CN103869084A - Anti-bovine interferon gamma double-antibody sandwich ELISA (Enzyme Linked Immunosorbent Assay) kit and manufacturing method thereof - Google Patents

Abstract

The anti-bovine interferon-gamma double antibody sandwich ELISA kit and its preparation method belong to the field of biotechnology detection. The anti-bovine interferon-gamma monoclonal antibody 3E1 is secreted from the hybridoma cell line BovinIFNgamma-3E1, and the monoclonal antibody 3E1 is obtained from the hybridoma cell line BovinIFNgamma-6E5. The anti-bovine interferon-gamma monoclonal antibody 6E5 was secreted, the anti-bovine interferon-gamma monoclonal antibody 3E1 was coated on a microtiter plate, and the enzyme was labeled on the anti-bovine interferon-gamma monoclonal antibody 6E5. In this way, a double-antibody sandwich ELISA kit for detecting bovine gamma interferon was prepared with an ELISA plate coated with anti-bovine gamma interferon monoclonal antibody 3E1 and an enzyme-labeled anti-bovine gamma interferon monoclonal antibody 6E5. It requires precision instruments and can be carried out in ordinary laboratories.

Description

Anti-bovine gamma interferon double antibody sandwich ELISA kit and preparation method thereof

technical field

The invention belongs to the field of biotechnology detection, in particular to a double-antibody sandwich ELISA kit for detecting bovine gamma interferon.

Background technique

Interferon is a group of active proteins (mainly glycoproteins) with multiple functions, generally induced by interferon inducers, it is a cytokine produced by monocytes and lymphocytes, and its types are divided into three categories , α-(leukocyte) type, β-(fibroblast) type, γ-(lymphocyte) type; they have broad-spectrum anti-virus, affect cell growth, differentiation, and regulate immune function on the same kind of cells. biological activity. Interferon can enhance the activity of natural killer cells (NK cells), macrophages and T lymphocytes, thereby playing an immune regulation role and enhancing antiviral ability.

As a broad-spectrum antiviral agent, interferon (IFN) does not directly kill or inhibit viruses, but mainly makes cells produce antiviral proteins through the action of cell surface receptors, thereby inhibiting virus replication. the

Bovine tuberculosis is a chronic wasting infectious disease caused by Mycobacterium bovis, which can be transmitted to humans or other animals through sick animals. In addition to infecting humans, tuberculosis can also infect more than 50 kinds of mammals and more than 20 kinds of birds. Bovine tuberculosis occurs in all countries in the world, and it is still a common multiple disease in my country. Its spread and prevalence not only affect the sustainable development of animal husbandry, but also seriously affect human health. At present, the testing methods used to diagnose bovine tuberculosis at home and abroad can be roughly divided into three categories: one is bacteriological testing methods, such as smear microscopy, bacterial culture and animal inoculation, etc.; the second category is using molecular biological testing methods , such as biological probe method, PCR method, etc.; the third category is the use of immunological methods for detection, such as intradermal allergy, ELISA and ELISPOT methods. Intradermal allergy has played an important role in the diagnosis and prevention of bovine tuberculosis, but the shortcoming of this method is that there are many factors that interfere with tuberculosis quarantine, including physical and chemical factors, and more of them are non-specific. The interference of typical mycobacteria, because each mycobacterium exhibits cross-allergic reactions, often causes non-specific reactions. All countries in the world are devoting themselves to the research of rapid diagnostic methods for bovine tuberculosis, and have established some new, rapid, accurate and reliable detection methods, and have made remarkable progress in this field. In the 1990s, Wood et al. proved that after Mycobacterium bovis infected the body, it would stimulate the body's lymphocytes to form memory lymphocytes, sensitize the lymphocytes, and use the specific antigen of Mycobacterium bovis to the aseptically collected bovine blood. (BPPD) re-stimulation will cause the sensitized lymphocytes in the whole blood to be activated and release a large amount of γ-interferon, so the detection of bovine tuberculosis can be achieved by detecting the γ-interferon in the bovine whole blood supernatant . This whole blood interferon-gamma assay has been shown to provide a rapid and sensitive means for the diagnosis of a range of diseases in animals such as humans, monkeys, cattle, buffaloes, sheep and goats, and extensive field trials of the bovine interferon-gamma assay have been performed. It has been completed in many countries in the world (including Australia, Ireland, New Zealand, Argentina, Spain, Italy and the United States), and is gradually replacing the combined intradermal test with mycocin. The sensitivity of the γ-interferon diagnostic method is 81.8% to 100%, and the specificity The sensitivity is 94% to 100%, which is more sensitive than the skin test. However, the key to establishing rapid, accurate, sensitive, specific and simple diagnostic methods is the acquisition of monoclonal antibodies with high specificity and affinity.

Contents of the invention

The purpose of the present invention is to provide a fast, accurate, sensitive, specific and simple diagnostic method for detecting bovine gamma interferon double antibody sandwich ELISA kit.

The present invention includes:

(1) Enzyme plates coated with anti-bovine interferon-γ monoclonal antibody 3E1 (capture antibody);

(2) Enzyme-labeled anti-bovine interferon-γ monoclonal antibody 6E5 (detection antibody);

Wherein, the anti-bovine interferon-gamma monoclonal antibody and the enzyme-labeled anti-bovine interferon-gamma monoclonal antibody coated on the microtiter plate are respectively aimed at different epitopes of bovine interferon-gamma. the

In addition, the kit of the present invention may also include one or more of the following reagents: dilution solution, washing solution, substrate chromogenic solution, stop solution and the like.

The natural bovine gamma interferon and the artificially expressed recombinant bovine gamma interferon can be detected by using the detection kit of the invention. The invention provides reliable means for bovine cellular immunity research and rapid detection of bovine tuberculosis. The kit of the invention is convenient to operate, has low use cost and good repeatability, and is suitable for large-scale popularization and application.

The present invention uses synthetic peptides and GST-bovine gamma interferon purified protein as antigens to prepare anti-bovine gamma interferon monoclonal antibodies, and obtains a series of monoclonal antibodies with different antigenic epitopes. After further screening, two strains targeting bovine gamma interferon are obtained as a result Hybridoma cell lines with different epitopes of interferon antigens, the monoclonal antibodies secreted by the cell lines have high titer, strong specificity, and good detection reactivity.

The invention provides a reliable means for rapid detection of bovine gamma interferon. The kit is easy and fast to operate, and can be used for bovine cellular immunology research and rapid detection of bovine tuberculosis. It is suitable for scientific research and detection of bovine tuberculosis in colleges and universities, scientific research units, veterinary departments above the municipal level, and entry-exit inspection and quarantine bureaus. The method requires low cost, significant economic benefits and wide application prospects.

The present invention also proposes the preparation method of above kit:

The anti-bovine interferon-γ monoclonal antibody 3E1 was secreted from the hybridoma cell line Bovin IFN gamma-3E1, and the anti-bovine interferon-γ monoclonal antibody 6E5 was secreted from the hybridoma cell line Bovin IFN gamma-6E5. The monoclonal antibody 3E1 was coated on the microtiter plate, and the anti-bovine gamma interferon monoclonal antibody 6E5 was enzyme-labeled.

The anti-bovine interferon-gamma monoclonal antibody 3E1 coated on the ELISA plate was obtained from the secretion of the hybridoma cell line Bovin IFN gamma-3E1 (classification name: hybridoma cell line, preservation number: CGMCC No.7105); enzyme-labeled anti-bovine interferon Gamma interferon monoclonal antibody 6E5 is secreted from hybridoma cell line Bovin IFN gamma-6E5 (classification name: hybridoma cell line, preservation number: CGMCC No.7104).

The enzyme-labeled anti-bovine gamma interferon monoclonal antibody 6E5 of the present invention is produced by labeling with horseradish peroxidase using a commercial enzyme labeling kit. the

The monoclonal antibody can be coated onto the microtiter plate as follows: dilute the monoclonal antibody to 0.35 μg/mL with sodium bicarbonate buffer, add 100 μL/well to the microtiter plate, place it at 4°C for 12-16 hours, and use After washing with PBST, the cells were blocked with 5% skimmed milk, 350 μL/well, at 37°C for 2 hours, and then washed with PBST.

Detailed ways

The following examples further illustrate the content of the present invention, but should not be construed as limiting the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.

Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

1. Anti-bovine interferon-γ monoclonal antibody cell line

Anti-bovine gamma interferon monoclonal antibody cell line Bovin IFN gamma-3E1, classification and name: hybridoma cell line, preserved by the General Microbiology Center of China Microbiological Culture Collection Management Committee, preservation number: CGMCC No.7105, preservation address: Beijing No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District.

Anti-bovine gamma interferon monoclonal antibody cell line Bovin IFN gamma-6E5, classification and name: hybridoma cell line, preserved by the General Microbiology Center of China Microbiological Culture Collection Management Committee, preservation number: CGMCC No.7104, preservation address: Beijing No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District.

The anti-bovine interferon-γ monoclonal antibody coated on the microtiter plate was secreted from the above-mentioned hybridoma cell line Bovin IFN gamma-3E1 (cell preservation number: CGMCC No.7105).

The enzyme-labeled anti-bovine interferon-γ monoclonal antibody is secreted from the above-mentioned hybridoma cell line Bovin IFN gamma-6E5 (cell deposit number: CGMCC No.7104).

2. Preparation of anti-bovine interferon-γ monoclonal antibodies 3E1 and 6E5

(1) Take out the preserved hybridoma cell line Bovin IFN gamma-3E1 (classification name: hybridoma cell line, preservation number: CGMCC No.7105) that can secrete anti-bovine interferon-γ monoclonal antibody hybridoma cell line from liquid nitrogen , resuscitated at 37°C, and prepared to prepare mouse ascites antibody after the cells grew well. That is, 10-week-old Balb/c female mice were injected intraperitoneally with paraffin oil, each injected with 0.5ml. After 10 days, the hybridoma cells were diluted with DMEM medium, and each mouse was injected with 3 million hybridoma cells intraperitoneally. Ascites was collected after the abdomen was significantly enlarged, and the collected ascites was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken and stored at -20°C. Purify the antibody with a protein G affinity column and determine the concentration and titer, aliquot and store at -70°C.

(2) Take out the preserved hybridoma cell line Bovin IFN gamma-6E5 (classification name: hybridoma cell line, preservation number: CGMCC No.7104) that can secrete anti-bovine interferon-γ monoclonal antibody hybridoma cell line from liquid nitrogen , resuscitated at 37°C, and prepared to prepare mouse ascites antibody after the cells grew well. That is, 10-week-old Balb/c female mice were injected intraperitoneally with paraffin oil, each injected with 0.5ml. After 10 days, the hybridoma cells were diluted with DMEM medium, and each mouse was injected with 3 million hybridoma cells intraperitoneally. Ascites was collected after the abdomen was significantly enlarged, and the collected ascites was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was taken and stored at -20°C. Purify the antibody with a protein G affinity column and determine the concentration and titer, aliquot and store at -70°C.

3. Enzyme Labeling of Anti-Bovine Interferon-γ Monoclonal Antibody 6E5

The purified monoclonal antibody 6E5, which can react with natural bovine interferon-γ, was labeled with the horseradish peroxidase labeling kit of Thermo Company. The specific steps are as follows: Dissolve 1mg of IgG in PBS with a volume of 0.5-1ml; then dissolve 1mg of lyophilized EZ-Link Plus Activated Peroxidase in 100ml of deionized water, and then add IgG to it; immediately add 10μl of Sodium Cyanoborohydride was placed in the reaction system and allowed to react at room temperature for 1 hour; then 20 μl of quenching Buffer was added and allowed to react at room temperature for 15 minutes; the labeled sample was purified with a Protein G column from GE.

4. Anti-bovine interferon-gamma monoclonal antibody 3E1 coated on the ELISA plate

(1) Dilute the anti-bovine interferon-γ monoclonal antibody 3E1 to 0.35 μg/mL with carbonate buffer, add 100 μL/well to the ELISA plate, coat at 4°C for about 12 hours, and then coat in a water bath at 37°C 30～60min;

(2) Discard the coating solution, wash once with 1×PBST, add 5% skim milk, bathe in 37°C water, and seal for 2 hours;

(3) Discard the blocking solution, wash 3 times with 1×PBST, and use it for detection.

5. Induced production of natural bovine interferon-γ

bovine whole blood culture

① blood collection

Aseptically collect at least 5 mL of bovine blood, anticoagulate, and mix thoroughly by gently inverting several times. Cultures were performed within 30 hours of blood collection. Note: Anticoagulated blood cannot be stored in the refrigerator.

② Blood culture

Gently invert and mix well before blood culture. Since this assay requires viable lymphocytes, it is necessary to minimize cell damage. Add anticoagulant blood to the 24-well culture plate, add two wells per animal, 1.5mL per well (see the table below). Work under sterile conditions with disposable automatic pipettes or pipettes.

Recommended procedure for pipetting blood or stimuli into 24-well culture plates

Animal No. 1 NIL A1 BPPD A2 Animal No. 2 NIL A3 BPPD A4 Animal No. 3 NIL A5 BPPD A6 Animal No. 4 NILB1 BPPD B2 Animal No. 5 NIL B3 BPPD B4 animal number 6 NIL B5 BPPD B6 animal number 7 NILC1 BPPD C2 animal number 8 NIL C3 BPPD C4 animal number 9 NIL C5 BPPD C6 animal number 10 NILD1 BPPD D2 Animal No. 11 NIL D3 BPPD D4 animal number 12 NIL D5 BPPD D6

Note: In the above table, NIL=negative antigen (PBS); BPPD=purified bovine tuberculin.

③ Add stimulant

Aseptically add 100 μL PBS (negative antigen control) and 100 μL BPPD to the corresponding wells. Antigen must be thoroughly mixed with blood.

④ Incubation

Incubate the plate containing blood and antigen in a 37°C, CO ₂ humidified incubator for 16-24 hours.

⑤Harvesting of plasma samples to be tested

Carefully draw the upper plasma sample with an adjustable pipette and transfer to a separate 1.5mL centrifuge tube.

NOTE: Try to avoid aspirating cells when aspirating plasma samples. If a small amount of red blood cells are aspirated, remove them by centrifugation.

⑥ Storage of samples

Samples can be stored at 2-8°C for 7 days and at -20°C for several months. Prior to storage, each storage tube must be sealed with a suitable cap. Samples must be returned to room temperature and thoroughly mixed before testing.

6. Kit detection steps:

(1) Add 100 μL of negative control solution to wells A1 and A2 of the ELISA plate coated with anti-bovine interferon-γ monoclonal antibody 3E1, add 100 μL of positive control solution to wells A3 and A4, and add plasma samples to be tested in the remaining wells (PBS stimulated plasma, BPPD stimulated plasma), 100 μL per well, 37 ° C water bath for 60 min ± 5 min.

(2) Discard the liquid in the wells, add washing solution to each well, do not allow cross-contamination between the wells, pat, discard the washing solution, pat on absorbent paper, and repeat washing 5 times.

(3) Add 100 μL of anti-bovine interferon-γ enzyme-labeled antibody (6E5) diluted to the working concentration to each well, and place in a 37°C water bath for 60 min±5 min.

(4) Discard the liquid in the wells, add washing solution to each well, do not allow cross-contamination between the wells, pat, discard the washing solution, pat on absorbent paper, and repeat washing 5 times.

(5) Add the prepared chromogenic solution under dark conditions, add 100 μL to each well, and develop color at 37°C for 30 minutes.

(6) After the color development is over, add 50 μL of stop solution to each well, and measure the OD450 value of the sample with a microplate reader.

(7) Results

Judgment of quality:

a. OD450<0.200 of bovine gamma-interferon negative control wells

b. The OD450 of bovine γ-interferon positive control wells>0.500

Note: If one of the criteria is not met, the test is invalid and the test must be repeated.

Result judgment:

If OD450 of BPPD-OD450 of PBS≥0.15, it is judged as positive.

If OD450 of BPPD-OD450 of PBS<0.15, it is judged as negative.

7. The characteristics of this detection method:

It only takes 3 hours to detect bovine interferon by this method, and it is more accurate for the detection of interferon secreted by bovine lymphocytes, with a sensitivity of 0.7ng/ml, especially when lymphocytes are stimulated by specific antigens such as tuberculin. Interferon can be detected under this method.

The monoclonal antibody secreted by this cell line only reacts with bovine interferon and does not react with interferon from other animals. This method is a double-sandwich ELISA method established by two monoclonal antibodies, rather than a conventional double-sandwich ELISA method established by polyclonal antibodies and monoclonal antibodies. The source of monoclonal antibody is more stable and easier than polyclonal antibody, and the specificity is stronger.

The method is easy to operate, does not require precision instruments, and can be carried out in ordinary laboratories.

Claims (2)

1. The anti-bovine gamma interferon double antibody sandwich ELISA kit is characterized in that it comprises: 1) ELISA plate coated with anti-bovine interferon-γ monoclonal antibody 3E1; 2) Enzyme-labeled anti-bovine interferon-γ monoclonal antibody 6E5; Among them, the anti-bovine interferon-gamma monoclonal antibody 3E1 and the enzyme-labeled anti-bovine interferon-gamma monoclonal antibody 6E5 coated on the microtiter plate are respectively aimed at different epitopes of bovine interferon-gamma. 2. the preparation method of kit as claimed in claim 1 is characterized in that: obtain anti-bovine gamma interferon monoclonal antibody 3E1 from the secretion of hybridoma cell line Bovin IFN gamma-3E1, from hybridoma cell line Bovin IFN gamma-6E5 The anti-bovine interferon-gamma monoclonal antibody 6E5 was secreted, the anti-bovine interferon-gamma monoclonal antibody 3E1 was coated on a microtiter plate, and the anti-bovine interferon-gamma monoclonal antibody 6E5 was enzyme-labeled.