CN114075551A - Monoclonal antibody of brucella salina lipopolysaccharide and application thereof - Google Patents
Monoclonal antibody of brucella salina lipopolysaccharide and application thereof Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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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- G—PHYSICS
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- 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
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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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- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- 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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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- 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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Abstract
The invention relates to the technical field of monoclonal antibody preparation, and particularly discloses a monoclonal antibody of Brucella lipopolysaccharide of Serratia species and application thereof. The method comprises the steps of extracting brucella sallinae seed LPS by a hot phenol water method, and thermally inactivating the brucella sallinae seed at 80 ℃ for 1h to immunize Balb/c mice; fusing splenocytes from the immunized mice with SP2/0 myeloma cells; a positive hybridoma cell strain is screened by a limiting dilution method and an indirect ELISA method, is named as 2H2, and the preservation number of the hybridoma cell strain 2H2Is CCTCC NO of C2021134; preparing 2H2 Serratia seriae Brucella LPS monoclonal antibody, wherein the heavy chain subtype is IgG1The 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 abortus Lipopolysaccharide (LPS) and application thereof.
Background
Brucella (Brucella) belongs to zoonosis pathogenic bacteria, and the most obvious symptoms of a patient after infection show wave heat, which is also called as Mediterranean relaxation heat or Maltese ear heat. The world animal health Organization (OIE) classifies Brucellosis (hereinafter referred to as "Brucellosis") as a B-type infectious disease, which is specified as a B-type infectious disease according to the infectious disease prevention and treatment Law of the people's republic of China. Can cause acute and chronic acute infections of human beings and various animals, and the infection of female animals with brucella is mainly manifested by reproductive capacity reduction, easy abortion and stillbirth. The male livestock can cause orchitis and sterility symptoms. The contact transmission mode between people is rare, and the brucellosis is mainly infected by contacting bacteria-carrying secretion of infected animals and entering into organisms through damaged skin mucous membranes, respiratory tracts and digestive tracts. The acute stage patient shows slight flu symptoms, the chronic stage patient shows symptoms of repeated fever (wave heat), anemia, arthralgia, splenic enlargement, testicular enlargement and the like, the serious patient loses labor force, and the serious patient has great threat to the social public health system.
The cell membrane of the Brucella is composed 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 consists of three parts, namely Outer Membrane Proteins (OMPs), Lipopolysaccharide (LPS), and phospholipids. Most antibodies produced following brucella infection are directed against Lipopolysaccharide (LPS). LPS is mainly composed of lipid A, core polysaccharide and O side chain, and Brucella is classified into smooth type (S type) or rough type (R type) according to the existence of O side chain.
According to related literature, an ELISA kit aiming at early stage antibody or main antibody of brucella is reported to have higher sensitivity and specificity in clinical application (Mantur et al 2007). The high-grade and medium-grade brucella melitensis and the Dingjiabo and the like respectively extract brucella melitensis (LPS) of sheep species and brucella suis S2 by a hot phenol water method, eliminate cross reaction, prepare monoclonal antibodies aiming at the brucella melitensis, and invent different ELISA methods to detect samples suspected of brucellosis on the basis (the high-grade and medium-grade 2014 of Chinese stephagia, the Dingjiabo and the like 2017). The competitive ELISA method of the dog brucella is invented by utilizing the dog brucella monoclonal antibody in the Zhuliang (Zhuliang et al 2020).
The method is established on the basis of a Brucella lipopolysaccharide monoclonal antibody of a species of Serratia at home and abroad literature and patent.
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 objective of the invention is to provide a monoclonal antibody secreted by hybridoma cell line 2H 2.
The last purpose 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, in particular to a brucella salina detection kit.
In order to achieve the purpose, the invention adopts the following technical measures:
the applicant extracts brucella abortus Lipopolysaccharide (LPS) of the species of the sarin by a hot phenol water method, and thermally inactivates the brucella abortus at 80 ℃ for 1h to immunize a Balb/c mouse; fusing splenocytes from the immunized mice with SP2/0 myeloma cells; screening a positive hybridoma cell strain named as 2H2 by a limiting dilution method and an indirect ELISA method, wherein the preservation number of the hybridoma cell strain 2H2 is CCTCC NO: C2021134; preparing 2H2 Serratia seriae Brucella LPS monoclonal antibody, wherein the heavy chain subtype is IgG1The light chain type is kappa type, the concentration is 8.775mg/mL, the titer is 1:512000, and the hybridoma cell strain 2H2 is delivered to the China center for type culture Collection at 6 months and 3 days 2021 and is classified and named: hybridoma cell line 2H2, accession number: CCTCC NO, C2021134, address: wuhan university in Wuhan, China.
The invention also comprises monoclonal antibody secreted by hybridoma cell strain 2H 2.
The hybridoma cell strain 2H2 or the monoclonal antibody secreted by the hybridoma cell strain is applied to the preparation of a brucella detection kit, in particular to the application of the brucella detection kit of the species Serratia.
Compared with the prior art, the invention has the following advantages:
the method selects the brucella arenicola to extract Lipopolysaccharide (LPS), and compares the homology of key enzyme genes of the brucella to synthesize Lipopolysaccharide (LPS), and the result shows that the homology is up to more than 99 percent, so the Lipopolysaccharide (LPS) of the brucella arenicola strain is selected as an antigen to prepare the monoclonal antibody. Meanwhile, the bacterium can effectively reduce the risk of infecting brucellosis when laboratory personnel operate.
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 IgG1The 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 developing the establishment of the brucella pathogeny and antibody detection method in the future.
Drawings
FIG. 1 shows the results of ELISA assay of antibody titer in immunized mice.
FIG. 2 shows the result of Western Blot identifying the specificity of 2H2 and 3B9 monoclonal antibodies to different bacteria.
FIG. 3 shows the confirmation result of the optimal blocking condition of 2H2 monoclonal antibody double-antibody sandwich ELISA.
FIG. 4 is the confirmation result of the optimal incubation condition of the 2H2 monoclonal antibody double-antibody sandwich ELISA monoclonal antibody sample.
FIG. 5 shows the results of 2H2 monoclonal antibody double antibody sandwich ELISA specific assay.
Detailed Description
The technical schemes of the invention are conventional schemes in the field if not particularly stated; the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
preparing a monoclonal antibody of brucella abortus lipopolysaccharide:
1. preparation of antigen Lipopolysaccharide (LPS)
Centrifuging 24h Brucella (B.neotomae) bacterial solution of Serratia sericata at 4 deg.C and 10000r/min for 15min, collecting bacterial sludge, washing with PBS for 2 times, centrifuging to collect bacterial sludge, weighing bacterial sludge, adding equal amount of distilled water preheated at 66 deg.C and saturated phenol according to the ratio of bacterial sludge to water of 1:4, mixing, stirring in 66 deg.C water bath for 30min, cooling at 4 deg.C, centrifuging at 10000r/min for 15min, adding 500mL saturated sodium acetate methanol solution pre-cooled at 4 deg.C to precipitate Lipopolysaccharide (LPS), incubating in 4 deg.C refrigerator for more than 2h, then centrifuging at 10000r/min for 10min, suspending the precipitate in 80mL sterile pure water, stirring at 4 ℃ for 18h, then centrifuging at 10000r/min for 10min, storing the supernatant in a refrigerator at 4 deg.C, resuspending the precipitate in 80mL sterilized pure water, stirring at 4 deg.C for 2h, centrifuging at 10000r/min for 15min, and mixing with the supernatant. Discarding the supernatant, adding 8g trichloroacetic acid (TCA) into 160mL crude Lipopolysaccharide (LPS) extract collected twice, stirring at room temperature for 10-15 min, centrifuging at 10000r/min for 15min, removing the precipitate, and collecting the supernatant. Dialyzing the supernatant with pure water, and changing the solution at least twice; (dialyzing with pure water for 3h each time, dialyzing for about 4 times) to obtain Lipopolysaccharide (LPS), pre-freezing the dialyzed sample in a refrigerator at-80 deg.C, placing in a vacuum freeze dryer, and freeze-drying overnight. Lyophilized Lipopolysaccharide (LPS) was weighed, suspended in carbonate buffer at a concentration of 1mg/mL, and then dispensed in 1mL vials for lyophilization, and stored at-80 ℃. The integrity of Lipopolysaccharide (LPS) was checked by electrophoresis on a 12% SDS-PAGE gel and silver staining.
2. Animal immunization
Mixing inactivated Brucella in Serratia species with Freund's complete adjuvant in equal volume, emulsifying, and performing subcutaneous multi-point injection to primarily immunize 5 SPF-level Balb/c female mice at a dose of 3 × 109CFU/only; after the initial immunization, a booster immunization was performed every two weeks for five times at a dose of 3X 109CFU/only.
The indirect ELISA method is adopted to detect the antibody titer of the immune mice, and the serum dilution titer of positive mice is more than 1:12800, wherein the serum dilution titer ratio of No. 4 mice is the highest (figure 1). For this purpose, mice No. 4 were selected for booster immunization at an injection dose of 6X 109CFU/mouse for cell fusion。
3. Cell fusion
a. Preparation of feeder cells
The growth of the hybridoma cells depends on a certain cell density, and moreover, the feeder cells can secrete certain cell growth factors and the like to help the growth of the hybridoma cells. Taking one female BALB/c mouse with the SPF of 8-10 weeks old, taking off eyeballs, bleeding and killing, collecting blood and separating serum to obtain negative serum. Soaking in 75% alcohol for 5min, transferring into sterile operation table, and the operation steps are the same as the preparation method of immune spleen cells. The middle part of the abdomen was cut with a small incision, the skin was torn open, 5mL of HAT-containing complete 1640 medium was aspirated with a 5mL syringe, the peritoneum was lifted with sterile forceps, the needle was carefully inserted, the culture was injected, massaged and withdrawn, repeated once, and the two aspirations were mixed. Mixing with ground and centrifugally resuspended splenocytes, adding HAT complete culture medium to 100-150 mL, laying 10 cell plates with 96 pores, each 100-150 μ L, and adding 5% CO2Incubator, 37 degrees overnight standby.
b. Preparation of immune spleen cells
Strengthening immune mice, bleeding from eye sockets, collecting positive serum, soaking in 75% alcohol for 5min, fixing the mice, taking spleen aseptically, adding into a 3mL basic 1640 homogenizer for grinding, supplementing basic 1640 to 10mL, standing for 2min, sucking the upper layer into a 50mL centrifugal tube, supplementing 10mL 1640 into the homogenizer, and washing twice.
c. Preparation of myeloma cells
The mice were sacrificed by cervical dislocation and sterilized by soaking in 75% ethanol for 5 min. The back of the mouse is fixed upwards and is fixed on an anatomical plate, the skin and the hair are stripped under the aseptic condition, and the tumor mass taken down is put into a homogenizer. Firstly adding 5mL of basic 1640 culture medium, slightly grinding, supplementing 10mL of basic 1640 culture medium after full grinding, uniformly mixing, standing for 2min, slightly sucking 5mL to 50mL of sterile centrifuge tube of supernatant, then supplementing 10mL of basic 1640 culture medium twice, uniformly mixing and standing for 2min after each supplement, sucking 10mL for the first time, sucking out all the liquid for the second time, properly discarding about 3mL of liquid at the bottom during the last suction, and avoiding sucking large tissue blocks during the suction. Centrifuging at 1000r/min for 10min, discarding supernatant, and resuspending cells in basic 1640 culture medium to keep the cell suspension volume at about 30 mL. And adding about 20mL of lymphocyte separation liquid into another 50mL centrifuge tube, slowly adding the cell suspension obtained in the previous step above the lymphocyte separation liquid in an adherent manner (the volume ratio of the cell suspension to the lymphocyte separation liquid is (1: 1-2: 1): 1200r/min, centrifuging for 10-15 min, sucking a white compact cell layer at the boundary of the liquid level, washing twice by using a basic 1640 culture medium, and finally, re-suspending the cells by using 10mL of the basic 1640 culture medium and counting.
d. Cell fusion
Mixing the separated myeloma cells and immune splenocytes at a ratio of 1:10 or 1:5, centrifuging to obtain myeloma cells (1-2 × 10)7) With immune spleen cells (1X 10)8) Mixing the mixture in a 50mL centrifuge tube, and centrifuging the mixture at 1200r/min for 10 min. And (3) completely sucking residual culture medium liquid on the wall of the centrifugal tube by using sterile filter paper to prevent the action concentration of the fusion agent from being influenced, flicking the bottom of the centrifugal tube to slightly loosen cell masses, and beating the mixed mass of the centrifuged splenocytes and myeloma cells into a cloud shape till the mixed mass is completely beaten into the cloud shape. The 50% PEG4000 fluxing agent was preheated in advance in an incubator at 37 ℃. The fusion process is carried out in a 37 ℃ water bath, 0.8mL of 50% PEG4000 preheated at 37 ℃ is taken out and added into a cell mass at the bottom of a centrifuge tube within 1min, the collision of a gun head on the cells is reduced as much as possible, after the cell mass is added while the gun head rotates at a constant speed, the disposable rubber head is used for stirring gently for 30s, and after the addition is finished, the rotation at a constant speed is carried out for 30s until the fusion agent is fully acted. The fusion reaction was terminated by slow addition of basal 1640 medium (pre-warmed at 37 ℃ C. in advance). Adding 2mL (namely 1mL corresponding to 60 s) in the first 2min, adding 3mL in the 3-4 min, adding 5mL in the 5min, and finally adding about 30mL to terminate the reaction, wherein the whole operation is completed within 10min as far as possible. Centrifuging at 1000r/min for 8min, removing supernatant, and placing cell sediment in a 37 ℃ incubator for 5-8 min. The cell pellet was gently resuspended in HAT medium (pre-warmed at 37 ℃ C.) containing pre-prepared feeder cells, and plated in 96-well cell plates for culture at 100. mu.L per well. Placing in an incubator (37 ℃, 5% CO)2) Culturing in medium.
4. Hybridoma cell selection
HAT complete 1640 medium was added 50. mu.L the first day after fusion, plates were removed as little as possible 3 days later, and cells were observed daily to check for contamination, pH and colony growth. If the pollution exists, the solution is immediately treated by strong acid or strong alkali. After fusion, 200. mu.L of HT complete 1640 medium was added at day 4, and 50. mu.L of HT complete 1640 medium was added at day 7, and wells of single or multiple cell clones were labeled. When the cell colony grows to about 1/4-1/3, the supernatant of the cell culture is detected by indirect ELISA. In the indirect ELISA for cell supernatant titer detection, 50. mu.L of horseradish peroxidase-labeled goat anti-mouse IgG (H + L) was aspirated from each well as a primary antibody at a dilution of 1: 10000. And respectively taking extracted brucella abortus Lipopolysaccharide (LPS) and extracted escherichia coli Lipopolysaccharide (LPS) as coating antigens, and screening holes with high positive values and low negative values by ELISA (enzyme-Linked immuno sorbent assay) detection to be regarded as positive hybridoma cells. Marking the corresponding positive hybridoma cells, supplementing HT culture medium for amplification, and preparing for subcloning.
And (3) carrying out secondary screening and third screening on the hybridoma cells screened for the first time by using the same screening method to finally obtain two positive hybridoma cell strains which can stably secrete brucella abortus Lipopolysaccharide (LPS) of the anti-sarin mouse, wherein the two positive hybridoma cell strains are named as 2H2 and 3B 9.
5. Preparation and purification of mouse ascites monoclonal antibody
And carrying out amplification culture on the established positive cell strains 2H2 and 3B9, continuously culturing for 3-4 generations, and detecting the titer of cell supernatant by an indirect ELISA method, wherein the titer is still higher, and the two cell strains are used for preparing ascites.
Pre-stimulation: female BALB/c mice 8-10 weeks old were selected, and 500. mu.L/mouse of Freund's incomplete adjuvant was first intraperitoneally injected. And injecting the hybridoma cells into the abdominal cavity after 5-7 days, re-suspending the expanded hybridoma cells by using a basic 1640 culture medium, and centrifuging at 1000r/min for 10 min. Approximately 5X 10 injections per mouse5～1×106Cells were injected at a volume of about 300. mu.L per mouse. Observing whether the abdominal cavity of the mouse is obviously expanded about 7 days after injection, wiping the abdominal cavity of the mouse with alcohol cotton when the mouse is inconvenient to move, inserting a 20mL syringe needle into the abdominal cavity of the mouse so as to facilitate the liquid in the abdominal cavity to flow out, and paying attention to the fact that the needle eye can infect the mouse and disinfecting. 10000rCentrifuging at 4 deg.C for 15min to obtain supernatant as ascites, removing upper oil and fat components, removing bottom red blood cells, and freezing at-80 deg.C.
The rProtein G Beads 4FF is loaded into a proper chromatographic column, and the chromatography is balanced by a combined Buffer with 5 times of the volume of the strain, so that the filler is in the same Buffer system with the target protein to play a role in protecting the protein. Adding the sample into balanced rProtein G Beads 4FF (ensuring the target protein to be fully contacted with the rProtein G Beads 4FF to improve the recovery rate of the target protein), and collecting the effluent. And (3) cleaning with a washing impurity Buffer with the volume of 10-15 times of the column volume, removing the non-specifically adsorbed impurity protein, and collecting the washing impurity solution. And (3) using an elution Buffer with the volume 5-10 times of that of the column, and collecting an eluent, namely the target protein component. The filler is balanced by using the combined Buffer with 3 times of column volume and the pure water with 5 times of column volume in sequence, and finally the filler is balanced by using 20 percent ethanol with 5 times of column volume, and then the filler is stored in the equal volume of 20 percent ethanol and is stored at 4 ℃ to prevent the filler from being polluted by bacteria. Dialyzing the eluate in PBS (pH7.0,0.01M) for 24 hr, collecting high purity eluate, and storing at-80 deg.C.
The ascites before and after the above purification were subjected to ELISA titer measurement, and the results were as follows:
ELISA (enzyme-Linked immunosorbent assay) measurement results of ascites titer before and after purification of hybridoma cell strain 2H2
Results of ELISA assay of ascites titer before and after purification of hybridoma cell line 3B9
6. Monoclonal antibody subtype identification
The kit was first brought to room temperature and the wash solution was then brought to working concentration with pure water (one part concentrated wash plus 19 parts pure water). And taking out the enzyme label plate. 8 wells were required for each sample, 8 wells for positive controlNegative control 8 wells. The redundant waste is preserved by a valve bag, and a drying agent is remembered to be put in. 50 muL of cell supernatant (or specific affinity purified antibody) is added into an enzyme-labeled microplate, 8 holes are added into each sample, and 50 muL of positive control and negative control are respectively added into 8 holes and each hole. Then 50. mu.L of the specimen dilution was added to each well. And (3) attaching a sealing plate film, and incubating for 30-40 min at 37 ℃. Discarding the liquid in the plate, washing the plate for 5 times, and then drying the plate on a water-absorbing material without fiber or washing the plate for 5 times by a machine. 8 enzyme-labeled secondary antibodies (IgG) were added to 8 wells of each sample1、IgG2a、IgG3、IgG2bIgA, IgM, Ig κ, Ig λ) 100 μ L each, as well as 8 wells of the universal positive control. And marking on the sample adding or enzyme labeling plate. And (4) pasting a sealing plate film, and incubating for 30-40 min in a dark place at 37 ℃. And (4) absorbing the liquid in the plate, washing the plate for 5 times, and then drying the plate on a water-absorbing material without fiber or washing the plate for 5 times by a machine. Adding 100 μ L of color-developing agent A and B into each well, and changing a new plate with sealing film to prevent light and develop color at 37 deg.C for 15 min. The kit has good specificity, and the result can be observed by naked eyes generally, 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 the color blue. Adding reaction stop solution (50. mu.L per well) to stop the reaction, and then using an enzyme-linked immunosorbent assay (OD)450nmThe absorbance was measured.
The results show that the heavy chain subtypes of brucella abortus lipopolysaccharide monoclonal antibodies 2H2 and 3B9 are IgG1The light chain types are all kappa types.
7. Monoclonal antibody concentration detection
And (3) detecting the concentration of the antibody by using the BCA protein concentration detection kit, and performing specific operation according to the kit instruction. The results showed that in step 5, the concentrations of the brucella abortus lipopolysaccharide monoclonal antibodies 2H2 and 3B9 of the sarin species were 8.775 and 4.256mg/mL, respectively, in the purified ascites.
8. Monoclonal antibody potency detection
The monoclonal antibodies 2H2 and 3B9 were diluted in a dilution medium (PBS) at 1000-fold and 500-fold ratios, respectively, and the titers were determined by an indirect ELISA assay. The results show that the titer is 1:512000 and 1:256000, the purity and the titer are high, the affinity is good, the binding capacity is strong, and the specificity is good.
9. Western Blot detection of monoclonal antibody specificity
The extracted brucella abortus Lipopolysaccharide (LPS) is used for coating an enzyme label plate, positive cell strains 2H2 and 3B9 aiming at the brucella abortus Lipopolysaccharide (LPS) are screened out through a three-time limiting dilution method, purified ascites is respectively used for dilution according to the ratio of 1:2000, a PVDF membrane is incubated overnight at the temperature of 4 ℃, IgG (H + L) is diluted by PBS, and the dilution is carried out according to the ratio of 1: 5000. The Western Blot result shows that the purified antibody can be specifically combined with Brucella and does not react with Escherichia coli, Salmonella enteritidis and human Ochrobactrum (figure 2).
The above results indicate that the monoclonal antibody secreted by hybridoma 2H2, either from protein concentration or from antibody titer, is significantly superior to the monoclonal antibody secreted by hybridoma 3B9, and therefore, hybridoma 2H2 was sent to the chinese type culture collection for collection at 2021, 6/3, under classification name: hybridoma cell line 2H2, accession number: CCTCC NO, C2021134, address: wuhan university in Wuhan, China.
10. Enzyme-labeled monoclonal antibody and potency assay
And (3) marking the purified brucella abortus lipopolysaccharide monoclonal antibody 2H2 by using horseradish peroxidase (HRP), wherein the result shows that the titer of the 2H2 monoclonal antibody after enzyme labeling reaches 1:64000 in the purified ascites.
The details are shown in the following table:
example 2:
determination of detection conditions for detecting Brucella in Serratia species by 2H2 monoclonal antibody ELISA:
1. determination of antibody coating concentration and enzyme-labeled monoclonal antibody dilution
The optimal coating concentration of the antibody and the dilution of the enzyme-labeled monoclonal antibody are determined by using a matrix titration test, and the result shows that when the coating dilution of the 2H2 monoclonal antibody of the Brucella Lipopolysaccharide (LPS) of the species Serratia arenicola is 1:128000 and the dilution of the enzyme-labeled 2H2 monoclonal antibody is 1:16000, the positive OD is measured450nmValue and negative OD450nmValue of valueRespectively 1.142 and 0.087. Considering the loss of the antibody in the using process, the dilution of the coating 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 enzyme-labeled monoclonal antibody dilution
2. Determination of optimal closure time
The result shows that the positive Brucella OD is obtained under the condition of sealing for 2.5h at 37 DEG C450nmValue and negative OD450nmThe ratio of values (P/N) was the highest and was 40.860, so blocking was selected at 37 ℃ for 2.5h (FIG. 3).
3. Determination of optimal incubation conditions for samples
The result shows that the OD of the positive Brucella is 1h at 37 DEG C450nmValue and negative OD450nmThe ratio of values (P/N) was the highest and was 31.239, so the antigen sample incubation time was 1h (FIG. 4).
4. Determination of the action time of a substrate
The results show that the OD of positive Brucella is 15min at 37 ℃ in dark450nmValue and negative OD450nmThe ratio of the values (P/N) was the highest and 26.800, so the action time was 15min, as shown in the following table:
5. determination of negative critical value of Brucella double-antibody sandwich ELISA
By judging 30 negative ox nose swab secretion OD450nmValue, processing test data by SPSS 17.0
Thus, the maximum negative cutoff value was determined to be 0.189, but the OD was taken into account450nmThe value was varied, so a negative cut-off value of 0.247 was set, and the determination of the negative cut-off value is shown in the following table:
in the above table, P is a positive control, and is an inactivated Brucella strain liquid of Serratia species with a concentration of 3 × 109CFU/mL。
Example 3:
the application of the 2H2 monoclonal antibody in preparing the Brucella ELISA detection kit comprises the following steps:
the antigen detection method of the double-antibody sandwich ELISA of the Brucella of the species Serratia arenicola comprises the following steps:
(1) coating: the 2H2 monoclonal antibody coating dilution was 1:100000, 100. mu.L/well, and incubated overnight at 4 ℃.
(2) Washing the plate: washing solution with a concentration of 250 μ L/hole for 4-5 times, each for 3 min.
(3) And (3) sealing: 2% BSA blocking solution 200. mu.L/well, incubated at 37 ℃ for 2.5 h.
(4) The same as (2).
(5) Adding the antigen or sample to be detected, incubating at 37 ℃ for 1h at a concentration of 100. mu.L/well.
(6) The same as (2).
(7) Enzyme-labeled monoclonal antibody: therefore, the dilution of the enzyme-labeled 2H2 monoclonal antibody is 1:15000, 100 mu L/hole, and the incubation is carried out for 1H at 37 ℃.
(8) The same as (2).
(9) Substrate: the TMB single-component developing solution is 100 mu L/hole, and is incubated for 15min at 37 ℃.
(10) Stopping liquid: stop buffer 50. mu.L/well.
(11) Reading: enzymeStandard instrument reading OD450nmAnd (4) processing the numerical value.
The judgment standard is as follows: when OD is reached450nmIf the value is more than 0.247, the brucella is positive.
Specific experiments:
and detecting Brucella sarmentsii, Brucella melitensis low virulent strain M5-90, Escherichia coli, Salmonella enteritidis, human ochrobactrum and PBS. The result shows that the kit only reacts specifically with Brucella, and the reaction on Brucella of Serratia species is optimal, which indicates that the specificity of the kit is better (figure 5). In the specificity experiment, the bacterial concentrations of the bacterial liquids to be detected are all 3 multiplied by 109CFU/mL。
And (3) sensitivity test:
culturing Brucella in Serratia species at a ratio of 3 × 109CFU/mL dilution to 3X 101CFU/mL, the result of detecting the antigen sensitivity shows that the lowest dilution of the bacterial antigen is 3 multiplied by 10 through the determination test of the negative critical value5CFU/mL。
Claims (5)
1. A hybridoma cell strain with the 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 as claimed in claim 1, and application of the hybridoma cell strain or the monoclonal antibody secreted by the hybridoma cell strain in preparation of a brucella detection kit.
4. The hybridoma cell strain or the monoclonal antibody secreted by the hybridoma cell strain as claimed in claim 1, and application of the hybridoma cell strain or the monoclonal antibody secreted by the hybridoma cell strain in preparation of a detection kit for detecting the Brucella species in Serratia arenicola.
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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