CN101788563B - Spotted deer gamma-interferon double-antibody sandwich ELISA detection method, kit thereof and application of kit - Google Patents

Abstract

The invention belongs to the fields of agricultural microbial genetic engineering and animal infectious diseases. The invention relates to a sika deer gamma-interferon double-sandwich ELISA detection method, a kit and an application. A cell line CerIFN-γ4C capable of stably secreting sika deer γ-interferon monoclonal antibody was obtained, and its preservation number is CCTCC NO: C200966. The invention also establishes a sika deer gamma-interferon double-sandwich ELISA detection method, the core of which is to construct a sika deer gamma-interferon monoclonal antibody, prepare a sika deer gamma-interferon polyclonal antibody, and the like. The kit of the invention comprises the monoclonal antibody of sika deer gamma-interferon, the polyclonal antibody of sika deer gamma-interferon, bovine tuberculosis specific three-gene fusion antigen protein RCE and other reagents. The invention also discloses the application of the detection method and the kit. The invention has strong specificity, high sensitivity, simple operation and rapid diagnosis.

Description

Sika Deer γ-Interferon Double Antibody Sandwich ELISA Detection Method, Kit and Application

technical field

The invention relates to the technical field of microbial genetic engineering and the technical field of animal infectious diseases. It specifically relates to a detection method, kit and application of sika deer gamma-interferon.

Background technique

Cervus Tuberculosis (CTB) is a chronic zoonotic disease mainly caused by Mycobacterium bovis. The disease is easily contagious and can occur throughout the year. It is prevalent worldwide. It used to be one of the diseases that caused the largest number of deaths in humans and animals. It was designated as a B-type animal infectious disease by the World Organization for Animal Health (OIE), and China listed it as a disease. It is a second-class animal disease. There are three kinds of mycobacteria that mainly cause tuberculosis in humans and animals: Mycobacterium tuberculosis human, Mycobacterium bovis, and Mycobacterium avian. Tuberculosis etiology surveys at home and abroad have found that bovine tuberculosis, human tuberculosis and deer tuberculosis are mutually infectable. Therefore, the control of bovine tuberculosis and human tuberculosis must take into account the prevention and control of tuberculosis in wild animals such as deer. Since no effective drugs and vaccines have been found so far for the prevention and treatment of deer tuberculosis, some countries generally adopt the "quarantine-hunting" policy, that is, all positive deer from quarantine are killed to achieve the control of deer tuberculosis. Therefore, accurate and timely diagnosis methods are particularly important for the prevention and treatment of this disease.

The only method recommended by OIE is bovine tuberculin skin test (TST). However, since tuberculin may contain the same non-specific antigen components as other mycobacteria, false positives are prone to occur during detection; tuberculin intradermal allergy is not sensitive to open tuberculosis and systemic tuberculosis in the late stage of infection. In addition, the TST procedure is cumbersome, time-consuming and labor-intensive, and the judgment of the results is highly subjective, which requires professionals to operate, which greatly reduces the implementation effect of the "quarantine-cull" policy, and it can only be used for in vivo tests, but not for preserved samples. Retrospective analysis is even more unsuitable for the general survey of bovine tuberculosis in wild animals and remote mountainous areas. Therefore, people are committed to developing a new diagnostic method that is simpler, faster, more specific, and more sensitive.

γ-interferon (IFN-γ for short) is a cytokine produced by CD4+Th1 cells, CD8+ cytotoxic T cells and NK cells under the stimulation of antigens (bacteria, viruses, etc.) and mitogens (ConA, PHA, etc.) . IFN-γ has anti-infection, anti-tumor activity and immunomodulatory effects, such as activating macrophages, increasing the expression of MHC class I and class II molecules, and promoting antigen presentation, etc., and plays an important role in the body's anti-infection and immunity. Based on the close relationship between IFN-γ and infection, pathogen-specific antigens are used in medicine and veterinary medicine to stimulate monocytes to produce IFN-γ in vitro for diagnosis of infectious diseases. The method has high sensitivity and specificity, showing great application prospects. The IFN-γ release assay in vitro is to stimulate the whole blood or peripheral mononuclear cells (PBMC) of the tested animal with mycobacterial-specific or non-specific antigens, so that a small number of T cells can be fully stimulated to secrete a large amount of IFN-γ, and then The cellular method of detecting IFN-γ concentration by enzyme-linked immunosorbent assay, IFN-γ release assay in vitro is of great significance for the early diagnosis of tuberculosis latent infection.

The higher specificity of IFN-γ test will help low-prevalence countries to conduct more accurate diagnosis of latent tuberculosis infection (LTBI) and provide targeted anti-tuberculosis treatment for detected individuals in a timely manner, reducing drug-resistant tuberculosis mycobacteria. Bacillus spread and control the abuse of antibiotics. Antigen-specific IFN-γ assay can be used to detect the effect of anti-TB treatment. According to the test principle of the antigen-specific IFN-γ assay, in the in vivo environment, when the effector lymphocytes that have recently been exposed to the TB-specific antigen encounter the same antigen again, it only takes a few hours to produce IFN-γ. Unlike effector lymphocytes, memory T lymphocytes often take several days to produce IFN-γ. Therefore, if the culture time of whole blood in the test is 24 hours or less, the detection is mainly of IFN-γ produced by effector lymphocytes, not by memory T lymphocytes. However, if the culture time is extended, the detection will be mainly IFN-γ produced by memory T cells, which may lead to positive results in patients who have been treated or the infection has been cleared. Therefore, the antigen-specific IFN-γ test can be used as an evaluation of the disease progression and therapeutic effect of TB organisms.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, and provide a sika deer gamma-interferon double antibody sandwich ELISA detection method, a kit and its application in the in vitro diagnosis of sika deer tuberculosis.

The gist of the present invention is to obtain the monoclonal antibody cell line with good specificity, sensitivity and stability, by determining the best working concentration of sika deer gamma-interferon (IFN-gamma) mono/polyclonal antibody, the best Determine the blocking concentration and time, determine the best antibody binding (incubation) time, determine the best plasma stimulation concentration and time to be tested, and determine the best cytokine (IFN-γ) concentration to be tested, and finally establish the sika deer IFN-γ Gamma tuberculosis detection method and special kit and application.

The present invention is realized through the following technical solutions:

The present invention transfers the constructed recombinant fusion vector plasmid pET-32a-IFN-γ into Escherichia coli BL21 competent cells, and induces expression to obtain a soluble IFN with anti-virus, anti-tumor activity, immune regulation and strong heat resistance - gamma protein. Purified IFN-γ protein was used to immunize mice, and positive hybridoma cells were screened with eukaryotic cell line stable secreted expression supernatant, and a high-titer monoclonal antibody was obtained. Using the obtained monoclonal antibody and rabbit anti-IFN-γ polyclonal antibody, a double-antibody sandwich ELISA detection method and a special kit for sika deer IFN-γ were established, and the detection sensitivity reached 24.9pg/ml.

The applicant sent a cell line CerIFN-γ4C, which can stably secrete sika deer interferon-γ monoclonal antibody, to the China Center for Type Culture Collection (CCTCC) in Wuhan University, Wuhan, Hubei Province on December 1, 2009 Deposited, the deposit number is CCTCC NO: C200966.

The applicant has established a double-antibody sandwich ELISA detection method for sika deer gamma-interferon, the steps of which include:

(1) Dilute the sika deer γ-interferon monoclonal antibody with the preservation number CCTCC NO: C200966 to the working concentration (50ng/well) with the coating solution and add it to the wells of the microplate, 100μl per well, and place it at 37°C for 1h. 4°C refrigerator overnight;

(2) Discard the liquid in the well, wash the microtiter plate with washing solution for 3 times, each time for 3 minutes, and pat dry with absorbent paper;

(3) Add 100 μl of blocking solution to the wells of each microplate well, act at 37° C. for 1 hour, and wash according to the method in step (2);

(4) Add the induced sika deer whole blood culture supernatant to be tested into the wells of the ELISA plate coated with sika deer gamma-interferon monoclonal antibody, 100 μl per well; meanwhile, separate the collected negative and positive control culture supernatants Add it to the well, act at 37°C for 1 hour, and repeat the method of step (2) for washing;

(5) Dilute the rabbit anti-sika deer gamma-interferon polyclonal antibody according to the working concentration (50ng/well) with the washing solution, add 100μl to each well, act at 37°C for 1h, and repeat the method of step (2) for washing;

(6) Dilute the horseradish peroxide-labeled goat anti-rabbit IgG with the washing liquid according to the working concentration (according to the volume ratio of 1:5000), add 100 μl to each well, act at 37° C. for 1 hour, and repeat the method of step (2) for washing;

(7) Add 100 μl of color development solution to each well of the microplate well, and develop color at room temperature for 10 minutes in the dark;

(8) Add 50 μl of stop solution to each well of the microplate well to terminate the reaction;

(9) Determining the light absorption value of step (8) at a wavelength of 630nm on an enzyme-linked immunoassay instrument;

in:

The coating liquid described in step (1) is prepared as follows: sodium carbonate 1.59g; sodium bicarbonate 2.93g; dilute to 1000ml with distilled water;

The preparation described in step (2) is as follows: sodium chloride 8.0g; Potassium chloride 0.2g; Disodium hydrogen phosphate 0.2g; Potassium dihydrogen phosphate 2.9g; Tween-200.5ml; .4;

The blocking solution described in step (3) is prepared as follows: 100ml of flushing solution; 10g of skimmed milk;

The chromogenic solution described in step (7) is prepared as follows: disodium hydrogen phosphate 7.16g; citric acid 1.92g; be fixed to 100ml with distilled water to obtain liquid A; disodium hydrogen phosphate 7.16g; citric acid 1.92g; Set the volume to 100ml, pH 5.0, obtain 100ml of 0.1mol/L phosphate-citrate buffer solution, add 40mg of o-phenylenediamine; 0.15ml of hydrogen peroxide at 30% concentration, obtain B liquid, mix the A liquid and B liquid, obtain the chromogenic liquid.

The termination solution described in step (8) was prepared as follows: 22.2ml of 2mol/L sulfuric acid, 177.8ml of distilled water.

The applicant also assembled a double-antibody sandwich ELISA kit for sika deer γ-interferon that is compatible with the above detection method, which includes: sika deer γ-interferon monoclonal antibody with the deposit number CCTCC NO: C200966, sika deer γ-interferon Interferon polyclonal antibody, preservation number is CCTCC NO: M208244 (patent application number is 2009100609131, patent publication number is CN101538578A) bovine tuberculosis-specific three-gene fusion antigen protein RCE, horseradish peroxide-labeled goat anti-rabbit IgG, color development solution and stop solution.

The components and preparation methods of the chromogenic solution and the stop solution described therein are as follows:

Chromogenic solution: disodium hydrogen phosphate 7.16g; citric acid 1.92g; distilled water to 100ml to obtain liquid A; disodium hydrogen phosphate 7.16g; citric acid 1.92g; distilled water to 100ml, pH 5.0 to obtain 0.1mol/L phosphate-citrate buffer solution 100ml, add o-phenylenediamine 40mg; 30% concentration of hydrogen peroxide 0.15ml, obtain B liquid, mix described A liquid and B liquid, obtain chromogenic liquid .

Stop solution: 22.2ml of 2mol/L sulfuric acid; 177.8ml of distilled water.

A more detailed technical solution is described in "Detailed Embodiments".

Description of drawings

Sequence listing SEQ ID NO: 1 is the DNA sequence of the sika deer IFN-γ gene fragment of the present invention.

Sequence Listing SEQ ID NO: 3-4 is the primer sequence for amplifying the sika deer IFN-γ gene fragment in the present invention.

Fig. 1: is the overall technical roadmap of the present invention.

Figure 2: is the map of the prokaryotic expression vector pET32a(+) used in the present invention.

Fig. 3 is the electrophoresis pattern of PCR amplified sika deer IFN-γ of the present invention. In the figure: M, DNA standard DL2000; 1, negative control; 2, RT-PCR product

Figure 4: SDS-PAGE detection pattern identification pattern of Merlot IFN-γ protein prepared by the present invention. In the figure: M, protein molecular mass standard; 1, CerIFN-γ supernatant protein; 2, purified CerIFN-γ protein.

Fig. 5 is a Western-Blot diagram of sika deer IFN-γ protein prepared by the present invention. In the figure 1, recombinant pET32α-CerIFN-γ; 2, negative quality control; M, protein standard.

Fig. 6 is a titer diagram of the detection of sika deer IFN-γ monoclonal antibody (McAb) by indirect ELISA detection in the present invention.

Fig. 7 is a titer diagram of detection of sika deer IFN-γ polyclonal antibody (PcAb) by indirect ELISA detection in the present invention.

Fig. 8 is a sensitivity titer chart of detection of sika deer IFN-γ monoclonal antibody (McAb) by indirect ELISA detection in the present invention.

Detailed ways

Example 1: Cloning of the target gene sika deer IFN-γ

1. Source of plasmid and host bacteria

The pET-32a(+) plasmid vector used in this example was purchased from Novagen. Escherichia coli BL21 (DE3) competent cells were purchased from Hubei Wuhan Life Technology Co., Ltd.

2. Primer design and synthesis

Prokaryotic expression primers were designed according to the sika deer IFN-γ cDNA sequence (GenBank accession No: X63079) published on GenBank. Primers were synthesized by Shanghai Sangon Biotechnology Co., Ltd.

The prokaryotic expression primer sequences are as follows: upstream primer: 5'ATAT GGATCC GATCTTATGGCCAGGGCCCA3' (underlined is the BamHI site); downstream primer: 5'TAAT AAGCTT TTACGTTGATGCTCTCCGGCCTCG3' (underlined is the HindIII site).

3. PCR amplification of the target gene

Sika deer jugular vein blood was collected aseptically, anticoagulated with heparin, 10 mL whole blood of sika deer was taken, and concanavalin A (English abbreviation: Con A, purchased from Sigma Company) 60 μg/mL was added at 37°C and 5% CO ₂ environment Cultivate for 24h. The total RNA of peripheral blood leukocytes stimulated by Con A was extracted as the template of RT-PCR, and the mature peptide sequence of sika deer IFN-γ was amplified by one-step method, as shown in the sequence table SEQ ID NO: 1. The full length of the sequence is 438bp.

The PCR amplification reaction system is: 10×Taq Buffer 5.0μL, 25mmol/L MgCl ₂ 1.0μl, 2mmol/L dNTPs 1.5μL, 20μmol/L upstream and downstream primers 1.0μL each, Taq DNA polymerase 1.0μL, template 3μL, Add sterile double distilled water to 50 μL. Amplification cycle parameters: pre-denaturation at 95°C for 5min; then denaturation at 95°C for 30s, annealing at 59°C for 30s, extension at 72°C for 45s, 30 cycles; final extension at 72°C for 10min. The amplified product was detected by 0.8% agarose gel electrophoresis, and the PCR product was purified with a gel recovery kit (purchased from Shanghai Sangon Bioengineering Technology Co., Ltd., refer to the kit instruction manual).

4. PCR product recovery:

Use the UNIQ-10 Column DNA Gel Recovery Kit produced by Shanghai Sangon Biotechnology Co., Ltd. to recover DNA fragments, and follow the steps provided in the instructions of the UNIQ-10 Column Centrifugal DNA Gel Recovery Kit. The specific operations are as follows:

Use 0.8% agarose gel electrophoresis to separate the target DNA fragment from other DNA as much as possible. Under the long-wave ultraviolet lamp, cut off the agar block containing the target DNA fragment with a surgical blade burned on the flame of the alcohol lamp, and put it in 1.5 mL sterile centrifuge tube.

Add 400 μL of Binding Buffer per 100 mg of agarose gel, and place in a water bath at 50-60 ° C for 10 minutes to completely melt the agarose gel (when heating the sol, mix it every 2 minutes).

Put the UNIQ-10 column into the collection tube, transfer the melted gel solution to the UNIQ-10 column, let it stand at room temperature for 2 minutes, and centrifuge at room temperature at 8000r/min for 1 minute.

Remove the UNIQ-10 column, discard the waste liquid in the collection tube, put the UNIQ-10 column into the same collection tube, add 500μL WashSolution, and centrifuge at room temperature at 8000r/min for 1min.

Then add 500μL Wash Solution, remove the UNIQ-10 column, discard the waste liquid in the collection tube, put the UNIQ-10 column into the same collection tube, and centrifuge at room temperature at 12000r/min for 15sec.

Put the UNIQ-10 column into a sterilized 1.5mL centrifuge tube, add 10-20μL Elution Buffer or ddH ₂ O to the center of the membrane at the bottom of the column according to the relative amount of PCR product, and place it at room temperature or 37℃ for 2min at room temperature 12000r /min and centrifuge for 1 min, the liquid in the centrifuge tube is the recovered DNA fragment, which can be used immediately or stored at -20°C for later use.

Example 2, construction of recombinant plasmid pET-32a-IFN-γ

1. Ligation of vector plasmid pET-32a and IFN-γ PCR recovery product

The prokaryotic expression vector pET32a and the recovered IFN-γ PCR product were simultaneously double-digested with restriction endonucleases BamH I and Hind III (purchased from Bao Biological Engineering (Dalian) Co., Ltd.), respectively, and the digested product was digested with 0.8% agarose Gel electrophoresis detection, gel recovery kit to purify the digested product. The purified digested product was ligated with T4 DNA Ligase (purchased from Fermentas), and connected in a water bath at 16°C overnight, and then transformed into Escherichia coli DH5α competent cells.

2. Conversion of Ligation Products

Take 100 μL of competent cell DH5α and add it to a sterilized 1.5mL EP tube, add 10 μL each of the ligated intermediate plasmid pET-32a-IFN-γ and mix well. After placing on ice for 30min, heat shock at 42°C for 90sec, and ice bath for 3-5min. Add 400 μL LB liquid medium (each liter contains 5 g of yeast extract, 10 g of tryptone, and 10 g of NaCl, adjust the pH to 7.5 with 10 mol/L NaOH, autoclave at 121 °C for 20 min, and store at 4 °C for later use. In each 100 ml of LB liquid Add 1.5g of agar powder to the medium to obtain solid LB medium, autoclave at 121°C for 20 minutes, store at 4°C for later use), shake and culture at 37°C at 200r/min for 45 minutes to recover. The recovered recombinant Escherichia coli suspension was centrifuged at 5000 r/min at 4°C for 10 min, discarded 400 μL of the supernatant, and used the remaining 100 μL of the resuspended pellet to spread on LB agar containing 25 μg/mL kanamycin (purchased from Invitrogen) flat. Proliferate at 37°C for 1 hour, then turn the plate over, and incubate at 37°C upside down for 14-16 hours until colonies appear.

3. Extraction of plasmid

Use alkaline lysis method (with reference to Sam Brook.J, Fritsch.E.F, Mani Atis.T edited, Molecular Cloning Experiment Guide, translated by Huang Peitang, third edition, Science Press, Beijing, the method of 2002 edition ), the specific operations are as follows:

Randomly pick several single colonies on the LB plate with a sterilized toothpick, inoculate them in 3 mL of 25 μg/mL kanamycin LB liquid medium, and cultivate overnight at 37°C on a constant temperature shaker at 200 r/min.

Transfer the bacterial solution into a 1.5mL centrifuge tube, centrifuge at 8000r/min at 4°C for 3min, discard the supernatant, then repeat the centrifugation of the remaining 1.5mL bacterial solution, and invert the centrifuge tube on absorbent paper to drain the liquid.

Add 100 μL of ice-cold solution I (0.05mol/L glucose, 0.025mol/L Tris-HCl (pH8.0), 0.01mol/L EDTA), vortex to fully suspend the bacteria, and then add 200μL of the newly prepared solution II (0.2mol/L NaOH, 1% SDS, ready-to-use), invert the centrifuge tube several times, ice-bath for 5min, and finally add 150μL ice-cooled solution III (5mol/L sodium acetate 60mL, glacial acetic acid 11.5mL , water 28.5mL, pH5.0), gently invert the centrifuge tube several times, and ice bath for 10min.

Centrifuge at 12000r/min at 4°C for 10min, pipette the supernatant into another 1.5mL centrifuge tube, add an equal volume of isopropanol, mix well, and let stand at room temperature for 5min.

Centrifuge at room temperature at 12,000 r/min for 10 min, discard the supernatant, rinse the precipitate with 75% cold ethanol, and dry it in vacuum or naturally.

The precipitate was dissolved in 200 μL TE (pH 8.0) containing 20 μL RNase (20 μg/mL), and was bathed in water at 56 °C for 30 min or at 37 °C for 1 h to remove RNA.

Add 100 μL of 7.5 mol/L NH ₄ Ac, let stand at room temperature for 5 minutes, and then centrifuge at room temperature at 12000 r/min for 5 minutes.

Pipette the supernatant into another 1.5mL EP tube, add 2 times the volume of cold absolute ethanol, and place in an ice bath for 10min.

Centrifuge at 12000r/min at 4°C for 10min, discard the supernatant, rinse the pellet with 75% cold ethanol, dry it in vacuum and dissolve it in 20μL ddH ₂ O or TE (1mmol/L EDTA, 10mmol/L Tris-Cl, pH8.0) , stored in a -20°C refrigerator for later use.

4. Enzyme digestion and identification of recombinant plasmid pET-32a-IFN-γ

Use restriction endonucleases BamH I and Hind III to carry out single and double digestion of the intermediate plasmid pET-32a-IFN-γ, respectively, and after digestion, foreign fragments and vector fragments of the expected size will appear, which will contain the intermediate plasmid The Escherichia coli DH5α bacterial liquid was sent to Shanghai Sangong Biotechnology Engineering Co., Ltd. for sequencing. The sequencing results were compared with the sequence of sika deer IFN-γ published in Genbank (sequence accession number: X63079). The recombinant plasmid was constructed successfully and can be used for expression in Escherichia coli.

Embodiment 3, expression and purification of target fusion gene in Escherichia coli

1. Induced expression of the target gene

The Escherichia coli strain DH5α containing the recombinant expression vector was inoculated in 3 mL LB liquid medium containing 25 μg/mL kanamycin, and cultured on a shaker at 37° C. until the OD ₆₀₀ reached 0.6-0.8. Take 100 μL of the cultured bacterial liquid and inoculate it into 10 mL of fresh LB liquid medium containing 25 μg/mL kanamycin, culture it with shaking at 37°C for about 3 hours, until the OD ₆₀₀ reaches 0.6-0.8, add isopropylthio -β-D-galactoside (IPTG, purchased from Invitrogen Co.) to a final concentration of 0.8 mmol/L, continued to culture for 3 hours, and then collected the bacteria.

2. SDS-PAGE electrophoresis analysis of expression products

2.1 Preparation of SDS-PAGE electrophoresis samples

The induced recombinant Escherichia coli was centrifuged at 8000r/min for 15min. The precipitate was resuspended with 1/10 volume of 50mM Tris-HCl (pH 8.0), and kept on ice for 30min. Ultrasonic crushing was carried out under ice bath conditions until the bacterial liquid was no longer viscous, centrifuged at 10000r/min for 30min. Take a small amount of lysed supernatant and precipitate respectively, add 2 × protein electrophoresis sample buffer (100mmol/L Tris-HCl (pH6.8), 200mmol/L dithiothreitol (DTT, purchased from Amresco company), 4 %SDS (electrophoresis grade), 0.2% bromophenol blue, 20% glycerol) 125μL, shake and mix, boil at 100°C for 10min, centrifuge at 12000r/min for 5min, and take the supernatant for SDS-PAGE electrophoresis analysis.

2.2 Preparation and electrophoresis of SDS polyacrylamide gel

15% separation gel preparation: purified water 1.6mL, 30% acrylamide solution 2.0mL, 1.5mol/L Tris-Base solution (pH8.8) 1.3mL, 10% SDS 0.05mL, 10% ammonium persulfate 0.05mL, TEMED 0.003mL; After the ingredients are added, mix quickly, add to the rubber plate, and add isobutanol on top.

5% stacking gel preparation: 2.1mL purified water, 0.5mL 30% acrylamide solution, 0.38mL 1.0mol/L Tris-Base solution (pH6.8), 0.03mL 10% SDS, 0.03mL 10% ammonium persulfate, TEMED 0.003mL; After adding the ingredients, mix quickly, add to the top of the separating gel of the rubber-making plate, insert the sampling comb after filling. After the stacking gel is solidified, remove the comb; fix the gel on the electrophoresis device, add a sufficient amount of Tris-glycine electrophoresis buffer, and add each sample to the sample well; the electrophoresis voltage is 80V, and when bromophenol blue reaches When separating the gel interface, change the voltage to 120V until bromophenol blue swims out of the bottom of the gel, and terminate the electrophoresis.

2.3 Polyacrylamide gel staining and decolorization

Unload the gel, stain with Coomassie Brilliant Blue R250 staining solution (45% methanol, 45% purified water, 10% glacial acetic acid, 0.25% Coomassie Brilliant Blue R250) for more than 4 hours, and then use destaining solution (45% methanol, 45% Purified water, 10% glacial acetic acid) were decolorized until the background color was completely removed, and the results were observed. Determine whether the protein of interest is soluble or expressed as inclusion bodies.

2.4 Purification of recombinant protein

The collected recombinant protein expressing bacteria BL/pET-32a-IFN-γ was resuspended with the buffer solution provided with the kit purchased from Novagen, ultrasonically disrupted, centrifuged at 12000g at 4°C for 15 minutes, and the supernatant was taken for loading. The target protein was purified using Ni-NTA His Band chromatography column (purchased from Novagen). Use Binding buffer and washing buffer Washing buffer (20mM Tris-HCl pH7.9, 15mM midazole, 0.5M NaCl) to wash the column until the OD value reaches near the baseline, then replace it with Elute buffer (20mM Tris-HCl pH7. 9, 40mM Imidazole, 0.5M NaCl) to elute the combined recombinant protein, and collect the peak part of the protein as purified recombinant protein for further analysis.

3. Determination of the optimal conditions for the expression of the target protein

According to the method of the above-mentioned step 2 (ie, SDS-PAGE electrophoresis analysis of the expression product), the detection shows that the recombinant fusion protein IFN-γ prepared by the present invention is soluble expression. Resuscitate and save the transformed Escherichia coli BL21(DE3) bacterial liquid with pET-32a-IFN-γ, and culture it on a shaker at 37°C until the OD ₆₀₀ reaches 0.6-0.8, according to the IPTG concentration of 0.4mM, 0.8mM and 1.0mM respectively , and samples were taken before induction and 3h, 4h, and 5h after induction, and analyzed by SDS-PAGE.

It was determined that the best expression condition of IFN-γ protein was IPTG 0.8mmol/L, and the highest expression level was induced at 37℃ for 3 hours.

4. Determination of optimal conditions for recombinant protein purification

In order to explore the best imidazole concentration in the Wash Buffer, the imidazole concentration in the Wash Buffer was diluted in three gradients according to the final concentration of 30mM, 40mM, and 50mM. The samples were received after three concentrations of Wash Buffer, and analyzed by SDS-PAGE.

Determine the optimal concentration of imidazole in Wash Buffer for IFN-γ protein purification is 30mM.

Embodiment 4: Preparation of recombinant protein IFN-γ mono/polyclonal antibody

1. Immune mice and rabbits with recombinant protein IFN-γ

The Balb/C mice and Japanese long-eared white rabbits used in this example were all purchased from the Hubei Provincial Center for Disease Control and Prevention, and 6 female Balb/C mice aged 4-6 weeks were subcutaneously inoculated and purified at 200 μg per mouse Recombinant IFN-γ, the same amount of recombinant antigen in Freund's complete adjuvant was first immunized, and the same amount of antigen in Freund's incomplete adjuvant was boosted 2 weeks later, and the same amount of antigen without adjuvant was injected subcutaneously again after 2 weeks. After an interval of at least 1 month, on the 3rd day before fusion, select Balb/C mice with high antibody titers and intraperitoneally inject them with a dose of 500 μg/antigen every day. After 3 days of continuous immunization, collect blood from the tail vein of the mice , Indirect ELISA to detect the titer of serum anti-CerIFN-γ antibody.

At the same time, the purified CerIFN-γ was immunized with three female Japanese long-eared white rabbits weighing more than 2.0 kg according to the above procedure, and the antigen immunization dose was 700 μg/rat. After the third immunization, the serum antibody titer was measured by indirect ELISA, and the antibody titer reached Bloodletting was performed after the request, and hyperimmune serum was purified by the saturated ammonium sulfate method.

2. Sika deer IFN-γ cell fusion

2.1 Activation of SP2/0 myeloma cells

The myeloma (SP2/0) cells used in this example were purchased from the China Veterinary Drug Administration. The revived SP2/0 cells were collected and suspended (0.5-1×106) in 0.5 mL RPMI-1640 base solution and injected with Balb/C cells. Subcutaneously on the back of the mouse. After 9-10 days of solid tumor growth, select the time to collect tumor cells according to the size.

2.2 Cell Fusion

Spleen cells of immunized mice and activated SP2/0 myeloma cells were taken separately, and the two were fused under the action of a fusion agent, and feeder cells were prepared at the same time to assist the growth of hybridoma cells. The method is briefly described as follows (Shi Liangru, 1984 ):

2.2.1 Preparation of SP2/0 tumor cells

If it is cultured in a cell bottle, it can be washed directly with RPMI-1640 base solution and collected by centrifugation. The method of preparing from mouse tumors is as follows: the mice were killed by neck dislocation, soaked in 75% alcohol for 5mim, and the tumors were taken in an aseptic state in an ultra-clean bench. Break into several small pieces, then move to a homogenizer, add 5mL RPMI-1640 base solution and grind thoroughly, add 10mL RPMI-1640 solution, let stand for 2min, wait for the larger tissue mass to sink to the bottom of the tube, draw The cell suspension in the upper layer was stored in another centrifuge tube for later use, and then 10 mL of RPMI-1640 solution was added, and repeated once to control the volume of the removed cell suspension to 30 mL. Add 15mL of lymphocyte separation medium to another 50mL centrifuge tube, gently add the cell suspension on top of the separation medium (ratio: 1:2 to 1:1), 1800-2000r/m for 20min, use a pipette to draw The dense white cell layer at the interface was washed twice with RPMI-1640 solution, counted and set aside.

2.2.2 Preparation of immune spleen cells:

(1) Take a Balb/C mouse after the final booster immunization, sacrifice it by bloodletting from the orbit (collect the serum, it will be positive serum), soak it in 75% alcohol for 5 minutes to disinfect.

(2) Fix the sterilized mouse on the dissecting board, fix the forelimbs, fix the hind limbs crossed (the left hind limb is on top), clamp the skin of the lower abdomen with tweezers, cut a small mouth, tear off the skin to expose the peritoneum, change a set of tweezers and Scissors, cut open the peritoneum to expose the spleen, then change a set of instruments, clamp the spleen with tweezers, use scissors to remove the adipose tissue of the adherent cells, cut the outer membrane of the spleen, and put it into a sterilized homogenizer.

(3) Add 3mL RPMI-RPMI-1640 base solution to the homogenizer, grind (not too vigorously, so as not to damage the splenocytes, a looser homogenizer can be used), squeeze out the splenocytes, take out the homogenizer, Add 7mL RPMI-1640 base solution, let it stand for 2min, draw the upper cell suspension into another sterile 50mL centrifuge tube, then add 10mL RPMI-1640 solution to the homogenizer, and repeat the same procedure twice.

(4) Centrifuge at 1000r/m for 10min to remove the supernatant, and count the cells after resuspending.

2.2.3 Preparation of feeder cells

(1) Take an unimmunized Balb/C mouse, bled from the orbit, and collect the serum as negative serum.

(2) After the limbs are fixed, the skin is torn open to expose the peritoneum, a small opening is cut on the peritoneum (in the center of the abdomen), and then 3mL RPMI-1640 solution is injected into the mouse abdominal cavity with a straw, sucked and beat several times, and then the liquid is sucked out and released. In a 50mL centrifuge tube, repeat again, this is the peritoneal macrophages.

(3) Prepare the spleen cell suspension by the same operation as above, and put it into the peritoneal macrophage tube.

(4) Centrifuge at 1000r/m for 10min to remove the supernatant, and count the cells after resuspending.

2.2.4 Cell Fusion

(1) Centrifuge the feeder cells at 1000 rpm for 5 minutes, remove the supernatant, and store them at 37°C for later use.

(2) Mix 1-2×107 SP2/0 and 108 immune cells (1:10-1:15) in a 50 mL centrifuge tube, centrifuge at 1500 r/m for 10 min.

(3) Empty the supernatant (it can be blotted dry with sterilized filter paper), and gently tap the bottom of the tube to loosen the cell pellet slightly.

(4) Place the centrifuge tube containing the cell mixture in a 37°C water bath. Then slowly drop in 50% PEG pre-warmed to 37°C within 1 min

0.8mL, add while gently stirring with a pipette tip for 1min.

(5) Then slowly add 10 mL of RPMI-1640 base solution pre-warmed at 37°C. The specific method is: drop 1 mL in the first minute. Add 1mL in the second minute, add 3mL in the 3rd to 4th minute, and add the remaining 5mL in the 5th minute. Add slowly and keep stirring gently every time you add. Finally, add 30mL RPMI-1640 solution, also need to add slowly.

(6) Centrifuge at 8000r/m for 5min, remove the supernatant and place at 37°C for 5-8min.

(7) Suspend with HAT medium, and at the same time, use HAT medium to suspend and feed splenocytes and mix with fused cells, add appropriate amount of HAT medium as needed, and seed in 96-well culture plates, about 250 μL/well. One fusion can inoculate 4-8 96-well plates. It can also be planted less according to the needs, generally calculated according to the number of SP2/0 cells, and the inoculation amount of each well contains about 10 ⁴ SP2/0 cells.

(8) Cultivate in a 37°C, 5% CO ₂ incubator.

(9) Start observation on the second day after fusion to see if there is any contamination, add 1 drop of HAT medium on the 4th day, suck out 100 μL of the medium and replace it with 100 μL of HT medium on the 8th to 10th day. When the fused cell colony grows to 1/4 of the culture well and the medium turns slightly yellow, carry out antibody detection.

2.2.5 Indirect ELISA method to detect hybridoma cell supernatant and screen positive hybridoma cells

Coat overnight at 4°C on the day before the measurement (gG: 50ng/well), wash three times with washing solution and pat dry, block at 37°C for 1 hour, wash three times with washing solution and pat dry, add hybridoma cell culture supernatant, and incubate at 37°C for 1 hour , washed three times with the washing solution and patted dry, added 1:10000 diluted goat anti-mouse IgG-HRP, incubated at 37°C for 45mins, washed 5 times, added 100μL of TMB substrate, developed color for 10min in the dark, and finally added 50μL of stop solution to terminate the reaction , Measure the OD value with a microplate reader at a wavelength of 630nm, and set the culture supernatant of SP2/0 cells as a negative control, and samples with an _OD630 greater than 2 times the negative control are positive.

2.2.6 Cloning of hybridoma cells (limiting dilution method)

(1) Prepare the mouse feeder cell layer before cloning or the day before.

(2) Gently blow off the hybridoma cells to be cloned from the culture well, and count the number of viable cells on a hemocytometer.

(3) Dilute the cells to 5, 10, 50 cells/ml with complete medium.

(4) The cell suspensions of the above three concentrations were respectively added to the 96-well culture plate of the prepared feeder cells, 100 μL/well, so that each corresponding well contained 0.5, 1 and 5 cells respectively.

(5) Change the medium once on the 4th day of culture, carefully observe the growth of the cells in each well on the 5th to 6th day, and record it.

(6) Detection of specific antibodies: 7-9 days after cloning, when the cell clones cover 1/3-1/2 field of view, it can be detected. If specific antibodies are detected in the cell growth wells, you can choose Antibody titer is high, grows as a single clone, and cell wells with good shape continue to be re-cloned or expanded in the same way.

(7) The cells in the positive wells can be moved to a 24-well culture plate, and another batch of culture medium should be added to the original wells to prevent simultaneous contamination of the two or cell death. When the cells in the 24-well plate grow well, they can be transferred to small square bottles, and more than 2 tubes of cells can be frozen.

Example 5: Identification of sika deer IFN-γ monoclonal antibody

1. Detection of chromosome number in hybridoma cells

After the cells were subcultured for 48 hours, colcemid was added to make the final concentration reach 0.04 μg/mL, and the culture was continued for 2-2.5 hours. After terminating the culture, all the adherent cells were blown down with a pipette, transferred into a 10 mL centrifuge tube, centrifuged at 1000 r/m for 10 min, and the supernatant was discarded. Add 5 mL of 0.075 mol/L potassium chloride hypotonic solution pre-warmed at 37°C, blow evenly with a straw, and place in a 37°C water bath for 15-20 minutes. Add 1 mL of freshly prepared fixative solution (methanol: glacial acetic acid = 3: 1) to each tube, mix well, centrifuge at 1000 r/m for 10 min, and remove the supernatant. Add 5mL of fixative to each tube, mix gently, let stand for 30min, centrifuge at 1000r/m for 10min, and discard the supernatant. Fix again, add 5mL of fixative, mix gently and let stand for 30min, centrifuge at 1000r/m for 10min, discard the supernatant, add 5mL of fixative in the same way, mix well, seal the tube and let stand at 4°C overnight. Gently suck off the supernatant, leave about 0.5mL of the supernatant, mix well to suspend the cells, draw 2-3 drops of the cell suspension with a dropper, and drop them on a clean glass slide soaked in ice water. Immediately blow off and run several passes over the flame to encourage the cells to spread out on the slide and let dry naturally. Dye with 10% Giemsa dye solution for 10min, wash off the lotion and dry naturally. Finally, observe and count under a microscope (Zhang Gusheng, Rong Bingpei, 1987).

2. Production and potency determination of monoclonal antibodies

After establishment, the hybridoma cells were expanded and cultured, the supernatant was collected, and the titer was determined by indirect ELISA. Mouse ascites can also be induced in mice to produce monoclonal antibodies. Take 8-10-week-old mice and inject 0.5mL of sterilized liquid paraffin intraperitoneally. After 7-10 days, inject 5×10 hybridoma cells intraperitoneally. ⁵ /mouse, after 7-10 days, extract the ascites of the mice, centrifuge to get the supernatant, measure the titer, and freeze it.

3. Western-blot analysis of the specificity of monoclonal antibodies

The purified recombinant fusion protein IFN-γ was subjected to SDS-PAGE electrophoresis.

Transfer: Cut out 6 pieces of Whatman 3M filter paper and 1 piece of nitrocellulose membrane (NC membrane). The size of the filter paper and membrane should be exactly equal to or slightly smaller than the size of the gel. The relative direction of the membrane and the gel after printing; soak the nitrocellulose membrane in purified water for 5 minutes; add a small amount of transfer buffer (39mmol/L glycine, 48mmol/L Tris base, 0.037% SDS (electrophoresis grade) , 20% methanol), soak 6 pieces of Whatman 3M filter paper in it. Then install the transfer electrophoresis tank according to the following method: lay the base (negative electrode) of the graphite electrode flat, and place 3 layers of 3M filter paper, polyacrylamide gel, nitrocellulose membrane and 3 layers of 3M filter paper in sequence. Thoroughly eliminate the air bubbles between the layers; buckle the upper cover of the transfer electrophoresis tank on the graphite electrode-transfer membrane glue complex; connect the power supply, and turn on the current according to the parameters of 0.65-1.0mA/ ^cm2 according to the area of the gel plate, and electrophoresis Transfer 0.5-2h.

Place the NC membrane in 1×TBST (10mmol/L Tris-HCl, 150mmol/L NaCl, 0.05% Tween-20, pH8.0) containing 1% BSA and 5% skim milk powder, and block overnight or at least 30min at room temperature ;

Membrane washing: Discard the blocking solution, wash the NC membrane 3 times with 1×TBST, 5 min each time;

Primary antibody incubation: put the NC membrane into the sika deer IFN-γ monoclonal antibody diluted with 1× TBST (volume ratio 1:3000), and incubate at 37°C for 1 h;

Membrane washing: take out the NC membrane and wash the membrane with 1×TBST for 3 times, 10min each time;

Secondary antibody incubation: transfer the membrane to 1×TBST diluted HRP (horseradish peroxidase)-labeled goat anti-bovine IgG (purchased from Sigma) antibody (volume ratio: 1:5000), and incubate at 37°C for 1 h;

Membrane washing: take out the NC membrane and wash the membrane with 1×TBST for 3 times, 10min each time;

Color development: Place the NC membrane in the newly prepared DAB color development solution and place it in a dark place for color development. After the color depth of the protein band reaches the requirement, rinse with 1×TBST to terminate the reaction.

The results showed that the sika deer IFN-γ monoclonal antibody exhibited high specificity, while the negative control group (pET32a(+) vector expression bacteria) had no reaction.

Example 6: Establishment of Sika Deer IFN-γ Double Antibody Sandwich ELISA Method

1. Determination of sensitivity of sika deer IFN-γ monoclonal antibody

The sensitivity of monoclonal antibody to IFN-γ in sika deer was determined by double-antibody sandwich ELISA. The monoclonal antibody was diluted 3000 times with the conservative value of coating solution (25mmol/L carbonate buffer, pH9.6), added to the wells of the ELISA plate at 100 μl per well, and coated overnight at 4°C. The next day, it was fully washed with PBST (PBS containing 0.05% Tween20, pH 7.4), and then 5% skimmed milk powder was added to block at 37°C for 45min. After fully washing with PBST, dilute the antigen (CerIFN-γ standard) with the washing solution to 50ng, 25.5ng, 12.75ng, 6.375ng, 3.1875ng, 1.593ng, 0.79ng, 0.398ng, 0.199ng, 0.099ng , 0.498ng, 0.025ng, 0.012ng, 0.006ng, 0.003ng and 0.002ng in 12 gradients, incubated at 37°C for 1h, and washed repeatedly. Rabbit anti-CerIFN-γ polyclonal antibody diluted 5000-fold with washing solution, 100 μl per well, incubated at 37°C for 45 minutes, and then fully washed. Add 1:5000 diluted HRP-goat anti-rabbit IgG, 100 μl per well, 37°C for 30 min, fully wash with 100 μl TMB/H ₂ O ₂ substrate solution per well, and react in the dark at room temperature for 10 min, then add 50 μl stop solution per well ( 0.25% hydrofluoric acid) to terminate the reaction, and read the optical density value (OD) at a wavelength of 630 nm.

2. Determination of the optimal working concentration of sika deer IFN-γ monoclonal/polyclonal antibody

The optimum working concentration was determined using square array indirect titration. Using sika deer IFN-γ as antigen, 50ng, 25.5ng, 12.75ng, 6.375ng, 3.1875ng, 1.593ng, 0.79ng, 0.398ng, 0.199ng, 0.099ng, 0.498ng, 0.025ng, 0.012ng, 0.006ng , 0.003ng and 0.002ng 12 gradients were used to coat 96-well ELISA plates at 100 μL per well, and coated overnight at 4°C. The next day, after fully washing with PBST, 5% skimmed milk powder was added to block at 37°C for 30min. CerIFN-γ monoclonal antibody diluted in 8 gradients of 200, 400, 800, 1600, 3200, 6400, 12800 and 25600 times with washing solution (or with washing solution 400, 800, 1600, 3200, 6400, 12800, 25600 and 51200 CerIFN-γ polyclonal antibody diluted 8 times in a gradient), 100 μL per well, incubated at 37°C for 30 min and then fully washed. Add 1:5000 dilution of HRP-goat anti-mouse IgG (or HRP-goat anti-rabbit IgG), 100 μl per well, 37°C for 30 minutes, after washing thoroughly, 100 μL TMB/H ₂ O ₂ substrate solution per well, and react in the dark at room temperature After 10 min, add 50 μL of stop solution (0.25% hydrofluoric acid) to each well to terminate the reaction, and read the optical density value (OD) at a wavelength of 630 nm

3. Specific release of IFN-γ from sika deer in vitro

Jugular venous blood was drawn from sika deer and anticoagulated with heparin. Add whole blood at 1.5ml/well to three wells of a 24-well cell culture plate, in which the positive control contains 60 μg concanavalin A (Con A), and the detection well contains 80 μg tuberculosis-specific protein Rv3872-CFP10-ESAT6 fusion Protein, blank control wells containing 50μ base 1640 culture solution. Slightly shake the culture plate to fully mix the reagents in each well with the blood, and incubate at 37°C for 16-24h in a cell culture incubator containing 5% CO ₂ . The culture supernatant was collected the next day, and the concentration of IFN-γ was detected by sandwich ELISA.

4. Establishment of sandwich ELISA for detection of sika deer IFN-γ

Properly dilute the monoclonal antibody with the coating solution, add 100 μl per well into the wells of the ELISA plate, and coat overnight at 4°C. The next day, after fully washing with PBST, 5% skimmed milk powder was added to block at 37°C for 45min. After fully washed with PBST, 100 μl of the above three supernatants were added to each well, incubated at 37°C for 1 h, and washed repeatedly. Rabbit anti-CerIFN-γ polyclonal antibody appropriately diluted with washing solution, 100 μl per well, incubated at 37°C for 45 minutes and then fully washed. Add 1:5000 diluted HRP-goat anti-rabbit IgG, 100 μl per well, 37°C for 30 min, fully wash with 100 μl TMB/H ₂ O ₂ substrate solution per well, and react in the dark at room temperature for 10 min, then add 50 μl stop solution per well ( 0.25% hydrofluoric acid) to terminate the reaction, and read the optical density value (OD) at a wavelength of 630 nm.

Example 7: Application of sika deer IFN-γ detection method for tuberculosis detection

1. Sample handling

Take 120 anticoagulated sika deer blood samples, add 1.5ml/well to three wells of a 24-well cell culture plate, wherein the positive control contains 60 μg concanavalin A (purchased from Sigma Company), and the detection well 80 μg preservation number It is CCTCC NO: M208244 (patent application number is 2009100609131, patent publication number is CN101538578A) bovine tuberculosis-specific three-gene fusion antigen protein RCE, and the blank control well contains 50 μ basic 1640 culture solution (see Appendix 1 for the formula). Slightly shake the culture plate to fully mix the reagents and blood in each well, culture in a cell culture incubator containing 5% CO2 at 37°C for 16-24h, and collect the culture supernatant the next day.

2. Detect the above samples with a commercialized ELISA kit (control test 1)

The commercial ELISA kit used in this example was purchased from Wuhan Keqian Animal Biological Products Co., Ltd. The method is as follows: use the bovine tuberculosis-specific three-gene fusion antigen protein RCE with the preservation number CCTCC NO: M208244 (patent application number 2009100609131, patent publication number CN101538578A), coat the microtiter plate at 4°C overnight, and wash 3 times with washing solution , blocked with 5% skimmed milk powder diluted with washing solution at 37°C for 1 h, washed the plate three times with washing liquid, then added 1:100 (volume ratio) diluted serum, 100 μL/well, repeated 3 times for each well, and reacted at 37°C for 30 min. After washing the plate three times, goat anti-bovine IgG diluted at a volume ratio of 1:10000 (volume ratio) was added, and reacted at 37°C for 30 minutes. After washing the plate 5 times, 100 μL of substrate solution (containing 1 mg/mL TMB and 0.03% H ₂ O ₂ ) was added, and 0.25% HF was added to stop the reaction after 10 min of color development in the dark, and read at OD ₆₃₀ . (Result Table 1)

3. Detect the above samples with commercial test strips (control test 2):

The commercial test strips used in this example were purchased from Korea Animal Genetics Corporation (English name of the company: Animal Genetics, Inc, trade name: Anigen Rapid Bovine TB Ab Test Kit). Operate according to the instructions of the test strip: take 100 μL of the blood sample of the sika deer to be tested and add it to the detection hole of the test strip, and observe the result after 10 minutes (result table 2).

4. Detect with the sika deer gamma-interferon double antibody sandwich ELISA method of the present invention:

The specific method is as follows: Dilute the monoclonal antibody at 50 ng/well with the coating solution (see Appendix 2), add 100 μl per well into the wells of the ELISA plate, and coat overnight at 4°C. The next day, wash thoroughly with washing solution (see Appendix 2) and add 5% skim milk powder to block at 37°C for 45min. After fully washing with washing solution, 100 μl of the above three supernatants (blood samples to be tested) were added to each well, incubated at 37° C. for 1 h, and washed repeatedly. Rabbit anti-sika deer gamma-interferon polyclonal antibody diluted with washing solution (50ng/well), 100μl per well, incubated at 37°C for 45min and then fully washed. Add 1:5000 dilution of horseradish peroxidase-labeled goat anti-rabbit IgG, 100 μl per well, 37°C for 30 minutes, after washing thoroughly, 100 μl of chromogenic solution per well, and react in the dark at room temperature for 10 minutes, then add 50 μl of stop solution per well (See Appendix 3) Terminate the reaction, and measure the absorbance value at a wavelength of 630nm on an enzyme-linked immunosorbent detection instrument.

Table 1120 test results of samples to be tested

Table 2120 test results of samples to be tested

5. Judgment of results

When the OD value difference between the positive and negative control wells is ≥0.25, and the OD value difference between the specific antigen (RCE) and the negative control wells is ≥0.1, it is judged to be positive for tuberculosis;

When the OD value difference between the positive and negative control wells is ≥0.25, and the OD value difference between the specific antigen (RCE) and negative control wells is <0.1, it is judged as tuberculosis negative;

When the difference in OD value between positive and negative control wells is <0.25, and the difference in OD value between specific antigen (RCE) and negative control wells is <0.1, the results cannot be determined.

Appendix 1

1. Preparation of RPMI1640 basal culture medium

(1) Measure 950ml of deionized water and place it in a certain container;

(2) Add 10 g of RPMI1640 powder (purchased from Shanghai Biyuntian Biotechnology Co., Ltd.) to deionized water at 15°C-30°C, and stir while adding to obtain a culture solution;

(3) add 2g sodium bicarbonate to the nutrient solution of step (2) by 1000ml nutrient solution;

(4) Add water to 1000ml, use CO ₂ to adjust the pH value of the culture solution to pH6.5-6.8, and close the container cork tightly before filtering;

(5) Immediately sterilize by positive pressure filtration with a microporous filter membrane with a pore size of 0.22 μm, and store in a refrigerator at 4°C for later use.

Appendix 2

(1) Flushing solution: 8.0g sodium chloride, 0.2g potassium chloride, 0.2g disodium hydrogen phosphate, 2.9g potassium dihydrogen phosphate, Tween-200.5ml, dilute to 1000ml with distilled water, pH7.4.

(2) Coating solution: 1.59g of sodium carbonate, 2.93g of sodium bicarbonate, and dilute to 1000ml with distilled water.

(3) Blocking solution: washing solution 100ml, skimmed milk 10g.

Appendix 3

Preparation of chromogenic solution:

Preparation of Chromogenic Solution A: Disodium hydrogen phosphate 71.6g, citric acid 19.2g, dilute to 1000ml with distilled water.

Preparation of chromogenic solution B: 100ml of 0.1mol/L phosphate-citrate buffer solution at pH5.0 (7.16g of disodium hydrogen phosphate, 1.92g of citric acid, distilled water to 100ml), 40mg of o-phenylenediamine , 0.15ml of 30% hydrogen peroxide.

Stop solution: 22.2ml of 2mol/L sulfuric acid, 177.8ml of distilled water.

Explanation: The "Sika deer γ-interferon" described in the present invention and the "Sika deer interferon-γ" named in the biological material sample preservation certificate are the same biological material sample. In this manual, the biological material sample is named "Sika deer interferon-γ". γ" was changed to "Sika deer γ-interferon" which is more suitable for Chinese expression habits.

sequence listing

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<120>Sika Deer γ-Interferon Double Antibody Sandwich ELISA Detection Method, Kit and Application

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Claims (4)

1. the cell line CerIFN-γ 4C that a strain can stably excreting sika deer gamma interferon monoclonal antibody is deposited in Chinese Typical Representative culture collection center, and deposit number is CCTCC NO:C200966.

2. monoclonal antibody specific by the sika deer gamma interferon of the described cell line of claim 1 secretion.

3. the double-antibody sandwich elisa detection kit of a sika deer gamma interferon, it is characterized in that, this kit comprises that deposit number is the sika deer gamma interferon monoclonal antibody of the cell line CerIFN-γ 4C secretion of CCTCC NO:C200966, sika deer IFN-γ polyclonal antibody, this polyclonal antibody is to be that Escherichia coli (Escherichia coli) BL21/pET-28a-RCE of CCTCC NO:M208244 is expressed by preserving number, horseradish peroxidase mark goat anti-rabbit igg, nitrite ion and stop buffer

Wherein said nitrite ion and stop buffer compound method are as follows:

Nitrite ion: sodium hydrogen phosphate 7.16g; Citric acid 1.92g; Supply distilled water to 100ml, obtain A liquid; Sodium hydrogen phosphate 7.16g; Citric acid 1.92g; Supply distilled water to 100ml, pH 5.0, obtain the phosphate-citrate salts damping fluid 100ml of 0.1mol/L, add o-phenylenediamine 40mg; The hydrogen peroxidase 10 .15ml of 30% concentration obtains B liquid, mixes described A liquid and B liquid, obtains nitrite ion;

Stop buffer: 2mol/L sulfuric acid (H ₂SO ₄) 22.2ml; Distilled water 177.8ml.

4. the application of monoclonal antibody claimed in claim 2 in preparation sika deer gamma interferon detection kit.

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