CN104450628A - Escherichia coli resistance O157:H7 monoclonal antibody and application thereof - Google Patents
Escherichia coli resistance O157:H7 monoclonal antibody and application thereof Download PDFInfo
- Publication number
- CN104450628A CN104450628A CN201410832762.8A CN201410832762A CN104450628A CN 104450628 A CN104450628 A CN 104450628A CN 201410832762 A CN201410832762 A CN 201410832762A CN 104450628 A CN104450628 A CN 104450628A
- Authority
- CN
- China
- Prior art keywords
- escherichia coli
- monoclonal antibody
- immunomagnetic beads
- chinese people
- japanese military
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides Escherichia coli resistance O157:H7 monoclonal antibody and application thereof, and belongs to the field of biotechnology. The preservation number of hybridoma cell strain 10G1A2 which generates the Escherichia coli resistance O157:H7 monoclonal antibody is CGMCC NO.9999. The invention further provides Escherichia coli resistance O157:H7 monoclonal antibody which is secreted by the hybridoma cell strain 10G1A2, and packaged immunomagnetic beads, as well as application of the immunomagnetic beads in detecting Escherichia coli O157:H7. The magnetic beads packaged by the Escherichia coli resistance O157:H7 monoclonal antibody is able to effectively gather Escherichia coli O157:H7 from samples of larger volumes, and then detect by a fluorescent quantitation PCR detection method; the detection sensitivity of Escherichia coli O157:H7 can be greatly increased, thus increasing the detection precision of Escherichia coli O157:H7.
Description
Technical field
The invention belongs to biological technical field, be specifically related to Chinese People's Anti-Japanese Military and Political College's enterobacteria O157:H7 monoclonal antibody and application thereof.
Background technology
It is one of food-safety problem and Sudden sensorineural hearing loss that the world today is on the increase that food borne pathogenic microorganism pollutes.Estimate according to WHO, the rate of failing to report of food origin disease is more than 95%, and the whole world has at least 2,000,000 people to die from food origin disease based on infectious diarrhea every year, and this kind of infection major part is because food and drinking-water are subject to caused by microbial contamination.In food borne pathogenic microorganism, enterohemorrhagic Escherichia coli (EHEC) occupies critical role.
EHEC mainly comprises O157:H7(and is also called Escherichia coli O 157: H7 or
e. colio157:H7), several serotype such as O26:H11 and O111, wherein O157:H7 is typical strain.Escherichia coli O 157: H7 infects can causing bleeding property colitis (Hemorrhagic colitis, HC), ecphyaditis, the serious gastrointestinal such as esophagostenosis and perforation of colon complication, hemolytic uremic syndrome (Hemolytic uremic syndrome also can be caused in children and the elderly, and thrombotic thrombocytopenic purpura (Thrombotic Thrombocytopenic purpura HUS), the severe complication such as TTP), severe one can cause death.Escherichia coli O 157: H7 resides in the enteron aisle of the domestic animals and fowls such as ox, sheep, pig, chicken for a long time, and ox is main reservoir host, young animal is easily caused to suffer from diarrhoea, and then pollute animal products, water source and farm crop etc., bring about great losses to agriculture-stock production, also grave danger is formed to the health of people simultaneously.Nineteen eighty-two, first this bacterium was found in the U.S., after this to distribute all over the world or local popular, 1996 at Osaka, Japan area happening and prevelence, patient exceedes ten thousand, dead 11 people, within 1999 ~ 2000 years, there occurs a lot of Diet_induced obesity O157:H7 event on the ground such as Jiangsu Province, China, Anhui, cause l77 people dead.The infection of Escherichia coli O 157: H7 has the features such as outbreak of epidemic trend, strong pathogenic and lethality and antibiotic therapy may aggravate one's illness, become global public health problem, worldwide health organization is also by Escherichia coli O 157: H7 is classified as new foodborne bacterial pathogens.The fluorescent quantitation method of existing detection Escherichia coli O 157: H7, detection sensitivity reaches 80CFU/ml, still can not meet the requirement of monitoring, the Escherichia coli O 157 polluted especially in the sample to which: when H7 is less, sample error and insufficient sensitivity high, cause recall rate not high.
Summary of the invention
The object of this invention is to provide the hybridoma cell strain of secretion Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody.
Another object of the present invention is to provide the Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody of above-mentioned hybridoma cell strain secretion, the high specificity of this monoclonal antibody, highly sensitive.
Another object of the present invention is to provide Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody at detection Escherichia coli O 157: the application in H7, can Escherichia coli O 157 effectively in enrichment testing sample: H7, combined with fluorescent quantitative PCR method, significantly improves sensitivity and accuracy.
Object of the present invention adopts following technical scheme to realize.
Produce a hybridoma cell strain 10G1A2 for Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody, its preserving number is CGMCC NO. 9999.
The present invention also provides a kind of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody of being secreted by described hybridoma cell strain 10G1A2.
The present invention also provides described Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody at detection Escherichia coli O 157: the application in H7.
The present invention also provides the immunomagnetic beads of described Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt and described immunomagnetic beads detecting Escherichia coli O 157: the application in H7.
In the present invention, the Escherichia coli O 157 in described immunomagnetic beads enrichment testing sample is adopted: H7, obtains pregnant solution; Employing fluorescence quantifying PCR method detects the Escherichia coli O 157 in described pregnant solution: H7.
In the present invention, in described enrichment testing sample, the method for Escherichia coli O 157: H7 is: added in testing sample by described immunomagnetic beads and adsorb, precipitate described immunomagnetic beads with magnetic frame, immunomagnetic beads described in wash-out, get elutriant as pregnant solution.
Compared with prior art, the present invention has following beneficial effect:
The full bacterial immunity of the present invention's Escherichia coli O 157: H7, uses Escherichia coli O 157 afterwards: H7 face grease polysaccharide selects monoclonal antibody specific, substantially increases this monoclonal antibody to Escherichia coli O 157: the specificity of H7 and sensitivity.
Compared with ordinary method, the magnetic bead of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt of the present invention can from the larger sample enrichment liquid of volume effective enrichment Escherichia coli O 157: H7, then adopt fluorescent quantitative PCR detection method, Escherichia coli O 157 can be improved greatly: the detection sensitivity of H7; In addition the magnetic bead of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt of the present invention can adsorb the Escherichia coli O 157 in pre-enrichment liquid specifically: H7, the interference of other miscellaneous bacterias to separation and Culture or fluorescence quantitative PCR detection can be got rid of, can Escherichia coli O 157 be improved: the accuracy that H7 detects.Because magnetic bead is coated with specific antibody, concentration effect and efficiency improve greatly, and it is combined by non covalent bond that magnetic bead is combined with bacterium, can not affect Escherichia coli O 157: the physiology and chemistry characteristic of H7.
Accompanying drawing explanation
The SDS-PAGE electrophorogram of Fig. 1 monoclonal antibody, wherein M-molecular weight Mark, 1-purifying Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 face grease Monoclonal Antibody against Polysaccharides.
Preservation information:
The biomaterial (strain) of ginseng Ju: 10G1A2;
Classification And Nomenclature: produce Escherichia coli O 157: the strain of H7 monoclonal antibody hybridoma cell;
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica;
Preserving number is CGMCC NO. 9999, and preservation date is: on November 18th, 2014.
Specific embodiments
0.05mol/L carbonate buffer solution (pH9.6): containing 0.05 mol/L Na
2cO
3with 0.05 mol/L NaHCO
3the aqueous solution, pH value is 9.6.
PBS damping fluid (0.01M, pH 7.4): get 137 mmol NaCl, 2.7 mmol KCl, 10 mmol Na
2hPO
4with 2 mmol KH
2pO4 is water-soluble, regulates pH to 7.4, is settled to 1L with water.
PBST damping fluid: adding final concentration in PBS damping fluid (0.01M, pH 7.4) is 0.05%(mass percentage concentration) tween 20.
Confining liquid: the PBST damping fluid containing mass concentration being 0.5% bovine serum albumin (BSA).
Sheep anti mouse HRP-IgG:HRP(horseradish peroxidase) sheep anti-mouse igg (abcam:ab6789) that marks.
Tmb substrate nitrite ion: comprise A liquid and B liquid.A liquid: be that to join 100 mL concentration be 0.1 mol/L, pH is be made in the phosphoric acid buffer of 6.0 for 3,3', 5,5'-tetramethyl benzidines (be abbreviated as TMB, the Zhengzhou four seasons chemical company) solution of 10 mg/mL by 1 mL concentration; The compound method of described concentration to be 0.1 mol/L, pH the be phosphoric acid buffer of 6.0 is: 0.1 mol/L biphosphate sodium water solution 87.7 mL and the 0.1 mol/L Sodium phosphate dibasic aqueous solution 12.3 mL mixes.B liquid: volumetric concentration is the H of 3%
2o
2the aqueous solution, 10 mL.A liquid and B liquid all need 4 DEG C of sealings, keep in Dark Place.During use every 10 mL A liquid add 50 μ L B liquid mixing use.
Stop buffer: the H of 2 mol/L
2sO
4solution.
Aminopterin-induced syndrome (A) stock solution compound method: get 1.76mg aminopterin-induced syndrome, add in 90mL ultrapure water, drip 1mol/L NaOH aqueous solution 0.5mL hydrotropy, until completely dissolved, add 1mol/L hydrochloric acid 0.5mL to neutralize, add ultrapure water and be settled to 100mL, 0.22um membrane filtration is degerming, packing in a small amount ,-20 DEG C of preservations.
Xanthoglobulin and thymidine (HT) stock solution compound method: get 136.1mg xanthoglobulin (H) and 38.8mg thymidine (T), add ultrapure water to 100mL, be placed in 45-50 DEG C of water-bath and make it to dissolve completely.0.22um membrane filtration is degerming, and packing in a small amount ,-20 DEG C of preservations, dissolve in 37 DEG C of water-baths before use.
L-glutaminate (LG) solution preparation method: get 2.92g L-glutaminate, be dissolved in 100mL ultrapure water, 0.22um membrane filtration is degerming, packing in a small amount ,-20 DEG C of preservations.
Dual anti-solution (mycillin solution) compound method: get penicillin (sodium salt) 1,000,000 unit and Streptomycin sulphate (vitriol) 1g, is dissolved in 100mL sterilizing ultrapure water, packing in a small amount ,-20 DEG C of preservations.
Perfect medium RPMI-1640: add final concentration (concentration expressed in percentage by volume) and be the LG solution of 1% and dual anti-solution in the incomplete RPMI-1640 substratum bought, add the new-born calve serum that final concentration (concentration expressed in percentage by volume) is 10%.
HT incubator: add the HT stock solution that final concentration (concentration expressed in percentage by volume) is 1% in perfect medium RPMI-1640
HAT substratum: adding final concentration (concentration expressed in percentage by volume) in perfect medium RPMI-1640 is the HT stock solution of 1% and the A stock solution of 1%.
The preparation of embodiment 1 Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody
The preparation of 1 antigen
e. colithe centrifugal 5min of O157:H7 nutrient solution 50mL, 10000rpm, gets bacterium mud, resuspended with physiological saline, adds the formaldehyde solution of bacteria suspension volume 0.5%, 37 DEG C of deactivation 24h, and after brine three times, adjustment bacterial concentration is 2.5 × 10
9individual/mL, obtains deactivation O157:H7 bacteria suspension, and-20 DEG C frozen for subsequent use.
Get deactivation O157:H7 bacteria suspension, use iNtRON Biotechnology company lipopolysaccharides to extract test kit LPS Extraction Kit(article No.: 17141), to extract Escherichia coli O 157: H7 face grease polysaccharide ,-20 DEG C frozen for subsequent use.
The foundation of 2 hybridoma cell strains
2.1 immune animal
Select female BAl BIc in 6 ~ 8 week age/c mouse 10.Every mouse peritoneal injection 0.2mL deactivation O157:H7 bacteria suspension (prepared by step 1), at interval of same dosage booster shots in 1 week once, immunity 5 times altogether, wherein last immunity is 3d before fusion, by tail vein injection same amount antigen booster immunization.
The preparation of 2.2 feeder cell
Carry out 24h before cytogamy, BALB/c mouse is extractd eyeball blood sampling, separation of serum, as negative control sera during antibody test; To be dislocated lethal mouse by neck simultaneously, in 75% alcohol, soak 10min, after belly is upwards fixing, aseptic abdominal cut skin, skin peeled off by tweezers, exposes peritonaeum, and cotton ball soaked in alcohol wiping peritonaeum is sterilized; With injector to inject 10mL HAT substratum to abdominal cavity, note avoiding penetrating intestinal tube, the right hand fixes syringe, makes syringe needle be retained in intraperitoneal, left hand holds cotton ball soaked in alcohol abdomen massage 1min gently, till the HAT substratum yellowing of the substratum repeatedly aspirating injection subsequently in inhalation syringe.The substratum of sucking-off at the centrifugal 10min of 1000r/min, abandon supernatant, with 5mL HAT substratum, sedimentation cell is suspended, use HAT substratum adjustment cell concn to reach 2 × 10
5individual/mL, obtains feeder cell suspension.Feeder cell suspension is added 96 orifice plates, and one, every hole (about 50 μ L), puts 37 DEG C, 5%CO
2cultivate in incubator.
The preparation of 2.3 myeloma cell SP2/0
Merge first two weeks and start recovery myeloma cell SP2/0(by Yangzhou University's veterinary college Infectious Diseases Lab present), enlarged culturing, guarantee during fusion that myeloma cell SP2/0 is in logarithmic phase, have good form, viable count is higher than 95%; Merge the same day, cannot be used up full RPMI-1640 substratum (Gibco article No.: 12633012) blown down gently from bottle wall by myeloma cell SP2/0, be collected in 50mL centrifuge tube, the centrifugal 10min of 1000r/min, supernatant discarded, add incomplete RPMI-1640 substratum 30mL, with the centrifugal supernatant discarded of method; Then by Cell resuspension in incomplete RPMI-1640 substratum 10mL, mixing, make myeloma cell SP2/0 suspension, and do viable count.
The preparation of 2.4 splenic lymphocyte
Female BAl BIc/c mouse in label taking topic 2.1 after last immune 3d, extracts eyeball blood sampling as positive serum; The lethal mouse of cervical dislocation, soaks 10min in 75% alcohol, puts on the dissection plate of super clean bench immediately, and belly upward, fix by four limbs.Aseptic taking-up spleen, the full RPMI-1640 substratum that cannots be used up washing for several times, and removes the reticular tissue of attachment removal with tweezers.Spleen is moved on 200 order micro-strainers, be placed in fresh incomplete RPMI-1640 substratum, grind gently with grinding rod, spleen is made single cell suspension, collect splenocyte suspension, the centrifugal 10min of 1000r/min, extracting spleen cell cannots be used up full RPMI-1640 centrifuge washing 1 ~ 2 time, is finally resuspended in by splenocyte in the incomplete RPMI-1640 of 10mL, mixing, obtain splenic lymphocyte suspension, and for subsequent use after doing viable count.
2.5 cytogamy
Merge according to a conventional method, get the myeloma cell SP2/0 after counting and be mixed in 50mL centrifugal bottle in the ratio that the ratio of cell count is 1:5 ~ 1:10 with splenic lymphocyte, fully mix, the centrifugal 10min of 1000r/min, abandons supernatant.Touch at the bottom of centrifugal bottle with palm, the cell of precipitation is broken up.To put into 40 ~ 41 DEG C of water-baths bottom centrifugal bottle, rotation limit, limit adds the PEG-4000 1mL merged, and adds in 1min, continues to rotate to add incomplete RPMI-1640 substratum 15mL simultaneously, and add in 90s, from slow to fast, front 5s adds 1mL.After room temperature leaves standstill 10min, the centrifugal 10min of 1000r/min, abandons supernatant, adds HAT substratum (HAT substratum has selectivity to hybridoma so be sometimes called HAT selective medium), suspension hybridoma.Hybridoma suspension is assigned to the 96 porocyte culture plates adding feeder cell, is placed in 37 DEG C, 5%CO
2incubator in cultivate.After 5d, to swap out half substratum with the HAT substratum of fresh preheating; To swap out HAT substratum with the HT substratum of preheating after 10d; Observe the growing state of hybridoma, when its cell culture supernatant turns yellow or clone is distributed to more than 1/10 of hole floorage, draw appropriate cell culture supernatant and carry out antibody test.
2.6 screening
2.6.1 the selection of the best antigen coated amount of indirect ELISA and serum dilution
The positive serum (title 2.4) of immune mouse eyeball blood sampling preparation before merging, as positive control during screening; The serum of Non immune mouse eyeball blood sampling preparation as negative control (title 2.2), hole of simultaneously returning to zero using PBST damping fluid as blank.Enzyme plate carries out square formation test, and determine that the best bag of detectable antigens is by the best weaker concn of concentration and the positive, negative control, concrete steps are:
(1) bag quilt: detectable antigens (in the present embodiment title 1 deactivation O157:H7 bacteria suspension) 0.05mol/L carbonate buffer solution (pH9.6) dilutes 10 times, 100 times, 1000 times, 10000 times respectively, each extent of dilution adds row from left to right, 100 μ L/ holes, 37 DEG C of effect rearmounted 4 DEG C of 2h spend the night;
(2) wash: discard the liquid in hole, PBST damping fluid washes 3 times, washs 5min at every turn, pats dry;
(3) close: every hole adds 150 μ L confining liquids, 37 DEG C of effect 2h;
(4) wash: method is the same;
(5) primary antibodie is added: primary antibodie (positive serum prepared by title 2.4) dilutes 100 times, 1000 times, 10000 times respectively with PBST damping fluid, and each extent of dilution adds a line, 100 μ L/ holes, 37 DEG C of effect 1h;
(6) wash: method is the same;
(7) ELIAS secondary antibody: the sheep anti mouse HRP-IgG adding working concentration (1:3000 dilution), 100 μ L/ holes, hatch 1h for 37 DEG C, and washing is the same;
(8) develop the color: add tmb substrate nitrite ion 100 μ L/ hole, 37 DEG C of reaction 10 ~ 15 min;
(9) termination reaction: add stop buffer, 50 μ L/ holes, termination reaction, measures each hole OD
450.
Result judges: with positive serum OD
450be worth about 1.0, negative serum OD
450value is less than 0.2, and positive serum OD
450value/negative serum OD
450(P/N value) is the best effort concentration of detectable antigens time maximum, and the serum dilution of this some correspondence is best serum dilution.Result: the best bag of detectable antigens (in title 1 deactivation O157:H7 bacteria suspension) is 1 × 10 by concentration
6individual/mL, the optimum dilution degree of positive serum is 1:1000; Negative optimum dilution degree is 1:1000.
Preparation is coated with the enzyme plate of detectable antigens:
(1) bag quilt: detectable antigens with best effort concentration bag by 96 hole polystyrene enzyme plates, 100 μ L/holes, 37 DEG C effect rearmounted 4 DEG C of 2h spend the night;
(2) wash: abandon coating buffer, wash 3 times with PBST damping fluid, wash 5min at every turn, pat dry;
(3) close: every hole adds 150 μ L confining liquids and closes, 37 DEG C of effect 2h;
(4) wash: according to the washing of step (2) method, obtain the enzyme plate being coated with detectable antigens ,-20 DEG C save backup.
Note: during indirect ELISA below detects, adopts the enzyme plate being coated with detectable antigens, and positive, negative serum all gets optimum dilution degree.
2.6.2 indirect ELISA test
In order to screen hybridoma cell strain, in label taking topic 2.5, cell culture supernatant 100 μ L adds and is coated with in the enzyme plate of detectable antigens, sets up the positive, feminine gender and blank simultaneously, hatches 1h for 37 DEG C, washs; Add the sheep anti mouse HRP-IgG of working concentration (1:3000 dilution), 100 μ L/ holes, hatch 1h for 37 DEG C, and the same washing pats dry; Add tmb substrate nitrite ion 100 μ L/ hole, 37 DEG C of reaction 10 ~ 15 min; Add stop buffer 50 μ L/ hole, termination reaction.Measure each hole OD
450.Judging criterion: the OD treating verify
450the OD of/negative hole
450>=2.1 can be judged to and treat that the strain of verify inner cell is for positive, can carry out next step test.
The cloning of 2.7 hybridomas
Limiting dilution assay is adopted to carry out cloning to the cell strain of double test positive.Mouse feeder cells is prepared before clone.Treat viable cell accurate counting in clone hole, become 20 cells/mL substratum with HT substratum 10 doubling dilution, join and be covered with in 96 porocyte culture plates of feeder cell, make every hole have 1 cell in theory, be placed in 37 DEG C, 5%CO
2incubator in cultivate.When Growth of Cells to hole floorage 1/10 time detect, strong positive hole is cloned by the same way again, so repeats 3 ~ 4 times, until positive rate reaches 100%, obtains the hybridoma cell strain of preliminary screening.By the hybridoma cell strain enlarged culturing of preliminary screening, freeze-stored cell, retains supernatant liquor simultaneously, adopts indirect ELISA to measure antibody titer, chooses higher some hybridoma cell strains of tiring.
The screening of 2.8 hybridoma cell strains
Get Escherichia coli O 157: H7 face grease polysaccharide carries out SDS-PAGE electrophoresis, and carry out immuning hybridization respectively with higher some hybridoma cell strain culture supernatant of tiring in title 2.7, choose the hybridoma cell strain of the best clone of immune response effect as secretion Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody, called after 10G1A2.
The specificity of 2.9 monoclonal antibodies
Strain subject comprises
e. Colio157:H7, EHEC O26:H11, EHEC O111, EPEC O127:H6, ETEC O148:H28,
e. colio25,
e. colio55,
e. colio78,
e. colio103,
e. colio138,
e. colio139,
e. colio141,
e. colio145,
e. colik88,
e. colik99,
e. colibL21,
e. colitop10, streptococcus aureus, bacillus dysenteriae, Klebsiella Pneumoniae, Pseudomonas aeruginosa, Aeromonas hydrophila, Vibrio parahaemolyticus, Salmonellas, Bacillus cereus, subtilis, citrobacter freundii, pasteurella multocida, Riemerlla anatipestifer, haemophilus parasuis, lactobacillus, Brucella, Listeria monocytogenes, fowl mycobacterium, suis, clostridieum welchii, Clostridium botulinum, fowl Podbielniak bacterium.By each strain subject according to method coated elisa plate in title 2.6.1, then carry out indirect ELISA detection, investigate the specificity of hybridoma cell strain 10G1A2 nutrient solution supernatant.Result shows, hybridoma cell strain 10G1A2 nutrient solution supernatant only with
e. Colithere is positive reaction in O157:H7, other each bacterium are negative reaction and (treat the OD of verify
450the OD of/negative hole
450>=2.1 are judged to be the positive).
3. the preparation of monoclonal antibody and character
The preparation of 3.1 monoclonal antibodies
Select the female BAl BIc/c mouse of more than body weight 20g in 8 ~ 10 week age, abdominal injection whiteruss, only, within 1 week, pneumoretroperitoneum injects hybridoma cell strain 10G1A2(2 × 10 to 0.5mL/
6~ 5 × 10
6cell/only), extract ascites when animal belly obviously increases, the centrifugal 10min of 3000r/min, gets supernatant liquor, and-20 DEG C are frozen for subsequent use.
Utilize HiTrap
tMprotein G affinity column (Sangon Biotech (Shanghai) Co., Ltd., article No. SD6606) carries out purifying to ascites supernatant liquor.In purge process, agents useful for same is chemical pure entirely, and the solvent of all solution is deionized water entirely, prepares rear use 0.45 μm of membrane filtration, the centrifugal 3min of 10000 r/min before ascites supernatant liquor purifying.Purifying concrete operation step is as follows:
(1) remove the stopper of chromatography column top, with shifting coupling, syringe and chromatography column are connected together.
(2) snap-off of chromatography column afterbody is removed.
(3) 10ml binding buffer(biotechnology (Shanghai) limited-liability company is drawn, article No. BSP048-2 with syringe), " dripping dripping " formula is injected, and enters chromatography column, injection rate 1ml/min with air-prevention.
(4) draw ascites supernatant liquor to be purified, be injected in chromatography column.
(5) 10ml binding buffer is drawn in chromatography column, wash-out foreign protein.
(6) with 5 ml elution buffer(0.1M citric acid solutions) wash post, prepare 1.5ml collection tube 5, often manage and add the Tris-HCl damping fluid 200 μ L that concentration is 1M, pH9.0 in advance, often 1ml collected by pipe.
After ascites supernatant liquor purifying, obtain purifying Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody, through Nanodrop1000(Thermo) to record protein content be 1.027mg/mL,-20 DEG C save backup, and with SDS-PAGE electrophoresis observation purification effect, visible target protein purity is very high, and main stripe size is slightly larger than 200kDa, as Fig. 1.
3.2 antibody titers measure
Adopt the enzyme plate (preparing in title 2.6.1) being coated with detectable antigens, ascites supernatant liquor cleer and peaceful on hybridoma cell strain 10G1A2 nutrient solution is cooked doubling dilution, adopt indirect ELISA method to detect and tire, nutrient solution supernatant is tired as 1:2430; Titer of ascites is >1:81000.
3.3 subclass measure
Subclass of antibody is detected according to Thermo scientific company monoclonal antibody subgroup identification test kit process specifications.The monoclonal antibody subclass that hybridoma cell strain 10G1A2 produces is IgG.
The affinity costant of 3.4 monoclonal antibodies
The affinity costant Kd=5.69*10 of the Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody that hybridoma cell strain 10G1A2 produces
-8.
Embodiment 2 prepares the immunomagnetic beads of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt
1. get 165 u L bead suspension (containing magnetic bead 5mg, be purchased from Life technologies, article No. 1309004) put in centrifuge tube, magnetic bead is precipitated (hereinafter referred to as magnetic force precipitation) with magnetic frame, concrete operation step is: be put in by centrifuge tube on magnetic frame, become after clarification until supernatant liquid and discard supernatant, retain the magnetic bead of bottom, remove magnetic frame;
2. add 1mL magnetic bead coupling buffer, resuspended magnetic bead, magnetic force precipitates;
3. successively add the magnetic bead in the purifying Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody (containing antibody 100ug) prepared in 75.7 μ L magnetic bead coupling buffers, 50 μ L embodiments 1 and step 2 after magnetic force precipitation, mixing, add 100 μ L treatment solutions again, mixing, put after 37 DEG C of shaking tables hatch 18h, magnetic force precipitates;
4. add the resuspended magnetic bead of 1mL confining liquid A, after 37 DEG C of shaking tables hatch 1h, magnetic force precipitates;
5. add 1mL storing solution, after vortex oscillation 5-10s, magnetic force precipitates; Repeat this operation once; Add 240uL storing solution mixing (overall solution volume 250uL), obtain the immunomagnetic beads of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt, in 4 DEG C of Refrigerator stores.
The borate buffer solution of magnetic bead coupling buffer: 1mol/L, its pH is 7.4;
Treatment solution: adding final concentration in the borate buffer solution (pH is 7.4) of 1mol/L is the ammonium sulfate of 3mol/L;
Confining liquid A:0.5g bovine serum albumin (BSA) is dissolved in the PBS damping fluid (0.02M, pH are 7.4) of 100mL;
Storing solution: 0.1g BSA is dissolved in 100mL PBS damping fluid (0.02M, pH are 7.4);
PBS damping fluid (0.02M, pH are 7.4): containing Na
2hPO
42.9g/L, KH
2pO
4the aqueous solution of 0.2g/L, NaCl 8.0g/L, KCl 0.2g/L, its pH is 7.4.
Embodiment 3 utilizes the immunomagnetic beads of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt to detect Escherichia coli O 157: H7
(1) enterorrhagia Bacillus coil 0157 in meat, meat product and other food is imported and exported according to SN/T0793-2010(: H7 detection method) carry out the pre-increasing bacterium of testing sample: testing sample is added in EC meat soup, under 42 DEG C of conditions, cultivate 18-24h, obtain enrichment liquid;
(2) in centrifuge tube, add the enrichment liquid 20-30mL that step (1) obtains, then add immunomagnetic beads (prepared by embodiment 2) the 50 μ L of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt, hatch 1h for 37 DEG C;
(3) magnetic bead is washed: be put in by centrifuge tube on magnetic frame and precipitated by magnetic bead, supernatant discarded after 3min, it is slowly resuspended to add 30mL PBS damping fluid (0.02M, pH are 7.4), is precipitated by magnetic bead, supernatant discarded with magnetic frame.Repeated washing like this 3 times;
(4) in the immunomagnetic beads after washing, adding 1mL concentration is 50mmol/L glycine solution, is interrupted vibration wash-out immunomagnetic beads 1h, is put in by centrifuge tube on magnetic frame, when immunomagnetic beads is all adsorbed on tube wall, takes out supernatant liquor, i.e. elutriant.This elutriant is Escherichia coli O 157: H7 pregnant solution.From Escherichia coli O 157: extracting directly sample DNA H7 pregnant solution, then detect in testing sample whether there is Escherichia coli O 157 with fluorescence quantitative PCR method: H7.
(5) fluorescence quantitative PCR detection
Sample thief DNA, use TaKaRa PCR kit for fluorescence quantitative (DRR390A), preparation reaction system is as follows: Premix Ex Taq (2 ×) 10ul, upstream primer (10uM) 0.4ul, downstream primer (10uM) 0.4ul, Taqman probe 0.8ul, ROX Reference Dye II (ROX reference dye II, 50 ×) () 0.2ul, template (sample DNA) 2ul, dH
2o 6.2ul.
Upstream primer (SEQ ID NO:1): 5 '-GCACTAAAAGCTTGGAGCAGTTC-3 '.
Downstream primer (SEQ ID NO:2): 5 '-AACAATGGGTCAGCGGTAAGGCTA-3 '.
Taqman probe: 5 '-FAM-CGTTGGCGAGGACC-TAMRA-3 '.FAM is reporter fluorescence group, TAMRA quenching fluorescence group.The nucleotide sequence of Taqman probe is as shown in SEQ ID NO:3.
The 20ul reaction system prepared, loads in quantitative fluorescent PCR reaction tubes, builds pipe lid, centrifugal, confirms to react without putting into ABI7500 quantitative real time PCR Instrument after drum in reaction tubes.
Response procedures is as follows: the first step: 95 DEG C of 30s; Second step 95 DEG C of 5s, 60 DEG C of 34s, repeat 40 circulations; Fluorescent signal is collected at 60 DEG C in reaction process.Net result: be positive with CT value≤35, needs revision test to verify as CT value equals 35; CT value > 35 is negative reaction.
Embodiment 4 utilizes the immunomagnetic beads of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt to detect the sensitivity of Escherichia coli O 157: H7
Prepare Escherichia coli O 157: H7 bacterium liquid, 10 times of doubling dilutions, coating sorbitol-MacConkey agar flat board (with reference to importing and exporting enterorrhagia Bacillus coil 0157 in meat, meat product and other food: H7 detection method) is to determine colony number.
Choose 10
1, 10
2, 10
3, 10
4, 10
5cFU/ml dilutes bacterium liquid, adopts method in embodiment 3 to carry out Escherichia coli O 157 respectively: H7 detects, and investigates the sensitivity of the inventive method.Result is 10
1cFU/ml-10
5cFU/ml respectively dilutes the equal test positive of bacterium liquid.Therefore, within the inventive method Sensitivity can reach 10CFU/mL.
Fluorescent quantitative PCR detection method is to Escherichia coli O 157: the Sensitivity of H7 is 80CFU/ml(Baoguang Li, Jin-Qiang Chen. Real-Time RCR Methodology for Selective Detection of Viable Escherichia coli O157:H7 Cells by Targeting Z3276 as a Genetic Marker [J].
applied and Environmental Microbiology, 2012,78 (15): 5297-5304).
Embodiment 5 utilizes the immunomagnetic beads of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt to detect the accuracy of Escherichia coli O 157: H7
1. interference test: (bacterial concentration is 10 to each 25ml to prepare the bacteria suspension (selecting bacterial strain to be non-O157:H7 bacterial strain in example 1 title 2.9) of each interference bacterial strain respectively
5/ mL), add Escherichia coli O 157: H7 and be about 1-10, carry out according to the operation steps in embodiment 3.Result is the positive.
Do not add Escherichia coli O 157: the bacteria suspension (selecting bacterial strain to be non-O157:H7 bacterial strain in example 1 title 2.9) of each interference bacterial strain of H7 is and detects feminine gender.
2. analog detection test: the random contact pollution modes that simulating nature infects, the random concentration (1CFU/g-10 of artificial preparation
3cFU/g) Escherichia coli O 157: H7 pollutes 100 parts, sample.Detect according to SN/T0793-2010, the inventive method (in embodiment 3 method) and fluorescence quantifying PCR method respectively.The verification and measurement ratio of discovery SN/T0793-2010 method (importing and exporting enterorrhagia Bacillus coil 0157 in meat, meat product and other food: H7 detection method) is 57%, and the inventive method recall rate is 98%, and fluorescence quantifying PCR method recall rate is 69%.Show that the present invention is by immunomagnetic beads and quantitative fluorescent PCR method for combined use, effectively the Escherichia coli O 157 in sample: H7 can be carried out enrichment, combined with fluorescent quantitative PCR method, substantially increases recall rate.The verification and measurement ratio of detection method is not only significantly higher than SN/T0793-2010 method, and is significantly higher than fluorescence quantifying PCR method.
SEQUENCE LISTING
<110> Propagation and Food Test Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau
<120> Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody and application thereof
<130> 20141229
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> artificial
<220>
<223> upstream primer
<400> 1
gcactaaaag cttggagcag ttc 23
<210> 2
<211> 24
<212> DNA
<213> artificial
<220>
<223> downstream primer
<400> 2
aacaatgggt cagcggtaag gcta 24
<210> 3
<211> 14
<212> DNA
<213> artificial
<220>
The nucleotide sequence of <223> Taqman probe
<400> 3
cgttggcgag gacc 14
Claims (7)
1. produce a hybridoma cell strain 10G1A2 for Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody, its preserving number is CGMCC NO. 9999.
2. a Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody of being secreted by hybridoma cell strain 10G1A2 described in claim 1.
3. Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody described in claim 2 is at detection Escherichia coli O 157: the application in H7.
4. the immunomagnetic beads of Chinese People's Anti-Japanese Military and Political College enterobacteria O157:H7 monoclonal antibody bag quilt described in claim 2.
5. immunomagnetic beads described in claim 4 is at detection Escherichia coli O 157: the application in H7.
6. apply according to claim 5, it is characterized in that adopting the Escherichia coli O 157 in immunomagnetic beads enrichment testing sample described in claim 4: H7, obtains pregnant solution; Employing fluorescence quantifying PCR method detects the Escherichia coli O 157 in described pregnant solution: H7.
7. apply according to claim 6, it is characterized in that the method for Escherichia coli O 157: H7 in described enrichment testing sample for: immunomagnetic beads described in claim 4 is added in testing sample and adsorbs, described immunomagnetic beads is precipitated with magnetic frame, immunomagnetic beads described in wash-out, gets elutriant as pregnant solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410832762.8A CN104450628B (en) | 2014-12-29 | 2014-12-29 | Anti- Escherichia coli O 157:H7 monoclonal antibodies and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410832762.8A CN104450628B (en) | 2014-12-29 | 2014-12-29 | Anti- Escherichia coli O 157:H7 monoclonal antibodies and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104450628A true CN104450628A (en) | 2015-03-25 |
| CN104450628B CN104450628B (en) | 2017-06-06 |
Family
ID=52897387
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201410832762.8A Active CN104450628B (en) | 2014-12-29 | 2014-12-29 | Anti- Escherichia coli O 157:H7 monoclonal antibodies and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104450628B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609398A (en) * | 2018-11-14 | 2019-04-12 | 自贡市疾病预防控制中心 | The immuno magnetic cell separation and detection method of Albert's Escherichia |
| CN113281137A (en) * | 2021-02-26 | 2021-08-20 | 中国计量科学研究院 | Method for removing magnetic beads of bacteria sample to be detected after enrichment of immunomagnetic beads |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990068868A (en) * | 1998-02-02 | 1999-09-06 | 김강권 | Monoclonal Antibodies and Uses for Detection of E. coli O157: H7 |
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
| CN103439496A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Escherichia coli O157:H7 enrichment and rapid detection method |
| CN103898065A (en) * | 2013-11-09 | 2014-07-02 | 中华人民共和国吉林出入境检验检疫局 | Hybridoma cell line of Escherichia-coli O157:H7 resistant monoclonal antibody |
-
2014
- 2014-12-29 CN CN201410832762.8A patent/CN104450628B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990068868A (en) * | 1998-02-02 | 1999-09-06 | 김강권 | Monoclonal Antibodies and Uses for Detection of E. coli O157: H7 |
| CN101504416A (en) * | 2008-05-20 | 2009-08-12 | 湖南工业大学 | Novel methods for detecting bacillus coli by gold-coating magnetic granule in-situ initiating high-sensibility chemical luminescence |
| CN103439496A (en) * | 2013-08-13 | 2013-12-11 | 南昌大学 | Escherichia coli O157:H7 enrichment and rapid detection method |
| CN103898065A (en) * | 2013-11-09 | 2014-07-02 | 中华人民共和国吉林出入境检验检疫局 | Hybridoma cell line of Escherichia-coli O157:H7 resistant monoclonal antibody |
Non-Patent Citations (2)
| Title |
|---|
| RILEY LW等: "Hemorrhagic colitis associated with a rare Escherichia coli serotype", 《N ENGL J MED》 * |
| 杨书豪等: "抗出血性大肠杆菌O157：H7单克隆抗体的制备及鉴定", 《中国人兽共患病杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609398A (en) * | 2018-11-14 | 2019-04-12 | 自贡市疾病预防控制中心 | The immuno magnetic cell separation and detection method of Albert's Escherichia |
| CN113281137A (en) * | 2021-02-26 | 2021-08-20 | 中国计量科学研究院 | Method for removing magnetic beads of bacteria sample to be detected after enrichment of immunomagnetic beads |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104450628B (en) | 2017-06-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jeong et al. | Pathotyping avian pathogenic Escherichia coli strains in Korea | |
| CN105175518B (en) | The bacteriocin and preparation method thereof that bacillus coagulans FM603 is generated | |
| CN102816850A (en) | Method for simultaneously detecting salmonella typhimurium, escherichia coli O157:H7 and listeria monocytogenesis | |
| Lupindu | Isolation and characterization of Escherichia coli from animals, humans, and environment | |
| CN102382888B (en) | Immunomagnetic bead -FQ-PCR detection method for Shewanella putrefaciens | |
| CN112029696B (en) | Edwardsiella piscicola derived from turbot and application thereof | |
| Breum et al. | Prevalence of Escherichia coli O157 and verocytotoxin producing E. coli (VTEC) on Danish beef carcasses | |
| Bi et al. | A novel SDS rinse and immunomagnetic beads separation combined with real-time loop-mediated isothermal amplification for rapid and sensitive detection of Salmonella in ready-to-eat duck meat | |
| CN104450628A (en) | Escherichia coli resistance O157:H7 monoclonal antibody and application thereof | |
| CN101381777A (en) | Listeria monocytogenes detection kit and detection method thereof | |
| CN103952487B (en) | Catch-dual real-time fluorescence PCR detection method of the magnetic of campylobacter jejuni and salmonella | |
| Chon et al. | Advanced methods for isolating from and confirming Campylobacter spp. in milk and dairy products | |
| CN102721812A (en) | Indirect ELISA (enzyme-linked immuno-sorbent assay) kit for detecting nephropathogenic avian infectious bronchitis virus and antibody thereof | |
| CN103160606A (en) | LAMP (loop-mediated isothermal amplification) detection kit of vibrio cholerae and detection method thereof | |
| CN101535811B (en) | For the method for in-vitro diagnosis PVL-producing staphylococcus aureus | |
| AU2020103778A4 (en) | Primer Set for Detection of Streptococcus agalactiae, Detection Kit and Multiplex PCR Detection Method | |
| CN104231076B (en) | The therapeutic monoclonal antibodies of anti-coli-infection, produce hybridoma cell strain of the monoclonal antibody and application thereof | |
| Xu et al. | Simultaneous detection of five pathogenic Vibrio species in seafood by a multiplex polymerase chain reaction coupled with high performance liquid chromatography assay | |
| CN108018228B (en) | Escherichia coli O157H 7 low virulent strain and application thereof | |
| CN102286630A (en) | Listeria monocytogenes (LM) immuno-capture and polymerase chain reaction (IC-PCR) detection reagent kit and preparation method | |
| RU2460801C1 (en) | Method for detecting sanitary indicator bacteria and starter culture in meat products | |
| Foroohi et al. | Evaluation and optimization of a loop-mediated isothermal amplification (LAMP) assay for the detection of Salmonella enterica serovar Typhi isolated from food samples | |
| Baker | Shiga Toxin-Producing Escherichia coli (STEC) Detection Strategies with Formalin-Fixed STEC Cells | |
| CN116925211A (en) | Salmonella typhimurium-resistant antibody, immunomagnetic beads marked by Salmonella typhimurium-resistant antibody and application of salmonella typhimurium-resistant antibody | |
| Khudhir | Immunomagnatic separation of E. coli O157: H7 from raw milk and locally produced soft cheese in Baghdad City |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |