higher than the ELISA level.

The term "ELISpot" is the abbreviation of enzyme-linked immunospot assay, and its greatest advantage is the detection of single cell level, and is the detection of living cell function, and is more sensitive than ELISA and limiting dilution method, etc., and can detect 1 cell secreting target protein from 20-30 ten thousand cells.

The traditional ELISA method for detecting bovine tuberculosis is detection at a population level, but if the level of a population cytokine is increased, the number of cells secreting the cytokine is increased, or the number of secreting cells is not increased and the single secretion level is increased. This distinction is important for studying the cellular immunity of the organisms, but the population-level detection cannot be distinguished, and only the single-cell level detection is adequate.

Before the present embodiments are further described, it is to be understood that the scope of the invention is not limited to the particular embodiments described below; it is also to be understood that the terminology used in the examples is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers.

When numerical ranges are given in the examples, it is understood that both endpoints of each of the numerical ranges and any value therebetween can be selected unless the invention otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In addition to the specific methods, devices, and materials used in the examples, any methods, devices, and materials similar or equivalent to those described in the examples may be used in the practice of the invention in addition to the specific methods, devices, and materials used in the examples, in keeping with the knowledge of one skilled in the art and with the description of the invention.

Unless otherwise indicated, the experimental methods, detection methods, and preparation methods disclosed herein all employ techniques conventional in the art of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related arts. These techniques are well described in the literature, and may be found in particular in the study of the MOLECULAR CLONING, Sambrook et al: a LABORATORY MANUAL, Second edition, Cold Spring Harbor LABORATORY Press, 1989and Third edition, 2001; ausubel et al, Current PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; wolffe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; (iii) METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol.119, chromatography Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999, etc.

Example 1 preparation of hybridoma cell lines and monoclonal antibodies

The preservation number is CCTCC NO: c2017283 hybridoma cell strain and preservation number of the hybridoma cell strain are CCTCC NO: and (3) obtaining the hybridoma cell strain of C2017282.

1. Animal immunization

The specific immunization program was as follows: the first immunization, 100 mu g of recombinant bovine gamma interferon purified protein fully emulsified by Freund's complete adjuvant is injected subcutaneously in multiple points at the abdomen, the second immunization is carried out by injecting 100 mu g of purified protein fully emulsified by Freund's incomplete adjuvant subcutaneously in multiple points at the abdomen after 2 weeks, the third immunization is carried out by injecting 100 mu g of purified protein without adjuvant in the abdominal cavity after two weeks, the blood sampling is carried out after 7d to determine the serum antibody titer, and the mice with higher titer are selected to carry out tail vein boosting immunization on 100 mu g of purified protein without adjuvant.

2. Cell fusion

The method comprises the following specific steps: after tail vein boosting immunization for 3d, a small amount of blood is collected, and serum is separated and frozen at the temperature of-20 ℃ to be used as a positive control in screening. Killing an immunized mouse according to a biological safety method, soaking the immunized mouse in 75% alcohol for 5min, aseptically taking spleen cells and myeloma cells SP2/0 in a logarithmic growth phase to fuse under the action of PEG, using ICR mouse abdominal cavity macrophages as feeder cells, suspending the fused cells and feeder cells by using HAT culture medium, subpackaging the cells into 96-well plates, and culturing in a 6% carbon dioxide incubator at 37 ℃. Adding fresh HAT culture medium after 5 days, culturing with HT culture medium after 10 days, periodically observing, changing culture medium, and detecting.

3. Establishment of indirect ELISA detection method

Positive cell clones were screened by indirect ELISA. The matrix test determines the coating concentration of the test antigen.

Detecting lateral gradient dilution of antigen coating buffer solution, coating an ELISA plate by 50 mu L per hole, and standing overnight at 4 ℃; PBST washing 3 times, each hole add 200 u L of confining liquid, 4 degrees C overnight; serum of immunized mice is longitudinally diluted by multiple times, each well is 50 mu L, and serum of normal mice is diluted by the same multipleAs a negative control, incubating at 37 ℃ for 2 h; washing with PBST for the third time, adding enzyme-labeled secondary antibody with working concentration, incubating at 37 deg.C for 1.5 hr with 50 μ L of each well, washing with PBST, developing OPD, and determining OD with enzyme-linked detector₄₉₀The optimal coating concentration of the detection antigen is determined.

4. Screening for Positive clones

And detecting the antibody condition secreted by the hybridoma cells by adopting a well-established indirect ELISA method. The specific method comprises the following steps: adding the culture supernatant of the hybridoma cells into an ELISA plate coated in advance, performing 50 mu L/hole treatment, taking the supernatant of SP2/0 cells as a negative control, taking immune multi-antiserum as a positive control, and performing water bath at 37 ℃ for 2 h; PBST wash 3 times; adding HRP-labeled goat anti-mouse IgG and IgM antibodies with working concentration, 50 mu L/hole, and performing water bath at 37 ℃ for 1.5 h; after washing, OPD is developed for 10-15 min, and OD is measured by an enzyme-labeling instrument after the display is ended₄₉₀And (6) reading. Measured hole OD₄₉₀More than two times of reading is judged to be positive when the reading is more than the negative control. The two positive clones were designated as positive cell clone 3E 9and positive cell clone 6F8, respectively.

5. Cloning of Positive hybridoma cells

And performing subcloning on the screened positive cell clones 3E 9and 6F8 for 2-3 times by using a limiting dilution method, and preserving. The corresponding preservation number of the positive cell clone 3E9 is CCTCC NO: c2017283, wherein the positive cell clone 6F8 has a preservation number of CCTCC NO: c2017282.

6. Preparation of ascites

Adopts a method of inducing ascites in vivo and is carried out according to a conventional method. Injecting 0.3-0.5 ml/mouse of liquid paraffin into the abdominal cavity of a healthy BALB/c mouse aged 10-12 weeks, and inoculating hybridoma cells 3E 9and 6F8 which are diluted by PBS and cultured to logarithmic phase into the abdominal cavity after 7-10 days⁵One cell/one; and 7d, collecting ascites, centrifuging to remove precipitates, collecting supernatant, measuring antibody titer by indirect ELISA, subpackaging, and storing at-70 ℃. The preservation number is CCTCC NO: the monoclonal antibody secreted by the hybridoma cell line of C2017283 or the subculture cell line thereof (corresponding to hybridoma cell 3E9) is marked as monoclonal antibody 3E9, and the preservation number is CCTCC NO: c2017282 hybridoma cell strain or subculture cell strain (corresponding hybrid) thereofHybridoma 6F8) was secreted and designated monoclonal antibody 6F 8.

7. Purification labeling of antibodies

The prepared 3E 9and 6F8 ascites were purified using Protein G affinity chromatography method, and the monoclonal antibody 6F8 was biotin-labeled.

And (3) marking the purified monoclonal antibody 6F8 by adopting a standard biotin marking method to obtain a biotin-marked bovine gamma interferon monoclonal antibody Bio-6F 8. Dissolving 2-10 mg of monoclonal antibody 6F8 protein in 1mL of phosphate buffer solution, and calculating the number of millimoles dissolved; balancing Biotin to room temperature, adding 2mg of Sulfo-NHS-Biotin into 100 mu L of ultrapure water, and adding Biotin with a certain concentration; room temperature for 30 minutes, or on ice for 2 hours; the purification column was pre-washed with 30mL of PBS, loaded, and 0.5mL or 1mL of the same buffer as the amount to be collected was collected in a separate tube, and the protein content of the monoclonal antibody was measured at an absorbance of 280 nm.

Monoclonal antibody 6F8 of the invention may also be labeled using other methods known in the art.

8. Detection of monoclonal antibody characteristics

Identification of monoclonal antibody subclasses

According to the specification of the monoclonal antibody subclass kit, an antigen-mediated ELISA method is adopted. Adding 50 mu L/hole of cell culture supernatant into the enzyme label plate coated with the antigen, washing for 3 times by PBST at 37 ℃ for 1h, and adding 1: 1000 dilution of goat anti-mouse IgG₁、IgG_2a、IgG_2b、IgG₃IgM subclass antibody 50 mu L/hole, 37 ℃ 0.5h, each monoclonal antibody adding each subclass two holes, PBST washing 3 times each time 5 min; adding 1: washing rabbit anti-sheep enzyme labeled second antibody (50 μ L/well) diluted by 5000 with PBST for 3 times at 37 ℃ for 15 min; adding 50 mu L of o-phenylenediamine (OPD) solution into the reaction kettle, keeping the reaction kettle in a dark place at 37 ℃ for color development for 10-15 min, and performing 2M H₂SO₄The reaction was terminated at 50. mu.L/well, and the subclass of the antibody was significantly higher than that of the antibody added to other wells as a monoclonal antibody subclass when observed with the naked eye.

The results showed that monoclonal antibody 3E9 subclass was IgG_2aMonoclonal antibody 6F8 subclass is IgG₁。

The identification result shows that the amino acid sequence of the complementarity determining region 1(CDR1) of the light chain variable region of the monoclonal antibody 3E9 is shown in SEQ ID NO.1, and specifically comprises the following steps:

SASSSVNYMH。

the amino acid sequence of the complementarity determining region 2(CDR2) in the light chain variable region of monoclonal antibody 3E9 is shown in SEQ ID NO.2, and specifically comprises:

STSNLAS。

the amino acid sequence of the complementarity determining region 3(CDR3) in the light chain variable region of monoclonal antibody 3E9 is shown in SEQ ID NO.3, and specifically comprises:

QQRNRYPLT。

the amino acid sequence of the light chain variable region of the monoclonal antibody 3E9 is shown in SEQ ID NO.4, and specifically comprises the following steps:

QIVLTQSPAFMSASPGEKVTITCSASSSVNYMHWFQQKPGTSPRLCIFSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRNRYPLTFGAGTKLELK。

the amino acid sequence of the light chain of monoclonal antibody 3E9 is shown in SEQ ID NO.5, and specifically comprises:

MHFQVQIFSFLLISASVIMSKGQIVLTQSPAFMSASPGEKVTITCSASSSVNYMHWFQQKPGTSPRLCIFSTSNLASGVPARFSGSGSGTSYSLTISRMEAEDAATYYCQQRNRYPLTFGAGTKLELK are provided. That is, the light chain of monoclonal antibody 3E9 contains 128 amino acids.

The amino acid sequence of the heavy chain variable region complementarity determining region 1(CDR1) of monoclonal antibody 3E9 is shown in SEQ ID NO.6, and specifically comprises:

SYTLH。

the amino acid sequence of the heavy chain variable region complementarity determining region 2(CDR2) of monoclonal antibody 3E9 is shown in SEQ ID NO.7, and specifically comprises:

YFNPYNDGIKYNEKFKN。

the amino acid sequence of the complementarity determining region 3(CDR3) of the heavy chain variable region of monoclonal antibody 3E9 is shown in SEQ ID NO.8, and specifically comprises:

ETYDAVDF。

the amino acid sequence of the heavy chain variable region of the monoclonal antibody 3E9 is shown as SEQ ID NO.9, and specifically comprises the following steps:

EVQLQQSGPELLKPGASVKMSCRASGYTFRSYTLHWVKQKPGQGLEWIGYFNPYNDGIKYNEKFKNKAKLTSDKSSSTVYMELNSLTSDDSAVYFCARETYDAVDFWGQGTTLTVSS。

the amino acid sequence of the heavy chain of the monoclonal antibody 3E9 is shown as SEQ ID NO.10, and specifically comprises the following steps:

MEWSWIFLFLLSGTAGVHSEVQLQQSGPELLKPGASVKMSCRASGYTFRSYTLHWVKQKPGQGLEWIGYFNPYNDGIKYNEKFKNKAKLTSDKSSSTVYMELNSLTSDDSAVYFCARETYDAVDFWGQGTTLTVSS are provided. That is, the heavy chain of monoclonal antibody 3E9 contains 136 amino acids.

Correspondingly, the nucleotide sequence of the complementarity determining region 1(CDR1) in the light chain variable region of monoclonal antibody 3E9 is shown in SEQ ID NO.11, specifically:

AGTGCCAGCTCAAGTGTAAATTACATGCAC。

the nucleotide sequence of the complementarity determining region 2(CDR2) in the light chain variable region of monoclonal antibody 3E9 is shown in SEQ ID NO.12, and specifically comprises:

AGCACATCCAACCTGGCTTCT。

the nucleotide sequence of the complementarity determining region 3(CDR3) in the light chain variable region of monoclonal antibody 3E9 is shown in SEQ ID NO.13, and specifically comprises:

CAGCAAAGGAATCGTTACCCGCTCACG。

the nucleotide sequence of the variable region of the light chain of the monoclonal antibody 3E9 is shown in SEQ ID NO.14, and specifically comprises the following steps:

CAGATTGTTCTCACCCAGTCTCCAGCATTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAATTACATGCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAGACTCTGCATCTTTAGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGTCGAATGGAGGCTGAGGATGCTGCCACTTATTACTGCCAGCAAAGGAATCGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA。

the nucleotide sequence of the light chain of the monoclonal antibody 3E9 is shown as SEQ ID NO.15, and specifically comprises the following steps:

ATGCATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATAATGTCCAAAGGACAGATTGTTCTCACCCAGTCTCCAGCATTCATGTCTGCATCTCCAGGGGAGAAGGTCACCATAACCTGCAGTGCCAGCTCAAGTGTAAATTACATGCACTGGTTCCAGCAGAAGCCAGGCACTTCTCCCAGACTCTGCATCTTTAGCACATCCAACCTGGCTTCTGGAGTCCCTGCTCGCTTCAGTGGCAGTGGATCTGGGACCTCTTACTCTCTCACAATCAGTCGAATGGAGGCTGAGGATGCTGCCACTTATTACTGCCAGCAAAGGAATCGTTACCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA。

that is, the nucleotides of the light chain of monoclonal antibody 3E9 contained 384 bases.

The nucleotide sequence of the heavy chain variable region complementarity determining region 1(CDR1) of monoclonal antibody 3E9 is shown in SEQ ID NO.16, and specifically comprises the following steps:

AGCTATACTTTGCAC。

the nucleotide sequence of the heavy chain variable region complementarity determining region 2(CDR2) of monoclonal antibody 3E9 is shown in SEQ ID NO.17, and specifically comprises the following steps:

TATTTTAATCCCTACAACGATGGTATTAAGTACAATGAGAAGTTTAAAAAC。

the nucleotide sequence of the heavy chain variable region complementarity determining region 3(CDR2) of monoclonal antibody 3E9 is shown in SEQ ID NO.18, and specifically comprises the following steps:

GAGACCTACGACGCGGTTGACTTC。

the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 3E9 is shown in SEQ ID NO.19, and specifically comprises the following steps:

ATGGAATGGAGTTGGATATTTCTCTTTCTCCTGTCAGGAACTGCAGGTGTCCACTCTGAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGTTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAGGGCTTCGGGATACACATTCAGGAGCTATACTTTGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATTTTAATCCCTACAACGATGGTATTAAGTACAATGAGAAGTTTAAAAACAAGGCCAAACTGACTTCAGACAAATCTTCCAGCACAGTCTACATGGAACTCAACAGCCTGACCTCTGATGACTCTGCGGTCTATTTCTGTGCAAGAGAGACCTACGACGCGGTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA。

the nucleotide sequence of the heavy chain of the monoclonal antibody 3E9 is shown as SEQ ID NO.20, and specifically comprises the following steps:

GAGGTCCAGCTGCAGCAGTCTGGACCTGAGCTGTTAAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAGGGCTTCGGGATACACATTCAGGAGCTATACTTTGCACTGGGTGAAGCAGAAGCCTGGGCAGGGCCTTGAGTGGATTGGATATTTTAATCCCTACAACGATGGTATTAAGTACAATGAGAAGTTTAAAAACAAGGCCAAACTGACTTCAGACAAATCTTCCAGCACAGTCTACATGGAACTCAACAGCCTGACCTCTGATGACTCTGCGGTCTATTTCTGTGCAAGAGAGACCTACGACGCGGTTGACTTCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA are provided. That is, the nucleotides of the heavy chain of monoclonal antibody 3E9 contained 351 bases.

The amino acid sequence of the complementarity determining region 1(CDR1) in the light chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.21, and specifically comprises:

KASQSVDYDGDSYMN。

the amino acid sequence of the complementarity determining region 2(CDR2) in the light chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.22, and specifically comprises:

DASNLES。

the amino acid sequence of the complementarity determining region 3(CDR3) in the light chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.23, and specifically comprises:

QQSNEDPLT。

the amino acid sequence of the light chain variable region of the monoclonal antibody 6F8 is shown in SEQ ID NO.24, and specifically comprises the following steps:

DIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPLTFGGGTRLELK。

the amino acid sequence of the light chain of the monoclonal antibody 6F8 is shown as SEQ ID NO.25, and specifically comprises the following steps:

METDTILLWVLLLWVPGSTGDIVLTQSPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYDASNLESGIPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPLTFGGGTRLELK are provided. That is, the light chain of monoclonal antibody 6F8 contained 131 amino acids.

The amino acid sequence of the heavy chain variable region complementarity determining region 1(CDR1) of monoclonal antibody 6F8 is shown in SEQ ID NO.26, and specifically comprises:

DYSMH。

the amino acid sequence of the heavy chain variable region complementarity determining region 2(CDR2) of monoclonal antibody 6F8 is shown in SEQ ID NO.27, and specifically comprises:

WINTETGEPTYANDFKG。

the amino acid sequence of the heavy chain variable region complementarity determining region 3(CDR3) of monoclonal antibody 6F8 is shown in SEQ ID NO.28, and specifically comprises:

NYGSSLAY。

the amino acid sequence of the heavy chain variable region of the monoclonal antibody 6F8 is shown in SEQ ID NO.29, and specifically comprises the following steps:

QIQLVQSGPELKKPGETVKISCKASDYTFTDYSMHWVKQAPGKGLKWMGWINTETGEPTYANDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARNYGSSLAYWGQGTLVTVSS。

the amino acid sequence of the heavy chain of the monoclonal antibody 6F8 is shown as SEQ ID NO.30, and specifically comprises the following steps:

MAWVWTLLFLMAAAQSIQAQIQLVQSGPELKKPGETVKISCKASDYTFTDYSMHWVKQAPGKGLKWMGWINTETGEPTYANDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCARNYGSSLAYWGQGTLVTVSS are provided. That is, the heavy chain of monoclonal antibody 6F8 contains 136 amino acids.

Correspondingly, the nucleotide sequence of the complementarity determining region 1(CDR1) of the light chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.31, specifically:

AAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAAC。

the nucleotide sequence of the complementarity determining region 2(CDR2) in the light chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.32, and specifically comprises:

GATGCATCCAATCTAGAATCT。

the nucleotide sequence of the complementarity determining region 3(CDR3) in the light chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.33, and specifically comprises:

GATGCATCCAATCTAGAATCT。

the nucleotide sequence of the variable region of the light chain of the monoclonal antibody 6F8 is shown in SEQ ID NO.34, and specifically comprises the following steps:

GACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTATCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGATGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGCTCACGTTCGGTGGTGGGACCAGGCTGGAGCTGAAA。

the nucleotide sequence of the light chain of the monoclonal antibody 6F8 is shown as SEQ ID NO.35, and specifically comprises the following steps:

ATGGAGACAGACACAATCCTGCTATGGGTGCTGCTGCTCTGGGTTCCAGGCTCCACTGGTGACATTGTGCTGACCCAATCTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTATCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGATGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCAAAGTAATGAGGATCCGCTCACGTTCGGTGGTGGGACCAGGCTGGAGCTGAAA are provided. That is, the nucleotides of the light chain of monoclonal antibody 6F8 contained 393 bases.

The nucleotide sequence of the complementarity determining region 1(CDR1) of the heavy chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.36, and specifically comprises:

GACTATTCAATGCAC。

the nucleotide sequence of the complementarity determining region 2(CDR2) of the heavy chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.37, and specifically comprises:

TGGATAAACACTGAGACTGGTGAGCCAACATATGCAAATGACTTCAAGGGA。

the nucleotide sequence of the complementarity determining region 3(CDR3) of the heavy chain variable region of monoclonal antibody 6F8 is shown in SEQ ID NO.38, and specifically comprises:

AACTACGGTAGTAGCTTGGCTTAC。

the nucleotide sequence of the heavy chain variable region of the monoclonal antibody 6F8 is shown as SEQ ID NO.39, and specifically comprises the following steps:

CAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGATTATACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAAATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGAAACTACGGTAGTAGCTTGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA。

the nucleotide sequence of the heavy chain of the monoclonal antibody 6F8 is shown as SEQ ID NO.40, and specifically comprises the following steps:

ATGGCTTGGGTGTGGACCTTGCTATTCCTGATGGCAGCTGCCCAAAGTATCCAAGCACAGATCCAGTTGGTGCAGTCTGGACCTGAGCTGAAGAAGCCTGGAGAGACAGTCAAGATCTCCTGCAAGGCTTCTGATTATACCTTCACAGACTATTCAATGCACTGGGTGAAGCAGGCTCCAGGAAAGGGTTTAAAGTGGATGGGCTGGATAAACACTGAGACTGGTGAGCCAACATATGCAAATGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCCTATTTGCAGATCAACAACCTCAAAAATGAGGACACGGCTACATATTTCTGTGCTAGAAACTACGGTAGTAGCTTGGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTTCA are provided. That is, the nucleotide of the heavy chain of monoclonal antibody 6F8 contained 408 bases.

② detection of ascites titer of monoclonal antibody

Diluting the detection antigen to a certain concentration by using a coating buffer solution, coating an ELISA plate strip by 50 mu L per hole, and standing overnight at 4 ℃; PBST was washed 3 times, 200. mu.L of blocking solution was added to each well, and the mixture was incubated at 4 ℃ forAt night; diluting the monoclonal antibody ascites in a multiple ratio, wherein each well is 50 mu L, and the diluted SP2/0 ascites in the same multiple ratio is used as a negative control, and incubating for 2h at 37 ℃; washing with PBST for 3 times, adding enzyme-labeled secondary antibody with working concentration, incubating at 37 deg.C for 1.5h, wherein each well contains 50 μ L of the enzyme-labeled secondary antibody; after PBST washing, OPD display, determination of OD by enzyme-linked Detector₄₉₀The value of (3) is determined by taking the P/N value more than or equal to 2.1 as a determination standard.

The results show that the titers of the monoclonal antibody 3E 9and the monoclonal antibody 6F8 both reach 1: 32768000.

identification of monoclonal antibody specificity

The specificity of the monoclonal antibody is identified by Dot-ELISA, and the specific steps are as follows: cutting an NC membrane with a certain size, soaking in deionized water, and airing; respectively sucking working concentration rHis-BoIL-4, rGST-BoIL-4, rHis-BoIFN-gamma, rGST-BoIFN-gamma, rHis-ChIFN-4, rGST-ChIFN-4, BL21(DE3) (pET) and BL21(pGEX-6P-1) on an NC membrane by using a pipettor, drying the supernatant at 37 ℃ for 30min, blocking the supernatant by using PBST containing 10% calf serum, and gently shaking the supernatant at room temperature overnight; after PBST washing, the NC membrane is immersed in cell culture supernatant or diluted ascites, and incubated for 2h at 37 ℃; PBST is washed for 3 times, each time is 10min, and then is immersed into goat anti-mouse HRP-Ig (G + M) enzyme-labeled antibody solution with working concentration, and is incubated for 1h at 37 ℃; PBST was washed 3 times for 10min, and DAB finally showed that the reaction was stopped with distilled water.

In the Dot-ELISA assay, monoclonal antibody 3E 9and monoclonal antibody 6F8 reacted only with rBoIFN-. gamma.and not with the other recombinant cytokines and controls described above, which were prokaryotically expressed.

Example 2 Assembly of bovine tuberculosis Gamma-interferon ELISpot detection kit

The bovine tuberculosis gamma-interferon ELISpot kit comprises the following assembly steps:

(1) preparation of biotin-labeled bovine gamma interferon monoclonal antibody (Bio-6F 8):

(2) Kit assembly

The method comprises the steps of putting bottle numbers of components such as a microporous filter membrane plate (such as a 96-pore filter membrane plate), a bovine gamma interferon monoclonal antibody 3E9 (a monoclonal antibody produced by a hybridoma cell line with a preservation number of CCTCC NO: C2017283 or a subcultured cell line thereof in a secretion mode), a biotin-labeled bovine gamma interferon monoclonal antibody Bio-6F8 (a monoclonal antibody produced by a hybridoma cell line with a preservation number of CCTCC NO: C2017282 or a subcultured cell line thereof in a secretion mode) into a kit plastic bracket according to the number of each kit, packaging and assembling the kit, putting a specification into the kit, and attaching an outer label and a side label.

Further, the kit is assembled with: avidin-alkaline phosphatase conjugate, NBT/BCIP substrate chromogenic solution, diluent, wash (20 ×), blocking solution, specific stimulator (e.g., CE protein), negative control (e.g., complete 1640 medium), and positive control (e.g., Phytolacca americana PWM).

In the kit, the microfiltration membrane plate and the bovine gamma interferon monoclonal antibody 3E9 (the monoclonal antibody secreted by hybridoma cell strain with the preservation number of CCTCC NO: C2017283 or a subcultured cell strain thereof) can also be replaced by the microfiltration membrane plate coated with the bovine gamma interferon monoclonal antibody 3E 9.

The preparation method of the microporous filter membrane plate coated with the bovine gamma interferon monoclonal antibody 3E9 comprises the following steps:

opening a 96-hole filter membrane plate in an aseptic mode, washing the filter membrane plate with 20 mu L/hole, 35% ethanol and a pre-wet PVDF membrane for 1min with PBS for 3 times, 250 mu L/hole and 1 min/time.

Adding 5 microgram/mL monoclonal antibody 3E9 (monoclonal antibody secreted by hybridoma cell strain with CCTCC NO: C2017283 or its passage cell strain) for capturing bovine gamma interferon into 96-hole filter membrane plate, coating at 4 deg.C overnight at 100 microgram/hole;

thirdly, abandoning the coating liquid, and washing the plate for 3 times and 1 min/time by using PBS containing sterilization;

adding complete 1640 culture medium containing 10% fetal calf serum, incubating and sealing at 37 ℃ for 2h at 200 mu L/hole;

abandoning the sealing liquid, washing the plate for 1 time by PBS, drying for 2h at 37 ℃, adding a drying agent to put the 96-hole filter membrane plate into a sealing bag together, vacuumizing and sealing, and storing at 4 ℃.

(3) Instructions for use of the kit

1) Take coated ELISpot plates (millipore filter plates coated with bovine gamma interferon monoclonal antibody 3E9) and add 50 μ L of specific stimulator (e.g. CE protein), positive control (e.g. pokeweed PWM), negative control (e.g. complete 1640 medium) to each well, respectively.

2) Adding 50 mu L of PBMC to be detected into each hole, flicking the ELISA plate or shaking by a micro-oscillator to uniformly mix the solution in the reaction plate.

3) Cover the cover and incubate at 37 ℃ for 24-48 h.

4) Wash each well 6 times 5 min/time with 250 μ L of 1 × wash. After each wash, the wells were drained to remove any remaining wash solution. Drying of the pore walls was avoided before the next reagent was added.

5) The enzyme-labeled antibody is diluted to 1.5. mu.g/mL (used as it is) with an enzyme-labeled antibody diluent after the transient ionization. mu.L of freshly prepared enzyme-labeled antibody was added to each well, capped, and incubated at 37 ℃ for 2 h.

6) Repeat step 4.

7) With diluent 1: Streptavidin-AKP was diluted 1000, 100. mu.L of freshly prepared enzyme was added to each well, capped, and incubated at 37 ℃ for 2 h.

8) Repeat step 4.

9) Adding 100 μ L substrate color developing solution into each well, attaching a sealing plate membrane, and developing at 37 deg.C in dark for 5-10 min.

10. After the termination of the rinsing with tap water, drying was carried out overnight at 37 ℃.

11) Count using ELISPOT plate reader.

TABLE 1 Components of kits according to the invention

Example 3 detection of bovine peripheral blood samples by the ELISpot assay kit based on non-specific Prostimogen PWM

1. Experimental Material

The bovine tuberculosis gamma-interferon ELISpot detection kit prepared in example 2 was used.

PBMC isolation, incubation and detection

Adding 5mL of bovine blood into a heparin sodium-containing blood collection tube in an aseptic manner, and inverting and uniformly mixing the collected blood to obtain anticoagulation blood;

② taking a lymphocyte separation tube to balance to room temperature, centrifuging for 3min at 800g to ensure that all separation liquid falls under the sieve plate, opening the tube cover, carefully superposing the blood sample on the sieve plate by a dropper, centrifuging for 20min at 800g at 18-22 ℃. After centrifugation, the liquid in the tube is divided into four layers, which are sequentially from top to bottom: a yellow clear plasma layer, a cloud cell layer, a colorless separation liquid layer and a red blood cell layer. Carefully sucking out the cloudy cell layer on the upper layer of the separation solution by using a suction pipe, placing the cloudy cell layer in another new centrifugal tube (into which the serum-free cell culture medium is added), uniformly mixing, and centrifuging at the temperature of 4 ℃ for 10min by using 500 g. The supernatant was discarded, the cells were resuspended in serum-free medium, 500g and centrifuged at 4 ℃ for 10 min. Discarding the supernatant, resuspending the cell suspension in a proper volume of 1640 completely, mixing the cell suspension and the cell suspension, taking the proper volume for counting, covering the centrifugal tube cover with the rest cell suspension, and placing the cell suspension on ice for later use.

③ adding the following reagents into the 96-hole filter membrane plate coated with the bovine gamma interferon monoclonal antibody 3E 9: each PBMC sample included 2 test wells, 50. mu.L of cell culture medium to negative control wells, 50. mu.L of diluted PWM to positive control wells (final concentration 10. mu.g/mL), 50. mu.L of bovine PBMC per well, 96-well filter plates placed at 37 ℃ and 5% CO₂Culturing in an incubator for 24-48 h;

and fourthly, taking out the 96-hole filter membrane plate, discarding the culture supernatant, washing the plate for 5 times and 5 min/time by using PBS, and spin-drying the plate after washing. Adding 1.5 mu g/mL bovine gamma interferon detection antibody Bio-6F8, 100 mu L/well, and incubating at 37 ℃ for 2 h;

washing the plate with PBS for 5 times, 5 min/time, drying after washing the plate, adding 1: 1000 dilution of avidin-alkaline phosphatase conjugate, 100. mu.L/well, incubated at 37 ℃ for 2 h;

sixthly, 100 mu L of substrate liquid nitrogen blue tetrazole and 5-bromine-4-chlorine-3-indole phosphoric acid are added into each hole, and the mixture is placed at room temperature and is shaded for color development. Adding purified water into a 96-hole filter membrane plate to terminate the reaction, removing liquid, and drying overnight at room temperature or in an oven at 37 ℃ for 2-3 h;

counting purple spots in each reaction hole by using an inverted microscope, wherein each spot represents a T cell secreting bovine gamma interferon; or putting the 96-hole filter membrane plate into an ELISpot instrument, and scanning, counting and analyzing the experimental results.

The results are shown in FIG. 1. The result shows that when a bovine PBMC sample is detected, the number of effective spots appearing in the positive control hole (A) is obviously more than that of the effective spots appearing in the negative control hole (B), and the result shows that the kit can be used for directly detecting bovine PBMC, and the kit has better sensitivity and specificity.

Example 4 comparison of the nonspecific stimulus PWM-based ELISpot assay kit with bovine gamma interferon ELISpot assay using 2G5 as the coating antibody and Bio-5E11 as the detection antibody for detecting bovine peripheral blood samples

1. Experimental Material

The bovine gamma interferon ELISpot detection kit was prepared according to the method of the present invention in example 3.

The difference lies in that:

the bovine gamma interferon monoclonal antibody 2G5 (secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2012107 or a passage cell line thereof) is used as a coating antibody, and the bovine gamma interferon monoclonal antibody Bio-5E11 (secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2012108 or a passage cell line thereof) is used as a detection antibody. Wherein the bovine gamma interferon monoclonal antibody 2G5 (secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2012107 or a passage cell line thereof) and the bovine gamma interferon monoclonal antibody 5E11 (secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2012108 or a passage cell line thereof) are disclosed in an authorized invention patent with the publication number of CN 102965343B.

2. Bovine gamma interferon ELISpot detection method using bovine gamma interferon monoclonal antibody 2G5 (secreted by hybridoma cell line with preservation number of CCTCC NO: C2012107 or passage cell line thereof) as coating antibody and bovine gamma interferon monoclonal antibody Bio-5E11 (secreted by hybridoma cell line with preservation number of CCTCC NO: C2012108 or passage cell line thereof) as detection antibody

1 healthy cow is selected, and a bovine gamma interferon ELISpot detection method using a bovine gamma interferon monoclonal antibody 2G5 (secreted by a hybridoma cell line with a preservation number of CCTCC NO: C2012107 or a passage cell line thereof) as a coating antibody and a bovine gamma interferon monoclonal antibody Bio-5E11 (secreted by a hybridoma cell line with a preservation number of CCTCC NO: C2012108 or a passage cell line thereof) as a detection antibody is adopted for detection.

3. Detection kit for bovine gamma interferon ELISpot

The same 1 healthy cow was selected and bovine interferon gamma detection was performed using the procedures of the bovine interferon gamma ELISpot detection kit prepared in example 2. The test results are compared and analyzed with the results of the bovine gamma interferon ELISpot detection method which takes a bovine gamma interferon monoclonal antibody 2G5 (secreted by a hybridoma cell strain with the preservation number of CCTCC NO: C2012107 or a passage cell strain thereof) as a coating antibody and a bovine gamma interferon monoclonal antibody Bio-5E11 (secreted by a hybridoma cell strain with the preservation number of CCTCC NO: C2012108 or a passage cell strain thereof) as a detection antibody. The results are shown in FIG. 2. As can be seen from the results in FIG. 2, according to the spot morphology of the image, the number of spots in the positive well of the detection antibody Bio-6F8 was found to be greater than that in the Bio-5E11 well, and the number of spots in the negative well of the capture antibody 2G5 was found to be greater than that in the negative well, and the background interference was found to be greater than that in 3E 9. Compared with the bovine gamma interferon ELISpot detection method which takes 2G5 (secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2012107 or a passage cell line thereof) as a coating antibody and Bio-5E11 (secreted by a hybridoma cell line with the preservation number of CCTCC NO: C2012108 or a passage cell line thereof) as a detection antibody, the ELISpot detection kit based on non-specific stimulus PWM in the embodiment 3 of the invention has the advantages of small negative background interference, large number of positive spots, round and fused spot forms and clear background.

Example 5 ELISpot assay kit and method based on non-specific Prostimogen PWM

Comparison of bovine tuberculosis gamma-interferon ELISA (enzyme-Linked immunosorbent assay) detection kit for detecting bovine peripheral blood mononuclear cell samples

1. Experimental Material

The bovine interferon gamma ELISpot detection kit prepared in example 3 was used.

ELISA kits (Mycobacterium bovis Gamma Interferon Test Kit for Cattle) were purchased from ThermoFisher.

2.

ELISA kit detection

Selecting 1 cow in a cow farm, and adopting commercialization

The ELISA kit is used for detection. The isolated PBMC were used as samples at concentrations of 3 x 10 respectively⁵Hole, 3 x 10⁴Hole, 3 x 10³Hole, 3 x 10²Hole/3 x 10 hole, two holes per hole. The kit was handled according to the instructions and the results were determined.

3. Detection kit for bovine gamma interferon ELISpot

The same 1 healthy cow in the cattle farm was selected, and the bovine tuberculosis was detected using the procedures of the bovine gamma interferon ELISpot detection kit prepared in example 3. The isolated PBMC were used at a concentration of 3 x 10 respectively⁵Hole, 3 x 10⁴Hole, 3 x 10³Hole, 3 x 10²Hole/3 x 10 hole, two holes per hole. Comparing the result of the test with

And (4) comparing and analyzing the results of the ELISA kit. The results are shown in tables 2-1 and 2-2.

TABLE 2-1 ELISpot method measurement results

Tables 2 to 2

Measurement results of ELISA kit

As can be seen from the results shown in tables 2-1 and 2-2, the number of ELISpot spots formed decreased with the decrease in the number of PBMCs under the same incubation conditions, when the number of PBMCs decreased to 3 x 10⁴At one/well, spots (about 80) can be formed by ELISpot detection method, and ELISA method OD₄₅₀The value was already close to the negative control level. The ELISpot detection method has higher sensitivity than the ELISA method under the same condition, and is more suitable for detecting the level of IFN-gamma secreted by living cells.

Example 6 comparison of the nonspecific stimulus PWM-based ELISpot assay kit with the Mabtech antibody for detecting bovine peripheral blood samples

1. Experimental Material

The bovine gamma interferon ELISpot detection method established in the research and the antibody pair (MT17.1, MT307) of Mabtech company detect the spot forming effect of the same PBMC. The detection method of the antibody pair (MT17.1, MT307) by Mabtech was carried out according to the instructions.

The results are shown in fig. 3, and it can be seen from fig. 3 that the ELISpot detection method established by the invention has the advantages of more positive hole spots, less negative hole spots, large difference of the number of positive negative spots, clear spot shape, bright background and good application value.

Example 7 bovine tuberculosis gamma-interferon ELISpot assay kit based on specific stimulus CE protein for detecting bovine peripheral blood samples

1. Experimental Material

Isolation of PBMC

Incubation and detection of PBMC

Adding the following reagents into the 96-well filter membrane plate coated with the bovine gamma interferon monoclonal antibody 3E 9: each PBMC sample included 3 test wells, 50. mu.L of cell culture medium to a negative control well, 50. mu.L of cell culture medium diluted PWM to a positive control well (final concentration of 5. mu.g/mL), and 50. mu.L of cell culture medium diluted specific stimulatory pro-CE protein to a test well (final concentration of 10. mu.g/mL). mu.L bovine PBMC was added to each well, and the 96-well filter plate was incubated at 37 ℃ with 5% CO₂Culturing in an incubator for 24-48 h;

② taking out the 96-hole filter membrane plate, abandoning the culture supernatant, washing the plate 5 times and 5 min/time by using PBS, and drying the plate after washing. Adding 1.5 mu g/mL bovine gamma interferon detection antibody Bio-6F8, 100 mu L/well, and incubating at 37 ℃ for 2 h;

washing the plate with PBS for 5 times and 5 min/time, drying after washing the plate, and adding 1: 1000 dilution of avidin-alkaline phosphatase conjugate, 100. mu.L/well, incubated at 37 ℃ for 2 h;

fourthly, 100 mul of liquid nitrogen blue tetrazole substrate and 5-bromo-4-chloro-3-indole phosphoric acid are added into each hole, and the mixture is placed at room temperature and is shaded for color development. Adding purified water into a 96-hole filter membrane plate to terminate the reaction, removing liquid, and drying overnight at room temperature or in an oven at 37 ℃ for 2-3 h;

3. Conditions for testing effectiveness

The number of spots in the PWM positive control hole is larger than that in the negative control hole, and the number of spots in the positive quality control PWM positive control hole is more than 20, so that the test is judged to be effective.

4. Bovine tuberculosis outcome determination

If the number of spots in the negative control well is less than 5, and the number of spots in the CE protein detection well minus the number of spots in the negative control well is greater than 6;

if the number of spots in the negative control hole is more than 6, the number of spots in the CE protein detection hole is more than 2 times of the number of spots in the negative control hole;

and if any detection result appears, judging the bovine tuberculosis to be positive, otherwise, judging the bovine tuberculosis to be negative.

The results are shown in FIG. 4. The result shows that the kit can effectively detect T lymphocytes secreting bovine gamma interferon in the peripheral blood of positive cattle with tuberculosis, the number of spots in positive quality control PWM positive control holes is more than 20(A), the number of spots in CE protein detection holes is more than 2 times of the number of spots in negative control holes (B and C).

Example 8 comparison of CE protein-based bovine tuberculosis ELISpot assay kit with comparative intradermal allergy test

1. Experimental Material

Comparative intradermal allergy test bovine PPD and avian PPD were purchased from the Chinese veterinary drug administration.

2. Comparative intradermal allergy test

36 cows were selected and a comparative skin test allergy test was carried out according to the national standard method (GB/T18645-.

3. Bovine tuberculosis gamma-interferon ELISpot detection kit

The 36 cows were selected and bovine tuberculosis was detected using the procedures of the bovine tuberculosis ELISpot detection kit prepared in example 7. The test results were compared and analyzed with comparative skin test allergy test results, as shown in table 3.

TABLE 3 CE protein-based ELISpot test kit for bovine tuberculosis and comparative skin test allergy test results (Unit: head)

Detecting 36 cattle in total, and calculating the positive coincidence rate by detecting the number of common positive cattle detected by the two kits/[ (the number of positive cattle detected by an ELISA kit + the number of positive cattle detected by an ELISpot detection kit)/2 ]; the number of common negative cattle detected by the two kits/[ (the number of negative cattle detected by the ELISA kit + the number of negative cattle detected by the ELISpot detection kit)/2 ] is calculated to obtain the negative coincidence rate. The total coincidence rate was calculated from (the number of positive cows detected together by the two kits + the number of negative cows detected together)/(the total number of detected cows).

Through statistical analysis, the positive coincidence rate of the bovine tuberculosis gamma-interferon ELISpot detection kit based on the CE protein and the skin test allergic reaction result is 76.9%, the negative coincidence rate is 72.7%, the total coincidence rate is 75%, the detection effect is good, and the diagnosis value of the kit in bovine tuberculosis diagnosis is good.

Example 9 CE protein-based bovine tuberculosis gamma-interferon ELISpot assay kit and

comparison of bovine tuberculosis Gamma-interferon ELISA detection kit

1. Experimental Material

A place: a cattle farm in Jiangsu.

Time: year 2017, month 10.

Number of test heads: 36 heads.

This test detects clinical cow PBMC samples and is commercially available

And comparing the ELISA kits to further confirm the effect of the CE protein as a stimulator of the bovine tuberculosis ELISpot detection kit. The results of detecting bovine peripheral mononuclear cell samples by the bovine tuberculosis gamma-interferon ELISpot detection kit are determined as described in example 7, and the results are shown in Table 4.

TABLE 4 CE protein-based bovine tuberculosis gamma-interferon ELISpot assayTest kit results and

ELISA kit result comparison (Unit: head)

"+" represents positive, and "-" represents negative

Detecting 36 suspected cattle with tuberculosis together to

The ELISA kit test result is used as reference, the cow which is detected as positive by the kit is judged as positive bovine tuberculosis, and the cow which is detected as negative by the kit is judged as negative bovine tuberculosis. The number of common positive cattle detected by the two kits/[ (the number of positive cattle detected by ELISA kit + the number of positive cattle detected by ELISpot detection kit)/2]Calculating the positive coincidence rate; the number of common negative cattle detected by the two kits/[ (the number of negative cattle detected by ELISA kit + the number of negative cattle detected by ELISpot detection kit)/2]And (5) calculating the negative coincidence rate. The total coincidence rate was calculated from (the number of positive cows detected together by the two kits + the number of negative cows detected together)/(the total number of detected cows).

Bovine tuberculosis gamma-interferon ELISpot detection kit based on CE protein through statistical analysis and detection method thereof

The ELISA kit has the positive coincidence rate of 88.2 percent, the negative coincidence rate of 89.5 percent and the total coincidence rate of 88.9 percent, has good detection effect, and shows that the kit has better diagnostic value in bovine tuberculosis diagnosis.

Example 10 comparison of CE protein-based bovine tuberculosis Gamma-interferon ELISpot assay kit with bovine tuberculosis antibody assay kit

1. Experimental Material

The bovine tuberculosis antibody detection kit is purchased from Han national anjie company.

2. Bovine tuberculosis antibody kit detection

Selecting 10 PPD positive cows in a tuberculosis positive cow farm, adopting a bovine tuberculosis antibody kit for testing, operating according to the kit operating instructions and judging the result.

3. Bovine tuberculosis gamma-interferon ELISpot detection kit

A positive cow of 10 PPD detection positive cows in a tuberculosis positive cow farm is selected, and the bovine tuberculosis is detected by using the steps of the bovine tuberculosis gamma-interferon ELISpot detection kit prepared in the embodiment 7. And comparing and analyzing the test result with the result of the bovine tuberculosis antibody kit. The results are shown in Table 5.

TABLE 5 CE protein-based results of the bovine tuberculosis gamma-interferon ELISpot assay kit compared to the results of the bovine tuberculosis antibody kit (unit: head)

Through statistical analysis, the positive coincidence rate of the results of the bovine tuberculosis gamma-interferon ELISpot detection kit and the bovine tuberculosis antibody detection kit based on the CE protein is 36.4%, the negative coincidence rate is 22.2%, and the total coincidence rate is 30.0%, thus showing that the ELISpot detection kit has good diagnostic value in bovine tuberculosis diagnosis.

Example 11 comparison of CE protein-based bovine tuberculosis Gamma-Interferon ELISpot assay kit with PPD-based bovine tuberculosis Gamma-Interferon ELISpot assay method

1. Experimental Material

PPD-based bovine tuberculosis gamma interferon ELISPOT detection method (Xuzhong, et al. establishment of bovine tuberculosis gamma interferon ELISPOT detection method. Bioengineering reports 2015,31(2): 183-194).

2. PPD-based bovine tuberculosis gamma interferon ELISpot detection method

Healthy cows and positive cows of a certain tuberculosis positive cow farm are selected and tested by a PPD-based bovine tuberculosis gamma interferon ELISpot detection method.

3. Bovine tuberculosis gamma-interferon ELISpot detection kit

1 healthy cow and 1 knot positive cow were selected, and bovine tuberculosis was detected using the procedures of the bovine tuberculosis ELISpot detection kit prepared in example 7. And comparing and analyzing the test result with the result of the PPD-based bovine tuberculosis gamma interferon ELISpot detection method. The results are shown in FIG. 5. The results show that both CE protein as a specific stimulator and PPD as a stimulator, speckles were formed in the test images of bovine blood. In the blood test chart of healthy cattle, when CE protein is used as a specific stimulant, spots are not formed, and because PPD components are complex, a plurality of contained proteins exist in pathogenic mycobacteria and environmental non-pathogenic mycobacteria at the same time, the conditions of positive tuberculosis infection, immune individuals, environmental mycobacteria infection and the like cannot be distinguished, cross reaction often occurs during serological detection, so that specificity is influenced to a certain extent, and spots are formed in healthy cattle blood. Therefore, the CE protein-based bovine tuberculosis gamma-interferon ELISpot detection kit eliminates the interference in the aspect and shows that the kit has good specificity. Has good detection effect, and shows that the kit has good diagnostic value in bovine tuberculosis diagnosis.

Example 12 detection of tuberculosis-free cattle herd by CE protein-based bovine tuberculosis gamma-interferon ELISpot detection kit

1. Experimental Material

2. Bovine tuberculosis gamma-interferon ELISpot detection kit

A tuberculosis-free bovine group was selected and bovine tuberculosis was detected using the procedures of the bovine tuberculosis ELISpot detection kit prepared in example 7. All experimental cows purified the herd of cattle for tuberculosis, for a total of 17. The results are shown in Table 7.

TABLE 7 detection results of tuberculosis-free herds of cattle

"+" represents positive, and "-" represents negative

The results show that in tuberculosis-purified cattle, specificity of the CE protein-based bovine tuberculosis gamma-interferon ELISpot detection kit is 100%. The kit can better distinguish the conditions of tuberculosis positive infection, immune individuals, environmental mycobacterium infection and the like, and cross reaction often occurs during serological detection, which shows that the kit has good specificity.

Example 13 CE protein-based bovine tuberculosis Gamma-interferon ELISpot assay kit for detecting IFN-Gamma negative bovine herd of positive bovine tuberculosis in skin test

1. Experimental Material

2. Bovine tuberculosis gamma-interferon ELISpot detection kit

A skin test positive bovine tuberculosis IFN- γ negative herd was selected, and bovine tuberculosis was detected using the procedures of the bovine tuberculosis ELISpot detection kit prepared in example 7, for a total of 4 experimental cows. The results are shown in Table 8.

TABLE 8 detection results of positive cattle in skin test

The results show that in the skin test positive cattle group, the cattle tuberculosis ELISpot detection kit is used for detection, the negative control is controlled to be less than 5, the number of spots formed by stimulation of the positive stimulation group is more than 20, and the test is established. Meanwhile, the number of spots formed by CE protein stimulation is less than 5, which indicates that the detection result is negative cattle.

In conclusion, the inventor uses sandwich ELISA method, uses the positive plasma containing natural BoIFN-gamma prepared by pokeweed mitogen (PWM) stimulation as screening antigen, and obtains 4 monoclonal antibodies with better capability of capturing natural BoIFN-gamma and 6 monoclonal antibodies with better capability of detecting natural BoIFN-gamma from at least 43 BoIFN-gamma monoclonal antibodies through the BoIFN-gamma monoclonal antibodies and the multi-antibody sandwich ELISA test.

And combining the 4 strains of capture antibodies and 6 strains of detection antibodies with better binding capacity to the natural BoIFN-gamma in the ELISA result by using an ELISpot method in a pairwise manner. And respectively using 4 strains of capture antibodies as coating antibodies in the ELISpot method, respectively using 6 strains of biotin-labeled detection antibodies as detection antibodies in the ELISpot method, forming 24 antibody pair combinations, and performing an ELISpot test to obtain 9 antibody pair combinations with a large number of spots.

Then, the combination of the 9 antibody pairs is synchronously compared with a Bovine IFN-gamma ELISpot antibody pair (MT17.1, MT307) of the company MABTECH AB, Sweden, and the principles of more positive hole spots, less negative hole spots, large difference of the number of positive negative spots, clear spot shapes, bright background and the like in the ELISpot test result are adopted. Finally, monoclonal antibody 3E 9and Bio-6F8 were selected as the capture antibody and the detection antibody of the bovine gamma interferon ELISpot detection method, respectively.

According to the invention, at least 43 BoIFN-gamma monoclonal antibodies are screened, and only 7 monoclonal antibodies finally show stronger immunoreactivity to natural BoIFN-gamma, which indicates that most of the monoclonal antibodies do not have the capability of recognizing natural BoIFN-gamma epitopes. Finally, monoclonal antibodies 3E 9and Bio-6F8 are selected as a capture antibody and a detection antibody of the bovine gamma interferon ELISpot detection method respectively, and a great deal of creative work is performed.

While the invention has been described with respect to a preferred embodiment, it will be understood by those skilled in the art that the foregoing and other changes, omissions and deviations in the form and detail thereof may be made without departing from the scope of this invention. Those skilled in the art can make various changes, modifications and equivalent arrangements, which are equivalent to the embodiments of the present invention, without departing from the spirit and scope of the present invention, and which may be made by utilizing the techniques disclosed above; meanwhile, any changes, modifications and variations of the above-described embodiments, which are equivalent to those of the technical spirit of the present invention, are within the scope of the technical solution of the present invention.

Sequence listing

<110> Yangzhou university

<120> an ELISpot detection kit for detecting bovine tuberculosis

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Gln Gln Arg Asn Arg Tyr Pro Leu Thr

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<213> Artificial Sequence (Artificial Sequence)

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Gln Ile Val Leu Thr Gln Ser Pro Ala Phe Met Ser Ala Ser Pro Gly

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Glu Lys Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Asn Tyr Met

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His Trp Phe Gln Gln Lys Pro Gly Thr Ser Pro Arg Leu Cys Ile Phe

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Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ala Arg Phe Ser Gly Ser

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Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Met Glu Ala Glu

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Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg Asn Arg Tyr Pro Leu Thr

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Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

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<211> 128

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met His Phe Gln Val Gln Ile Phe Ser Phe Leu Leu Ile Ser Ala Ser

1 5 10 15

Val Ile Met Ser Lys Gly Gln Ile Val Leu Thr Gln Ser Pro Ala Phe

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Met Ser Ala Ser Pro Gly Glu Lys Val Thr Ile Thr Cys Ser Ala Ser

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Ser Ser Val Asn Tyr Met His Trp Phe Gln Gln Lys Pro Gly Thr Ser

50 55 60

Pro Arg Leu Cys Ile Phe Ser Thr Ser Asn Leu Ala Ser Gly Val Pro

65 70 75 80

Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile

85 90 95

Ser Arg Met Glu Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Arg

100 105 110

Asn Arg Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

115 120 125

<210> 6

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Ser Tyr Thr Leu His

1 5

<210> 7

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Tyr Phe Asn Pro Tyr Asn Asp Gly Ile Lys Tyr Asn Glu Lys Phe Lys

1 5 10 15

Asn

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<211> 8

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Glu Thr Tyr Asp Ala Val Asp Phe

1 5

<210> 9

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Leu Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Thr Phe Arg Ser Tyr

20 25 30

Thr Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Tyr Phe Asn Pro Tyr Asn Asp Gly Ile Lys Tyr Asn Glu Lys Phe

50 55 60

Lys Asn Lys Ala Lys Leu Thr Ser Asp Lys Ser Ser Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Glu Thr Tyr Asp Ala Val Asp Phe Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 10

<211> 136

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 10

Met Glu Trp Ser Trp Ile Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly

1 5 10 15

Val His Ser Glu Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Leu Lys

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Arg Ala Ser Gly Tyr Thr Phe

35 40 45

Arg Ser Tyr Thr Leu His Trp Val Lys Gln Lys Pro Gly Gln Gly Leu

50 55 60

Glu Trp Ile Gly Tyr Phe Asn Pro Tyr Asn Asp Gly Ile Lys Tyr Asn

65 70 75 80

Glu Lys Phe Lys Asn Lys Ala Lys Leu Thr Ser Asp Lys Ser Ser Ser

85 90 95

Thr Val Tyr Met Glu Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val

100 105 110

Tyr Phe Cys Ala Arg Glu Thr Tyr Asp Ala Val Asp Phe Trp Gly Gln

115 120 125

Gly Thr Thr Leu Thr Val Ser Ser

130 135

<210> 11

<211> 30

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

agtgccagct caagtgtaaa ttacatgcac 30

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

agcacatcca acctggcttc t 21

<210> 13

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

cagcaaagga atcgttaccc gctcacg 27

<210> 14

<211> 318

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cagattgttc tcacccagtc tccagcattc atgtctgcat ctccagggga gaaggtcacc 60

ataacctgca gtgccagctc aagtgtaaat tacatgcact ggttccagca gaagccaggc 120

acttctccca gactctgcat ctttagcaca tccaacctgg cttctggagt ccctgctcgc 180

ttcagtggca gtggatctgg gacctcttac tctctcacaa tcagtcgaat ggaggctgag 240

gatgctgcca cttattactg ccagcaaagg aatcgttacc cgctcacgtt cggtgctggg 300

accaagctgg agctgaaa 318

<210> 15

<211> 384

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

atgcattttc aagtgcagat tttcagcttc ctgctaatca gtgcctcagt cataatgtcc 60

aaaggacaga ttgttctcac ccagtctcca gcattcatgt ctgcatctcc aggggagaag 120

gtcaccataa cctgcagtgc cagctcaagt gtaaattaca tgcactggtt ccagcagaag 180

ccaggcactt ctcccagact ctgcatcttt agcacatcca acctggcttc tggagtccct 240

gctcgcttca gtggcagtgg atctgggacc tcttactctc tcacaatcag tcgaatggag 300

gctgaggatg ctgccactta ttactgccag caaaggaatc gttacccgct cacgttcggt 360

gctgggacca agctggagct gaaa 384

<210> 16

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

agctatactt tgcac 15

<210> 17

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

tattttaatc cctacaacga tggtattaag tacaatgaga agtttaaaaa c 51

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gagacctacg acgcggttga cttc 24

<210> 19

<211> 408

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

atggaatgga gttggatatt tctctttctc ctgtcaggaa ctgcaggtgt ccactctgag 60

gtccagctgc agcagtctgg acctgagctg ttaaagcctg gggcttcagt gaagatgtcc 120

tgcagggctt cgggatacac attcaggagc tatactttgc actgggtgaa gcagaagcct 180

gggcagggcc ttgagtggat tggatatttt aatccctaca acgatggtat taagtacaat 240

gagaagttta aaaacaaggc caaactgact tcagacaaat cttccagcac agtctacatg 300

gaactcaaca gcctgacctc tgatgactct gcggtctatt tctgtgcaag agagacctac 360

gacgcggttg acttctgggg ccaaggcacc actctcacag tctcctca 408

<210> 20

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

gaggtccagc tgcagcagtc tggacctgag ctgttaaagc ctggggcttc agtgaagatg 60

tcctgcaggg cttcgggata cacattcagg agctatactt tgcactgggt gaagcagaag 120

cctgggcagg gccttgagtg gattggatat tttaatccct acaacgatgg tattaagtac 180

aatgagaagt ttaaaaacaa ggccaaactg acttcagaca aatcttccag cacagtctac 240

atggaactca acagcctgac ctctgatgac tctgcggtct atttctgtgc aagagagacc 300

tacgacgcgg ttgacttctg gggccaaggc accactctca cagtctcctc a 351

<210> 21

<211> 15

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 21

Lys Ala Ser Gln Ser Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn

1 5 10 15

<210> 22

<211> 7

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 22

Asp Ala Ser Asn Leu Glu Ser

1 5

<210> 23

<211> 9

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 23

Gln Gln Ser Asn Glu Asp Pro Leu Thr

1 5

<210> 24

<211> 111

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 24

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser Val Asp Tyr Asp

20 25 30

Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Ser Gly Ile Pro Ala

50 55 60

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Asn Ile His

65 70 75 80

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Ser Asn

85 90 95

Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Arg Leu Glu Leu Lys

100 105 110

<210> 25

<211> 131

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 25

Met Glu Thr Asp Thr Ile Leu Leu Trp Val Leu Leu Leu Trp Val Pro

1 5 10 15

Gly Ser Thr Gly Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala

20 25 30

Val Ser Leu Gly Gln Arg Ala Thr Ile Ser Cys Lys Ala Ser Gln Ser

35 40 45

Val Asp Tyr Asp Gly Asp Ser Tyr Met Asn Trp Tyr Gln Gln Lys Pro

50 55 60

Gly Gln Pro Pro Lys Leu Leu Ile Tyr Asp Ala Ser Asn Leu Glu Ser

65 70 75 80

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

85 90 95

Leu Asn Ile His Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Cys

100 105 110

Gln Gln Ser Asn Glu Asp Pro Leu Thr Phe Gly Gly Gly Thr Arg Leu

115 120 125

Glu Leu Lys

130

<210> 26

<211> 5

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 26

Asp Tyr Ser Met His

1 5

<210> 27

<211> 17

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 27

Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asn Asp Phe Lys

1 5 10 15

Gly

<210> 28

<211> 8

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 28

Asn Tyr Gly Ser Ser Leu Ala Tyr

1 5

<210> 29

<211> 117

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 29

Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu

1 5 10 15

Thr Val Lys Ile Ser Cys Lys Ala Ser Asp Tyr Thr Phe Thr Asp Tyr

20 25 30

Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu Lys Trp Met

35 40 45

Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala Asn Asp Phe

50 55 60

Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys

85 90 95

Ala Arg Asn Tyr Gly Ser Ser Leu Ala Tyr Trp Gly Gln Gly Thr Leu

100 105 110

Val Thr Val Ser Ser

115

<210> 30

<211> 136

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 30

Met Ala Trp Val Trp Thr Leu Leu Phe Leu Met Ala Ala Ala Gln Ser

1 5 10 15

Ile Gln Ala Gln Ile Gln Leu Val Gln Ser Gly Pro Glu Leu Lys Lys

20 25 30

Pro Gly Glu Thr Val Lys Ile Ser Cys Lys Ala Ser Asp Tyr Thr Phe

35 40 45

Thr Asp Tyr Ser Met His Trp Val Lys Gln Ala Pro Gly Lys Gly Leu

50 55 60

Lys Trp Met Gly Trp Ile Asn Thr Glu Thr Gly Glu Pro Thr Tyr Ala

65 70 75 80

Asn Asp Phe Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser

85 90 95

Thr Ala Tyr Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr

100 105 110

Tyr Phe Cys Ala Arg Asn Tyr Gly Ser Ser Leu Ala Tyr Trp Gly Gln

115 120 125

Gly Thr Leu Val Thr Val Ser Ser

130 135

<210> 31

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

aaggccagcc aaagtgttga ttatgatggt gatagttata tgaac 45

<210> 32

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

gatgcatcca atctagaatc t 21

<210> 33

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 33

gatgcatcca atctagaatc t 21

<210> 34

<211> 333

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 34

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 60

atctcctgca aggccagcca aagtgttgat tatgatggtg atagttatat gaactggtat 120

caacagaaac caggacagcc acccaaactc ctcatctatg atgcatccaa tctagaatct 180

gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 240

cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtaatga ggatccgctc 300

acgttcggtg gtgggaccag gctggagctg aaa 333

<210> 35

<211> 393

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 35

atggagacag acacaatcct gctatgggtg ctgctgctct gggttccagg ctccactggt 60

gacattgtgc tgacccaatc tccagcttct ttggctgtgt ctctagggca gagggccacc 120

atctcctgca aggccagcca aagtgttgat tatgatggtg atagttatat gaactggtat 180

caacagaaac caggacagcc acccaaactc ctcatctatg atgcatccaa tctagaatct 240

gggatcccag ccaggtttag tggcagtggg tctgggacag acttcaccct caacatccat 300

cctgtggagg aggaggatgc tgcaacctat tactgtcagc aaagtaatga ggatccgctc 360

acgttcggtg gtgggaccag gctggagctg aaa 393

<210> 36

<211> 15

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 36

gactattcaa tgcac 15

<210> 37

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 37

tggataaaca ctgagactgg tgagccaaca tatgcaaatg acttcaaggg a 51

<210> 38

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 38

aactacggta gtagcttggc ttac 24

<210> 39

<211> 351

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 39

cagatccagt tggtgcagtc tggacctgag ctgaagaagc ctggagagac agtcaagatc 60

tcctgcaagg cttctgatta taccttcaca gactattcaa tgcactgggt gaagcaggct 120

ccaggaaagg gtttaaagtg gatgggctgg ataaacactg agactggtga gccaacatat 180

gcaaatgact tcaagggacg gtttgccttc tctttggaaa cctctgccag cactgcctat 240

ttgcagatca acaacctcaa aaatgaggac acggctacat atttctgtgc tagaaactac 300

ggtagtagct tggcttactg gggccaaggg actctggtca ctgtctcttc a 351

<210> 40

<211> 408

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 40

atggcttggg tgtggacctt gctattcctg atggcagctg cccaaagtat ccaagcacag 60

atccagttgg tgcagtctgg acctgagctg aagaagcctg gagagacagt caagatctcc 120

tgcaaggctt ctgattatac cttcacagac tattcaatgc actgggtgaa gcaggctcca 180

ggaaagggtt taaagtggat gggctggata aacactgaga ctggtgagcc aacatatgca 240

aatgacttca agggacggtt tgccttctct ttggaaacct ctgccagcac tgcctatttg 300

cagatcaaca acctcaaaaa tgaggacacg gctacatatt tctgtgctag aaactacggt 360

agtagcttgg cttactgggg ccaagggact ctggtcactg tctcttca 408

Claims

1. An ELISpot detection kit for bovine gamma interferon, the kit comprising: (1) any one of the following:

1) Support medium and capture antibody;

2) Support medium coated with capture antibody;

The capture antibody is a first monoclonal antibody, the first monoclonal antibody includes a light chain and a heavy chain, the light chain includes a CDR region whose amino acid sequence is shown in SEQ ID NO. 1 to 3, and the heavy chain Including the CDR region whose amino acid sequence is shown in SEQ ID NO.6～8;

(2) Detection antibody; the detection antibody is a labeled second monoclonal antibody, the second monoclonal antibody includes a light chain and a heavy chain, and the light chain includes an amino acid sequence as shown in SEQ ID NO. 21-23; The CDR region shown in the heavy chain includes the CDR region whose amino acid sequence is shown in SEQ ID NO. 26-28.

2. the ELISpot detection kit of bovine gamma interferon according to claim 1, is characterized in that, described first monoclonal antibody, the aminoacid sequence of its light chain variable region is SEQ ID NO.4, its heavy chain The amino acid sequence of the variable region is SEQ ID NO.9.

3. the ELISpot detection kit of bovine gamma interferon according to claim 1, is characterized in that, described first monoclonal antibody, is secreted by the hybridoma cell strain or its passage cell strain whose deposit number is CCTCC NO:C2017283 produce.

4. the ELISpot detection kit of bovine gamma interferon according to claim 1, is characterized in that, described second monoclonal antibody, the aminoacid sequence of its light chain variable region is SEQ ID NO.24, its heavy chain The amino acid sequence of the variable region is SEQ ID NO.29.

5. The ELISpot detection kit of bovine gamma interferon according to claim 1, wherein the second monoclonal antibody is secreted by the hybridoma cell strain whose preservation number is CCTCC NO: C2017282 or its passage cell strain produce.

6. The ELISpot detection kit of bovine interferon gamma according to claim 1, wherein the detection antibody is a second monoclonal antibody labeled with biotin or alkaline phosphatase.

7. The ELISpot detection kit for bovine interferon gamma according to claim 1, wherein the kit further comprises one or more of the following reagents: 1) avidin-alkaline phosphatase Conjugate; 2) Substrate chromogenic solution; 3) Diluent; 4) Washing solution; 5) Blocking solution; 6) Negative control; 7) Positive control; 8) Specific stimulator; 9) Non-specific stimulator.

8 . The ELISpot detection kit according to claim 7 , further comprising any one or more of the following features: (1) the specific stimulator is CE protein; (2) the non-specific stimulant is CE protein; 8 . The heterosexual stimulant is PWM.

9. The application of the ELISpot detection kit according to any one of claims 1-8 in the detection of Mycobacterium bovis infection for non-diagnostic purposes.

10. Uses of the first hybridoma cell line that secretes bovine interferon gamma monoclonal antibody and the second hybridoma cell line that secretes bovine interferon gamma monoclonal antibody in the preparation of a detection reagent or a diagnostic reagent for cattle blight, the said section The deposit number of one hybridoma cell line is CCTCC NO: C2017283, and the deposit number of the second hybridoma cell line is CCTCC NO: C2017282.

11. Use of a first hybridoma cell line secreting bovine interferon gamma monoclonal antibody and a second hybridoma cell line secreting bovine interferon gamma monoclonal antibody in the preparation of an ELISpot detection kit for bovine interferon gamma, said The deposit number of the first hybridoma cell line is CCTCC NO: C2017283, and the deposit number of the second hybridoma cell line is CCTCC NO: C2017282.