CN114075551B - Monoclonal antibody of brucella lipopolysaccharide of sarin mouse species and application - Google Patents
Monoclonal antibody of brucella lipopolysaccharide of sarin mouse species and application Download PDFInfo
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- CN114075551B CN114075551B CN202110656015.3A CN202110656015A CN114075551B CN 114075551 B CN114075551 B CN 114075551B CN 202110656015 A CN202110656015 A CN 202110656015A CN 114075551 B CN114075551 B CN 114075551B
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- C—CHEMISTRY; METALLURGY
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1221—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Brucella (G)
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
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- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
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Abstract
The invention relates to the technical field of monoclonal antibody preparation, and particularly discloses a monoclonal antibody of brucella lipopolysaccharide of sarin mice species and application thereof. The invention uses a hot phenol water method to extract brucella LPS of sarin mouse species, and 1h heat-inactivated brucella of sarin mouse species is used for immunizing Balb/c mice at 80 ℃; fusing spleen cells of the immunized mice with SP2/0 myeloma cells; screening a positive hybridoma cell strain by a limiting dilution method and an indirect ELISA method, wherein the positive hybridoma cell strain is named as 2H2, and the preservation number of the hybridoma cell strain 2H2 is CCTCC NO: C2021134; preparation of 2H2 strain sarin murine Brucella LPS monoclonal antibody, heavy chain subtype IgG 1 The light chain type is kappa type, the concentration is 8.775mg/mL, the titer is 1:512000, the purity and the titer are high, the affinity is good, the binding capacity is strong, and the specificity is good.
Description
Technical Field
The invention relates to the technical field of monoclonal antibody preparation, in particular to a monoclonal antibody of brucella Lipopolysaccharide (LPS) of sarin mice and application thereof.
Background
Brucella (Brucella) belongs to pathogenic bacteria of human and animal co-occurrence, and the most obvious symptoms of patients after infection show wave heat, which is also called 'Mediterranean achalasia heat' or 'Malta ear heat'. The world animal health Organization (OIE) lists Brucellosis (Brucellosis for short) as a B-type infectious disease, and prescribes it as a B-type infectious disease according to the infectious disease control method of the people's republic of China. Can cause acute and chronic acute infections of human beings and various animals, and the female animals infected with Brucella mainly show hyporeproductive capacity, abortion and stillbirth. Male animals can cause orchitis and infertility. The human-to-human contact transmission mode is rare, and the brucellosis infection mainly contacts bacterial secretion of infected animals and enters the organism through damaged skin mucous membrane, respiratory tract and digestive tract. Patients in the acute stage show slight influenza symptoms, patients in the chronic stage show symptoms such as repeated fever (wave heat), anemia, pain of joints, splenomegaly and testicle enlargement, and serious patients lose labor force, thus forming a great threat to the public health system of society.
The cell membrane of Brucella consists of three layers of membranes, namely a cytoplasmic membrane, a peripheral cytoplasmic membrane and an outer membrane from inside to outside. The outer membrane of brucella comprises three parts, respectively Outer Membrane Proteins (OMPs), lipopolysaccharides (LPS), phospholipids. Most antibodies raised after brucella infection are directed against Lipopolysaccharide (LPS). LPS is mainly composed of three parts of lipoid A, core polysaccharide and O side chains, and the brucella is classified into smooth type (S type) or rough type (R type) according to the existence of the O side chains.
ELISA kits for early or primary Brucella antibodies are reported in the relevant literature to have higher sensitivity and specificity for clinical application (Mantur et al 2007). The method comprises the steps of extracting brucella melitensis Lipopolysaccharide (LPS) of sheep species and S2 brucella melitensis Lipopolysaccharide (LPS) of pig species by using a hot phenol water method respectively, preparing monoclonal antibodies aiming at the brucella melitensis lipopolysaccharide by excluding cross reaction, and then inventing different ELISA methods to detect samples suspected brucellosis (2014, ding Gubo and other 2017 of the step high). Zhu Liangquan A competition ELISA method of Brucella canis is invented by using a Brucella canis monoclonal antibody (Zhu Liang full 2020).
The patent is not searched out at home and abroad, and a detection method is established on the basis of the brucella lipopolysaccharide monoclonal antibody of the sarin mouse species.
Disclosure of Invention
The invention aims to provide a hybridoma cell strain 2H2, wherein the preservation number of the hybridoma cell strain 2H2 is CCTCC NO: C2021134.
Another object of the present invention is to provide a monoclonal antibody secreted by hybridoma cell line 2H 2.
The final object of the invention is to provide the application of the hybridoma cell strain 2H2 or the monoclonal antibody secreted by the hybridoma cell strain in preparing a brucella detection kit, particularly a brucella serrata detection kit.
In order to achieve the above object, the present invention adopts the following technical measures:
the applicant uses a hot phenol water method to extract brucella Lipopolysaccharide (LPS) of sarin mouse species, and the brucella of the sarin mouse species is heat-inactivated for 1h at 80 ℃ to immunize Balb/c mice; fusing spleen cells of the immunized mice with SP2/0 myeloma cells; screening a positive hybridoma cell strain by a limiting dilution method and an indirect ELISA method, wherein the positive hybridoma cell strain is named as 2H2, and the preservation number of the hybridoma cell strain 2H2 is CCTCC NO: C2021134; preparation of 2H2 strain sarin murine Brucella LPS monoclonal antibody, heavy chain subtype IgG 1 The light chain type is kappa type, the concentration is 8.775mg/mL, the titer is 1:512000, the hybridoma cell strain 2H2 is sent to China center for type culture collection for storage on 6-month 3 of 2021, and the classification and the naming are that: hybridoma cell line 2H2, accession number: CCTCC NO: C2021134, address: university of martial arts in chinese.
The invention also provides monoclonal antibodies secreted by hybridoma cell line 2H 2.
The hybridoma cell strain 2H2 or the monoclonal antibody secreted by the hybridoma cell strain can be applied to preparation of a brucella detection kit, in particular to a brucella detection kit of sarin mouse species.
Compared with the prior art, the invention has the following advantages:
the method selects the brucella of the sarin species to extract Lipopolysaccharide (LPS), compares the homology of key enzyme genes of the brucella to synthesize the Lipopolysaccharide (LPS), and results show that the homology is up to more than 99 percent, so that the Lipopolysaccharide (LPS) of the brucella of the sarin species is selected as an antigen to prepare the monoclonal antibody. Meanwhile, the strain can effectively reduce the risk of infection of brucellosis during operation of laboratory personnel.
A hybridoma cell strain 2H2 is successfully prepared by utilizing a hybridoma technology; the subtype of the monoclonal antibody 2H2 secreted by the hybridoma cell strain is IgG 1 The light chain type is kappa type; the concentration is 8.775mg/mL, the titer is 1:512000, the purity and the titer are high, the affinity is good, the binding capacity is strong, and the specificity is good. Can lay a good experimental foundation for the establishment of a Brucella pathogen and antibody detection method in the future.
Drawings
FIG. 1 shows the results of ELISA detection of antibody titers of immunized mice.
FIG. 2 shows the results of Western Blot identification of specific assays of 2H2 and 3B9 mAbs against different bacteria.
FIG. 3 shows the results of confirmation of optimal blocking conditions of a 2H2 mAb sandwich ELISA.
FIG. 4 shows the confirmation of the optimal incubation conditions for 2H2 mAb double antibody sandwich ELISA mAb samples.
FIG. 5 shows the results of a 2H2 mab sandwich ELISA specificity assay.
Detailed Description
The technical scheme of the invention is a conventional scheme in the field unless specifically stated; the reagents or materials, unless otherwise specified, are commercially available.
Example 1:
preparation of monoclonal antibodies to brucella lipopolysaccharide from sarin mice:
1. preparation of antigen Lipopolysaccharide (LPS)
Taking a brucella (B.neotame) bacterial solution of a sarin mouse species cultured for 24 hours, centrifuging for 15 minutes at 4 ℃ and 10000r/min, collecting bacterial sludge, washing for 2 times by using PBS, centrifuging to collect bacterial sludge, weighing bacterial sludge, adding equal amounts of distilled water preheated at 66 ℃ and saturated phenol according to the ratio of the bacterial sludge to water of 1:4, uniformly mixing, stirring for 30 minutes by using a water bath at 66 ℃, cooling at 4 ℃, centrifuging for 15 minutes at 10000r/min, adding 500mL of saturated sodium acetate methanol solution precooled at 4 ℃ in advance to precipitate Lipopolysaccharide (LPS), placing in a refrigerator at 4 ℃ for incubation for more than 2 hours, centrifuging for 10 minutes at 10000r/min, re-suspending the precipitate in 80mL of sterilized pure water, stirring for 18 hours at 4 ℃, centrifuging for 10 minutes at 10000r/min, storing supernatant in the refrigerator at 4 ℃, suspending the precipitate in 80mL of sterilized pure water again, stirring for 2 hours at 4 ℃, centrifuging for 15 minutes at 10000r/min, and mixing with the supernatant. The supernatant was discarded, then 8g of trichloroacetic acid (TCA) was added to 160mL of the total crude Lipopolysaccharide (LPS) extract collected twice, and after stirring at room temperature for 10 to 15min, the precipitate was removed by centrifugation at 10000r/min for 15min, and the supernatant was collected. Dialyzing the supernatant with pure water, and changing the solution at least twice; the Lipopolysaccharide (LPS) is obtained by crude extraction (dialysis with pure water for 3h each time and about 4 times each time), and the dialyzed sample is pre-frozen in a refrigerator at-80 ℃ and placed in a vacuum freeze dryer for freeze drying overnight. The lyophilized Lipopolysaccharide (LPS) was weighed, suspended in carbonate buffer at a concentration of 1mg/mL, and then sub-packaged in penicillin bottles at 1mL for lyophilization and storage at-80 ℃. The Lipopolysaccharide (LPS) integrity was checked by electrophoresis on a 12% SDS-PAGE gel and silver staining.
2. Immunization of animals
Mixing the inactivated brucella with Freund's complete adjuvant in equal volume, emulsifying, subcutaneously injecting 5 SPF-class Balb/c female mice for primary immunization, and administering 3×10 dose 9 CFU/only; after primary immunization, booster immunization was carried out every two weeks for five times at a dose of 3×10 9 CFU/CFU.
The indirect ELISA method is adopted to detect the antibody titer of the immunized mice, and the positive mouse serum dilution titer reaches more than 1:12800, wherein the serum dilution titer of the No. 4 mouse is the highest (figure 1). For this purpose, mice # 4 were selected for booster immunization at an injection dose of 6X 10 9 CFU/alone for cell fusion.
3. Cell fusion
a. Preparation of feeder cells
Hybridoma cell growth depends on a certain amount of cell density, and in addition, feeder cells can secrete some cell growth factors and the like to assist in hybridoma cell growth. One female BALB/c mouse with the age of 8-10 weeks of SPF is taken, the female BALB/c mouse is subjected to eyeball bloodletting and is sacrificed, blood is collected, and serum is separated, so that the female BALB/c mouse is negative serum. Soaking in 75% alcohol for 5min, transferring into aseptic console,the procedure is the same as the preparation method of immune spleen cells. The skin was torn open by cutting a small incision in the middle of the abdomen, 5mL of complete 1640 medium containing HAT was aspirated with a 5mL syringe, the peritoneum was lifted with sterile forceps, the needle was carefully inserted, the medium was injected, and the aspiration was repeated once and the two aspirates were mixed. Mixing with spleen cells after grinding and centrifugal resuspension, adding HAT complete culture medium to 100-150 mL, then spreading 10 96-well cell plates, each well being 100-150 mu L, placing 5% CO 2 Incubator, 37 ℃ overnight for use.
b. Preparation of immune spleen cells
Enhancing immunity of mice, collecting positive serum by orbital bleeding, soaking in 75% alcohol for 5min, fixing the mice, aseptically taking spleen, adding 3mL of basic 1640 homogenizer for grinding, adding basic 1640-10 mL, standing for 2min, sucking the upper layer into a 50mL centrifuge tube, adding 10mL of 1640 into the homogenizer, and washing twice repeatedly.
c. Preparation of myeloma cells
Mice were sacrificed by cervical spine removal and sterilized by soaking in 75% alcohol for 5min. The dissecting plate is fixed on the back of the mouse upwards, the fur is peeled off under the aseptic condition, and the removed tumor mass is placed into the homogenizer. Firstly adding 5mL of basic 1640 culture medium, lightly grinding, fully adding 10mL of basic 1640 culture medium after grinding, uniformly mixing, standing for 2min, lightly sucking 5mL to 50mL of sterile centrifuge tube of supernatant, then adding 10mL of basic 1640 culture medium twice, uniformly mixing and standing for 2min after each addition, sucking 10mL for the first time, sucking all for the second time, and properly discarding about 3mL of liquid at the bottom during the last sucking, so as to avoid sucking large tissue blocks during the sucking. Centrifugation at 1000r/min for 10min, supernatant was discarded, cells were resuspended in basal 1640 medium, and the cell suspension volume was kept at about 30 mL. Adding about 20mL of lymphocyte separation liquid into another 50mL centrifuge tube, slowly adding the cell suspension in the previous step above the lymphocyte separation liquid by adherence (the volume ratio of the cell suspension to the lymphocyte separation liquid is (1:1-2:1)), centrifuging for 10-15 min at 1200r/min, sucking the white compact cell layer at the boundary of the liquid level, washing twice with a basic 1640 culture medium, and finally re-suspending cells with 10mL of the basic 1640 culture medium and counting.
d. Cell fusion
The isolated myeloma cells and the immune spleen cells were thoroughly mixed at a ratio of 1:10 or 1:5, centrifuged, and the myeloma cells (1 to 2×10 7 ) With immune spleen cells (1X 10) 8 ) Mix in 50mL centrifuge tube and centrifuge for 10min at 1200 r/min. Residual culture medium liquid on the wall of the centrifugal tube is sucked by sterile filter paper so as to prevent the concentration of the fusion agent from being influenced, the bottom of the centrifugal tube is flicked to slightly loosen the cell mass, and the mixed mass of the centrifuged spleen cells and myeloma cells is required to be beaten into a cloud form and is required to be thoroughly beaten into Yun Wuzhuang. The 50% PEG4000 fluxing agent was preheated in advance in a 37℃incubator. The fusion process is carried out in a water bath at 37 ℃, 50% PEG4000 preheated at 37 ℃ is taken out to 0.8mL,1min is added into a cell mass at the bottom of a centrifuge tube, collision between cells by a gun head is reduced as much as possible, after the gun head is added while rotating at a constant speed, the gun head is gently stirred for 30s, and after the gun head is added, the gun head is rotated at a constant speed for 30s until the fusion agent fully acts. The fusion reaction was terminated by slow addition of basal 1640 medium (pre-warmed up at 37 ℃ in advance). 2mL (namely 60s corresponds to 1 mL) is added in the first 2min, 3mL is added in the 3 rd to 4 th min, 5mL is added in the 5 th min, and finally about 30mL is added to terminate the reaction, and the whole operation is completed within 10min as much as possible. Centrifuging at 1000r/min for 8min, discarding the supernatant, and placing the cell sediment in a 37 ℃ incubator for 5-8 min. The cell pellet was lightly resuspended in HAT medium (pre-warmed at 37 ℃ in advance) containing pre-prepared feeder cells and plated in 96 well cell plates for culture at 100 μl per well. Placing in incubator (37 ℃,5% CO) 2 ) Is cultured.
4. Hybridoma cell screening
50. Mu.L of HAT complete 1640 medium was added the first day after fusion, and the plates were moved as little as possible for 3 days later, and cells were observed daily to check contamination, pH and colony growth. If contaminated, the treatment is immediately carried out with a strong acid or base. After the fusion, 200. Mu.L of HT complete 1640 medium was changed per well on day 4, 50. Mu.L of HT complete 1640 medium was added on day 7, and wells of single and multiple cell clones were labeled, respectively. When the cell colonies grow to about 1/4 to 1/3 of the culture well, the supernatant of the cell culture is detected by indirect ELISA. In the indirect ELISA assay of cell supernatant titers, 50. Mu.L of primary antibody was aspirated from each well, and the secondary antibody was horseradish peroxidase-labeled goat anti-mouse IgG (H+L) at a dilution of 1:10000. The extracted brucine mouse species brucella Lipopolysaccharide (LPS) and escherichia coli Lipopolysaccharide (LPS) are respectively used as coating antigens, and holes with high positive values and low negative values are screened by ELISA detection and are regarded as positive hybridoma cells. Marking the corresponding positive hybridoma cells, supplementing HT culture standard for expansion, and preparing subclones.
And (3) carrying out second screening and third screening on the hybridoma cells screened for the first time by using the same screening method, and finally obtaining two positive hybridoma cell strains which can stably secrete brucella brucei Lipopolysaccharide (LPS) of the anti-sarin mouse species, wherein the positive hybridoma cell strains are named as 2H2 and 3B9.
5. Preparation and purification of mouse ascites monoclonal antibody
The established positive cell strains 2H2 and 3B9 are subjected to expansion culture, and after continuous culture for 3 to 4 generations, the cell supernatant titer is detected by an indirect ELISA method, and the cell strains are used for preparing ascites when the cell titer is still higher.
Pre-stimulation: female BALB/c mice of 8-10 weeks of age were selected, and first, 500. Mu.L/mouse Freund's incomplete adjuvant was injected intraperitoneally. After 5-7 days, injecting hybridoma cells into the abdominal cavity, and re-suspending the enlarged hybridoma cells by using a basic 1640 culture medium, and centrifuging at 1000r/min for 10min. Each mouse was injected approximately 5×10 5 ～1×10 6 The amount of cells injected was controlled to about 300. Mu.L per mouse. After the injection, the abdominal cavity of the mouse is observed to be obviously enlarged about 7 days, when the mouse is inconvenient to move, the abdominal cavity of the mouse is firstly wiped by alcohol cotton, then a 20mL syringe needle is inserted into the abdominal cavity of the mouse to facilitate the outflow of the abdominal cavity liquid, meanwhile, the existence of the needle eye is noted to cause the infection to the mouse, and the disinfection work is performed. 10000 Centrifuging at 4deg.C for 15min at r/min to obtain supernatant, removing upper oil component, removing possible red blood cell impurities, and freezing at-80deg.C.
The rProtein G Beads 4FF is loaded into a proper chromatographic column, and chromatography is balanced by using a combination Buffer with 5 times of the plant volume, so that the filler is under the same Buffer system as the target protein, and the function of protecting the protein is achieved. And (3) adding the sample into the balanced rProtein G Beads 4FF (ensuring that the target protein is fully contacted with the rProtein G Beads 4FF, and improving the recovery rate of the target protein), and collecting effluent. And (3) cleaning by using a impurity-cleaning Buffer with the volume of 10-15 times of the column volume, removing nonspecifically adsorbed impurity proteins, and collecting impurity-cleaning liquid. And (3) eluting Buffer with a volume which is 5-10 times that of the column, and collecting eluent, namely the target protein component. Sequentially using 3 times of column volume of combined Buffer and 5 times of column volume of pure water to balance the filler, finally balancing with 20% ethanol of 5 times of column volume, then preserving in 20% ethanol of equal volume, and preserving at 4deg.C to prevent the filler from being polluted by bacteria. Dialyzing the eluate in PBS (pH 7.0, 0.01M) for 24h, collecting eluate with higher purity, and storing at-80deg.C.
ELISA titers were determined for the pre-and post-purification ascites as described above, with the following results:
ELISA determination results of ascites titers of hybridoma cell line 2H2 before and after purification
ELISA determination results of ascites titers before and after purification of hybridoma cell line 3B9
6. Monoclonal antibody subtype identification
The kit was first brought to room temperature, and then the cleaning liquid was prepared to a working concentration (one part of concentrated cleaning liquid plus 19 parts of pure water) with pure water. And taking out the ELISA plate. Each sample required 8 wells, positive control 8 wells, negative control 8 wells. The excess is kept in a self-sealing bag, remembering to put in a desiccant. 50. Mu.L of the cell supernatant (or specifically affinity purified antibody) was added to the microplate, 8 wells were added to each sample, and the positive control and negative control were also 50. Mu.L per well of each 8 wells. Then 50. Mu.L of specimen diluent was added to each well. Attaching a sealing plate film to incubate for 30-40 min at 37 ℃. The liquid in the plate is discarded, and after the plate is washed for 5 times, the plate is beaten dry or washed by a machine for 5 times on the water absorbing material without fiber. Then 8 enzyme-labeled secondary antibodies (IgG) are respectively added into 8 holes of each specimen 1 、IgG 2a 、IgG 3 、IgG 2b IgA, igM, ig κ, igλ) of 100 μl each, as well as 8 wells of the universal positive control. Marking on the sample-adding or enzyme label plate. Attaching a sealing plate film to the substrate, and incubating the substrate for 30 to 40 minutes at 37 ℃ in a dark place. The liquid in the plate is sucked and discarded, and the plate is washed 5 times and then is beaten dry or washed 5 times on the water absorbing material without fiber. And adding 100 mu L of the color reagent A and the color reagent B into each hole respectively, and replacing a new sealing plate film plate with a light-proof color developing plate at 37 ℃ for 15min. The kit has good specificity, the result can be observed by naked eyes, and the Ig class or subclass of the sample can be known by looking at the enzyme-labeled secondary antibody corresponding to the hole with blue color. After the reaction was terminated by adding a reaction termination solution (50. Mu.L per well), the reaction was terminated by using an ELISA reader OD 450nm The absorbance was measured.
The results show that the heavy chain subtypes of the brucella lipopolysaccharide monoclonal antibodies 2H2 and 3B9 strains of the sarin mice are IgG 1 The light chain types are all kappa type.
7. Monoclonal antibody concentration detection
The antibody concentration is detected using a BCA protein concentration measurement kit, and specific operations are performed according to the kit instructions. The results show that in the purified ascites in the step 5, the concentrations of the brucella lipopolysaccharide monoclonal antibodies 2H2 and 3B9 strains of the sarin mice are 8.775mg/mL and 4.256mg/mL respectively.
8. Monoclonal antibody titer detection
Monoclonal antibodies 2H2 and 3B9 were diluted by a dilution (PBS) from 1000-fold to 500-fold, respectively, and titers were detected by an indirect ELISA assay. The results show that the titers are 1:512000 and 1:256000, the purity and the titers are high, the affinity is good, the binding capacity is strong, and the specificity is good.
9. Monobutyric-specific Western Blot detection
The ELISA plate is coated with extracted brucella brucei Lipopolysaccharide (LPS), positive cell strains 2H2 and 3B9 aiming at the brucella Lipopolysaccharide (LPS) are screened out through a three-time limiting dilution method, purified ascites are respectively used for dilution according to 1:2000, PVDF membranes are incubated overnight at 4 ℃, and IgG (H+L) is diluted by PBS according to 1:5000. Western Blot results showed that the purified antibodies all bind specifically to Brucella and do not react with E.coli, salmonella enteritidis and human pallidum (FIG. 2).
The above results show that the monoclonal antibody secreted by the hybridoma cell 2H2 is significantly better than the monoclonal antibody secreted by the hybridoma cell 3B9 from the protein concentration and the antibody titer, so that the hybridoma cell 2H2 is sent to the China center for type culture Collection for type culture collection, and the classification and the naming are that: hybridoma cell line 2H2, accession number: CCTCC NO: C2021134, address: university of martial arts in chinese.
10. ELISA monoclonal antibody and potency determination
Horse Radish Peroxidase (HRP) marks the purified brucella lipopolysaccharide monoclonal antibody 2H2, and the result shows that the titer of the 2H2 monoclonal antibody after enzyme labeling reaches 1:64000.
The specific table is shown below:
example 2:
determination of detection conditions for detecting brucella in sarin murine species by 2H2 mab ELISA:
1. determination of antibody coating concentration and ELISA monoclonal antibody dilution
The optimal coating concentration of the antibody and the dilution of the enzyme-labeled monoclonal antibody are determined by using a square matrix titration test, and the result shows that positive OD is measured when the coating dilution of 2H2 monoclonal antibody of brucella brucei Lipopolysaccharide (LPS) of sarin mice is 1:128000 and the dilution of the enzyme-labeled 2H2 monoclonal antibody is 1:16000 450nm Value and negative OD 450nm The values were 1.142 and 0.087, respectively. Considering that the loss exists in the using process of the antibody, the dilution of the coated 2H2 monoclonal antibody is 1:100000, and the dilution of the enzyme-labeled 2H2 monoclonal antibody is 1:15000.
Determination of antibody coating concentration and ELISA monoclonal antibody dilution
2. Determination of optimal closing time
The results showed that, under blocking at 37℃for 2.5h, brucella OD was positive 450nm Value and negative OD 450nm The ratio of values (P/N) was highest and was 40.860, so the closure was chosen at 37℃for 2.5h (FIG. 3).
3. Determination of optimal incubation conditions for samples
The results show that the OD of positive Brucella is under the condition of acting for 1h at 37 DEG C 450nm Value and negative OD 450nm The ratio of values (P/N) was highest and was 31.239, so the incubation time for the antigen samples was 1h (FIG. 4).
4. Determination of the action time of the substrate
The results show that the OD of the positive Brucella is under the condition of light-shielding at 37 ℃ for 15min 450nm Value and negative OD 450nm The ratio (P/N) of the values was highest and was 26.800, so the action time was 15min, as shown in the following table:
5. determination of Brucella double antibody sandwich ELISA negative threshold
By determining 30 negative bovine nasal swab secretions OD 450nm Value, test data were processed by SPSS 17.0 Thus, the maximum threshold of negative was determined to be 0.189, but the OD was measured 450nm Since there is fluctuation in the values, the negative threshold is set to 0.247, and the determination of the negative threshold is shown in the following table:
in the above table, P is positive control, is inactivated brucella solution of sarin murine species, and has a concentration of 3×10 9 CFU/mL。
Example 3:
application of 2H2 monoclonal antibody in preparation of Brucella ELISA detection kit:
the antigen detection method of the brucella double-antibody sandwich ELISA of the sarin mice comprises the following steps:
(1) Coating: the 2H2 mab coating dilution was 1:100000, 100. Mu.L/well, incubated overnight at 4 ℃.
(2) Washing the plate: washing liquid is 250 mu L/hole, and washing is carried out for 4-5 times for 3min each time.
(3) Closing: 200. Mu.L/well of 2% BSA blocking solution was incubated at 37℃for 2.5h.
(4) And (2) as well.
(5) The antigen or sample to be detected is added, 100. Mu.L/well and incubated at 37℃for 1h.
(6) And (2) as well.
(7) ELISA monoclonal antibody: therefore, the dilution of the enzyme-labeled 2H2 monoclonal antibody is 1:15000, 100 mu L/well, and the incubation is carried out for 1H at 37 ℃.
(8) And (2) as well.
(9) A substrate: TMB single-component color development solution 100. Mu.L/well was incubated at 37℃for 15min.
(10) Stop solution: stop solution 50. Mu.L/well.
(11) Reading: ELISA reader reading OD 450nm Is a numerical value.
The judgment standard is as follows: when OD is 450nm When the value is more than 0.247, the Brucella is positive.
Specificity experiments:
detecting brucella, brucella ovis, brucella attenuated strain M5-90, escherichia coli, salmonella enteritidis, pallidobacter hominis and PBS. The results showed that specific reaction with Brucella only, against sarinThe best response of brucella murine showed better specificity of the kit (fig. 5). In the specificity experiment, the bacterial concentration of the bacterial liquid to be detected is 3 multiplied by 10 9 CFU/mL。
Sensitivity test:
from 3×10 brucella cells of sarin murine species 9 CFU/mL dilution to 3X 10 1 CFU/mL, the result of detecting antigen sensitivity shows that the minimum dilution of the bacterial antigen is 3×10 through the determination test of the negative critical value 5 CFU/mL。
Claims (5)
1. A hybridoma cell strain has a preservation number of CCTCC NO: C2021134.
2. The monoclonal antibody secreted by the hybridoma cell line of claim 1.
3. The hybridoma cell strain or the monoclonal antibody secreted by the hybridoma cell strain, which is disclosed in claim 1, is applied to preparation of a brucella detection kit, wherein the brucella is brucella sandline and/or brucella ovis.
4. Use of the hybridoma cell strain of claim 1 or a secreted monoclonal antibody thereof in the preparation of a brucella serrata detection kit.
5. The use according to claim 3 or 4, wherein the detection kit is a double antibody sandwich ELISA detection kit.
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