CN104450628B - Anti- Escherichia coli O 157:H7 monoclonal antibodies and its application - Google Patents
Anti- Escherichia coli O 157:H7 monoclonal antibodies and its application Download PDFInfo
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Abstract
The present invention provides anti-Escherichia coli O 157:H7 monoclonal antibodies and its application, belong to biological technical field.Produce anti-Escherichia coli O 157:The hybridoma cell strain 10G1A2 of H7 monoclonal antibodies, its preserving number is CGMCC NO.9999.The present invention also provides a kind of anti-Escherichia coli O 157 secreted by the hybridoma cell strain 10G1A2:H7 monoclonal antibodies, coated immunomagnetic beads and the immunomagnetic beads are in detection Escherichia coli O 157:Application in terms of H7.The anti-Escherichia coli O 157 of the present invention:The coated magnetic bead of H7 monoclonal antibodies effectively can be enriched with Escherichia coli O 157 from the larger sample enrichment liquid of volume:H7, then using fluorescent quantitative PCR detection method, can greatly improve Escherichia coli O 157:The detection sensitivity of H7, improves Escherichia coli O 157:The accuracy of H7 detections.
Description
Technical field
The invention belongs to biological technical field, and in particular to anti-Escherichia coli O 157:H7 monoclonal antibodies and its application.
Background technology
Food borne pathogenic microorganism pollution growing number of food-safety problem and sudden public is defended in the world today
Make trouble one of part.Estimate that more than 95%, at least 2,000,000 people die from the rate of failing to report of food origin disease every year in the whole world according to WHO
Food origin disease based on infectious diarrhea, this kind of infection major part is because food and drinking-water are by caused by microorganism pollution.
In food borne pathogenic microorganism, EHEC（EHEC）Occupy critical role.
EHEC mainly includes O157:H7（It is also called Escherichia coli O 157:H7 orE. coliO157:H7）, O26:H11 and
Several serotypes such as O111, wherein O157:H7 is typical strain.Escherichia coli O 157:H7 infection can causing bleeding property colitis
(Hemorrhagic colitis, HC), appendicitis, the serious gastrointestinal complication such as lemostenosis and perforation of colon, in children
With can also cause hemolytic uremic syndrome (Hemolytic uremic syndrome, HUS) and thrombotic blood in the elderly
The severe complications such as platelet reduction property purpura (Thrombotic Thrombocytopenic purpura, TTP), severe one can draw
Play death.Escherichia coli O 157:H7 is resided 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, easily causes young animal diarrhoea, and then pollutes animal products, water source and crops etc., is made to agriculture-stock production
Into massive losses, while also the health to people constitutes grave danger.Nineteen eighty-two, the bacterium was found in the U.S. first, hereafter alive
Boundary various regions distribute or place is popular, exceed ten thousand, dead 11 people, 1999 ~ 2000 in Osaka, Japan area happening and prevelence, patient within 1996
Year there occurs a lot of Diet_induced obesity O157 on Jiangsu Province, China, Anhui and other places:H7 events, cause l77 people dead.Escherichia coli
O157:The infection of H7 may aggravate disease with outbreak of epidemic trend, strong pathogenic and lethal and antibiotic therapy
The features such as feelings, it has also become global public health problem, worldwide health organization is also by Escherichia coli O 157:H7 is classified as newly
Foodborne bacterial pathogenses.Existing detection Escherichia coli O 157:The fluorescent quantitation method of H7, detection sensitivity reaches 80CFU/ml,
Still the requirement of monitoring, the Escherichia coli O 157 for especially polluting in the sample can not be met:When H7 is less, sample error and
Insufficient sensitivity is high, causes recall rate not high.
The content of the invention
It is an object of the invention to provide the anti-Escherichia coli O 157 of secretion:The hybridoma cell strain of H7 monoclonal antibodies.
It is a further object of the present invention to provide the anti-Escherichia coli O 157 of above-mentioned hybridoma cell strain secretion:H7 monoclonals resist
Body, the high specificity of the monoclonal antibody, sensitivity are high.
Another object of the present invention is to provide anti-Escherichia coli O 157:H7 monoclonal antibodies are in detection Escherichia coli O 157:
Application in terms of H7, can effectively be enriched with the Escherichia coli O 157 in testing sample:H7, combined with fluorescent quantitative PCR method, significantly
Improve sensitivity and accuracy.
The purpose of the present invention adopts the following technical scheme that realization.
One kind produces anti-Escherichia coli O 157:The hybridoma cell strain 10G1A2 of H7 monoclonal antibodies, its preserving number is
CGMCC NO. 9999。
The present invention also provides a kind of anti-Escherichia coli O 157 secreted by the hybridoma cell strain 10G1A2:H7 monoclonals
Antibody.
The present invention also provides the anti-Escherichia coli O 157:H7 monoclonal antibodies are in detection Escherichia coli O 157:H7 aspects
Application.
The present invention also provides the anti-Escherichia coli O 157:The coated immunomagnetic beads of H7 monoclonal antibodies and the immune magnetic
Pearl is in detection Escherichia coli O 157:Application in terms of H7.
In the present invention, using the Escherichia coli O 157 in immunomagnetic beads enrichment testing sample:H7, is enriched with
Liquid；Escherichia coli O 157 in the pregnant solution is detected using fluorescence quantifying PCR method:H7.
In the present invention, Escherichia coli O 157 in the enrichment testing sample:The method of H7 is：The immunomagnetic beads is added
To enter adsorbed in testing sample, the immunomagnetic beads is precipitated with magnetic frame, elute the immunomagnetic beads, take eluent as richness
Liquid collecting.
Compared with prior art, the present invention has the advantages that：
Present invention Escherichia coli O 157:The full bacterial immunities of H7, use Escherichia coli O 157 afterwards:H7 surfaces lipopolysaccharides selects spy
Specific monoclonal antibodies, substantially increase the monoclonal antibody to Escherichia coli O 157:The specificity of H7 and sensitivity.
Compared with conventional method, anti-Escherichia coli O 157 of the invention:The coated magnetic bead of H7 monoclonal antibodies can be from volume
Escherichia coli O 157 is effectively enriched with larger sample enrichment liquid:H7, then using fluorescent quantitative PCR detection method, Ke Yiji
Big raising Escherichia coli O 157:The detection sensitivity of H7；In addition anti-Escherichia coli O 157 of the invention:H7 monoclonal antibodies are coated with
Magnetic bead can specifically adsorb Escherichia coli O 157 in pre- enrichment liquid:H7, can exclude other miscellaneous bacterias to being separately cultured or glimmering
The interference of Fluorescent Quantitative PCR detection, can improve Escherichia coli O 157:The accuracy of H7 detections.Because magnetic bead is coated with specificity
Antibody, concentration effect and efficiency are greatly improved, and magnetic bead is combined with bacterium to be combined by non-covalent bond, does not interfere with Escherichia coli
O157:The physiology and biochemical characteristic of H7.
Brief description of the drawings
The SDS-PAGE of Fig. 1 monoclonal antibodies, wherein M- molecular weight Mark, 1- purify anti-Escherichia coli O 157:
H7 face grease Monoclonal Antibody against Polysaccharides.
Preservation information：
Join the biomaterial of Ju（Strain）：10G1A2；
Classification And Nomenclature：Produce Escherichia coli O 157:The strain of H7 monoclonal antibody hybridoma cells；
Depositary institution：China Committee for Culture Collection of Microorganisms's common micro-organisms center（CGMCC）, address：Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica；
Preserving number is CGMCC NO. 9999, and preservation date is：On November 18th, 2014.
Specific embodiment
0.05mol/L carbonate buffer solutions（pH9.6）：Contain 0.05 mol/L Na2CO3With 0.05 mol/L NaHCO3
The aqueous solution, pH value is 9.6.
PBS（0.01M, pH 7.4）：Take 137 mmol NaCl, 2.7 mmol KCl, 10 mmol Na2HPO4
With 2 mmol KH2PO4 is dissolved in water, adjusts pH to 7.4, and 1L is settled to water.
PBST buffer solutions：In PBS（0.01M, pH 7.4）Middle addition final concentration of 0.05%（Quality percentage is dense
Degree）Tween-20.
Confining liquid：It is 0.5% bovine serum albumin(BSA) containing mass concentration（BSA）PBST buffer solutions.
Sheep anti mouse HRP-IgG: HRP（Horseradish peroxidase）The sheep anti-mouse igg of mark（abcam：ab6789）.
Tmb substrate nitrite ion：Including A liquid and B liquid.A liquid：By the 3,3' that 1 mL concentration is 10 mg/mL, 5,5'- tetramethyls
Benzidine（Be abbreviated as TMB, the Zhengzhou four seasons chemical company）It is that 0.1 mol/L, pH is 6.0 that solution is added to 100 mL concentration
It is made into phosphate buffer；The concentration is that the compound method of the phosphate buffer that 0.1 mol/L, pH is 6.0 is：0.1 mol/
The mL of the L biphosphates sodium water solution 87.7 and mL of the 0.1 mol/L disodium hydrogen phosphates aqueous solution 12.3 mixing.B liquid：Volume is dense
Spend the H for 3%2O2The aqueous solution, 10 mL.A liquid and B liquid are both needed to 4 DEG C of sealings, keep in dark place.Every 10 mL A liquid plus 50 μ L when using
B liquid is mixed to be used.
Terminate liquid：The H of 2 mol/L2SO4Solution.
Aminopterin-induced syndrome（A）Storage liquid making method：1.76mg aminopterin-induced syndromes are taken, is added in 90mL ultra-pure waters, 1mol/ is added dropwise
L NaOH aqueous solution 0.5mL hydrotropies, until completely dissolved, plus 1mol/L hydrochloric acid 0.5mL is neutralized, plus ultra-pure water is settled to
100mL, 0.22um membrane filtration are degerming, dispense in a small amount, -20 DEG C of preservations.
Hypoxanthine and thymidine（HT）Storage liquid making method：Take 136.1mg hypoxanthine（H）With
38.8mg thymidines（T）, plus ultra-pure water is to 100mL, is placed in 45-50 DEG C of water-bath and is allowed to be completely dissolved.0.22um films
Filtration sterilization, dispenses in a small amount, -20 DEG C of preservations, is dissolved in 37 DEG C of water-baths before use.
Glu（L·G）Solution compound method：2.92g Glus are taken, are dissolved in 100mL ultra-pure waters,
0.22um membrane filtrations are degerming, dispense in a small amount, -20 DEG C of preservations.
Dual anti-solution（Mycillin solution）Compound method：Take penicillin（Sodium salt）1000000 units and streptomysin（Sulfuric acid
Salt）1g, is dissolved in 100mL sterilizing ultra-pure waters, dispenses in a small amount, -20 DEG C of preservations.
Complete medium RPMI-1640：Final concentration is added in the incomplete RPMI-1640 culture mediums of purchase（Volume hundred
Divide concentration）It is 1% LG solution and dual anti-solution, plus final concentration（Concentration expressed in percentage by volume）It is 10% newborn calf serum.
HT incubators：Final concentration is added in complete medium RPMI-1640（Concentration expressed in percentage by volume）HT for 1% is stored
Liquid
HAT culture mediums：Final concentration is added in complete medium RPMI-1640（Concentration expressed in percentage by volume）HT for 1% stores
Liquid storage and 1% A storage liquid.
The anti-Escherichia coli O 157 of embodiment 1:The preparation of H7 monoclonal antibodies
The preparation of 1 antigen
E. coliO157:H7 nutrient solutions 50mL, 10000rpm centrifugation 5min, takes bacterium mud, resuspended with physiological saline, adds
The formalin of bacteria suspension volume 0.5%, 37 DEG C of inactivation 24h, after brine three times, adjustment bacterial concentration is 2.5 ×
109Individual/mL, obtains inactivating O157:H7 bacteria suspensions, -20 DEG C freeze it is standby.
Take inactivation O157:H7 bacteria suspensions, use iNtRON Biotechnology companies lipopolysaccharides extracts kits LPS
Extraction Kit（Article No.：17141）, extract Escherichia coli O 157:H7 surfaces lipopolysaccharides, -20 DEG C freeze it is standby.
The foundation of 2 hybridoma cell strains
2.1 immune animals
Selection 6 ~ 8 week old female BAl BIcs/c mouse 10.Every mouse peritoneal injection 0.2mL inactivates O157:H7 bacteria suspensions（Step
Rapid 1 prepares）, at interval of the booster shots of 1 week same dosage once, it is immunized 5 times altogether, wherein it is before fusion that last time is immune
3d, by tail vein injection same amount antigen booster immunization.
The preparation of 2.2 feeder cells
24h before cell fusion is carried out, BALB/c mouse is extractd into eyeball blood sampling, separate serum, during as antibody test
Negative control sera；Dislocated lethal mouse by neck simultaneously, soak 10min in 75% alcohol, it is aseptic after belly is fixed upwards
Abdominal cut skin, tweezers peel off skin, expose peritonaeum, and cotton ball soaked in alcohol wipes peritonaeum sterilization；10mL HAT are injected with syringe
Culture medium notes avoiding penetrating intestinal tube to abdominal cavity, and the right hand fixes syringe, is retained in syringe needle intraperitoneal, and left hand holds alcohol swab
Ball gently abdomen massage 1min, then aspirates the culture medium of injection until the HAT culture medium yellowing in inhalation syringe is repeatedly
Only.The culture medium for suctioning out is centrifuged 10min in 1000r/min, supernatant is abandoned, sedimentation cell is suspended with 5mL HAT culture mediums, made
Cell concentration is adjusted with HAT culture mediums reach 2 × 105Individual/mL, obtains feeder cells suspension.Feeder cells suspension is added 96
Orifice plate, drips per hole one（About 50 μ L）, put 37 DEG C, 5%CO2Cultivated in incubator.
The preparation of 2.3 myeloma cell SP2/0
Fusion the last fortnight starts recovery myeloma cell SP2/0（Presented by Yangzhou University's veterinary college Infectious Diseases Lab
Give）, Amplification Culture, ensure during fusion myeloma cell SP2/0 be in exponential phase, have good form, viable count
Higher than 95%；On the fusion same day, cannot be used up full RPMI-1640 culture mediums（Gibco article No.s：12633012）By myeloma cell
SP2/0 is gently blown down from bottle wall, is collected in 50mL centrifuge tubes, and 1000r/min centrifugation 10min, supernatant discarded is added incomplete
RPMI-1640 culture medium 30mL, supernatant discarded is centrifuged with method；Then by Cell resuspension in incomplete RPMI-1640 culture mediums
10mL, mixes, and is made myeloma cell's SP2/0 suspensions, and do viable count.
The preparation of 2.4 SPLs
Take and the female BAl BIc/c mouse after 3d are immunized in title 2.1 for the last time, extract eyeball blood sampling as positive serum；
The lethal mouse of cervical dislocation, soaks 10min in 75% alcohol, is put into immediately on the dissection plate in super-clean bench, belly upward, four
Limb is fixed.Aseptic taking-up spleen, cannots be used up full RPMI-1640 culture mediums washing for several times, and with tweezers except the connective group of attachment removal
Knit.Spleen is moved on 200 mesh micro-strainers, is placed in fresh incomplete RPMI-1640 culture mediums, gently ground with grinding rod
Mill, single cell suspension is made by spleen, collects splenocyte suspension, and 1000r/min centrifugation 10min, extracting spleen cell cannots be used up entirely
, finally be resuspended in splenocyte in the incomplete RPMI-1640 of 10mL by RPMI-1640 centrifuge washings 1 ~ 2 time, mixes, and obtains spleen pouring
Bar cell suspension, and do standby after viable count.
2.5 cell fusions
Merge according to a conventional method, it is 1 by the ratio between cell number to take the myeloma cell SP2/0 after counting with SPL:5
~1:10 ratio is mixed in 50mL centrifugal bottles, is fully mixed, 1000r/min centrifugation 10min, abandons supernatant.With palm touch from
Heart bottom of bottle, the cell of precipitation is broken up.Centrifugal bottle bottom is put into 40 ~ 41 DEG C of water-baths, fusion is added in rotation
PEG-4000 1mL, add in 1min, continue rotation and add incomplete RPMI-1640 culture mediums 15mL simultaneously, add in 90s
Complete, from slow to fast, preceding 5s adds 1mL.After being stored at room temperature 10min, 1000r/min centrifugation 10min abandon supernatant, add HAT culture mediums
（HAT culture mediums have selectivity so occasionally referred to as HAT selective mediums to hybridoma）, suspension hybridoma is thin
Born of the same parents.Hybridoma suspension is assigned to 96 porocyte culture plates for adding feeder cells, 37 DEG C, 5%CO is placed in2Temperature
Cultivated in case.After 5d, swapped out half culture medium with the HAT culture mediums of fresh preheating；Swapped out with the HT culture mediums of preheating after 10d
HAT culture mediums；The growing state of hybridoma is observed, treats that its cell culture supernatant turns yellow or clone is distributed to bottom hole area
More than 1/10 when, drawing appropriate cell culture supernatant carries out antibody test.
2.6 screenings
2.6.1 the selection of the optimal antigen coat amount of indirect ELISA and serum dilution
The positive serum that before fusion prepared by the blood sampling of immune mouse eyeball（Title 2.4）, as positive control during screening；It is non-to exempt from
Serum prepared by the blood sampling of epidemic disease eyeball of mouse is used as negative control（Title 2.2）, while being adjusted as blank using PBST buffer solutions
Zero hole.Square formation experiment is carried out on ELISA Plate, it is determined that detection antigen optimal coating concentration and the positive, negative control it is optimal dilute
Concentration is released, is concretely comprised the following steps：
（1）Coating：Detection antigen（O157 is inactivated in the present embodiment title 1:H7 bacteria suspensions）It is slow with 0.05mol/L carbonate
Fliud flushing（pH9.6）10 times, 100 times, 1000 times, 10000 times are diluted respectively, and each dilution factor adds a row, 100 μ L/ from left to right
Hole, 37 DEG C of rearmounted 4 DEG C of 2h of effect are overnight；
（2）Washing：The liquid in hole is discarded, PBST buffer solutions are washed 3 times, and 5min is washed every time, are patted dry；
（3）Closing：150 μ L confining liquids, 37 DEG C of effect 2h are added per hole；
（4）Washing：Method is the same；
（5）Add primary antibody：Primary antibody（Positive serum prepared by title 2.4）With PBST buffer solutions dilute respectively 100 times, 1000
Again, 10000 times, each dilution factor adds a line, 100 μ L/ holes, 37 DEG C of effect 1h；
（6）Washing：Method is the same；
（7）ELIAS secondary antibody：Add working concentration (1：3000 dilution) sheep anti mouse HRP-IgG, 100 μ L/ holes, 37 DEG C incubation
1h, washing is the same；
（8）Colour developing：Add the μ L/ holes of tmb substrate nitrite ion 100,37 DEG C of 10 ~ 15 min of reaction；
（9）Terminating reaction：Terminate liquid, 50 μ L/ holes, terminating reaction is added to determine each hole OD450。
Result judgement：With positive serum OD450Value is in 1.0 or so, negative serum OD450Value is less than 0.2, and positive serum
OD450Value/negative serum OD450(P/N values) is the best effort concentration for detecting antigen, the point corresponding serum-dilution when maximum
It is optimal serum dilution to spend.As a result：Detection antigen（O157 is inactivated in title 1:H7 bacteria suspensions）Optimal coating concentration for 1 ×
106Individual/mL, the optimum dilution degree of positive serum is 1：1000；Negative optimum dilution degree is 1:1000.
Preparation is coated with the ELISA Plate of detection antigen：
（1）Coating：Detection antigen is coated with 96 hole polystyrene ELISA Plates, 100 μ L/hole, 37 DEG C of works with best effort concentration
With rearmounted 4 DEG C of 2h overnight；
（2）Washing：Coating buffer is abandoned, is washed 3 times with PBST buffer solutions, 5min is washed every time, patted dry；
（3）Closing：150 μ L confining liquids are added to be closed per hole, 37 DEG C of effect 2h；
（4）Washing：According to step（2）Method is washed, and obtains being coated with the ELISA Plate of detection antigen, and -20 DEG C save backup.
Note：In following indirect ELISA detection, using the ELISA Plate for being coated with detection antigen, positive, negative serum takes
Optimum dilution degree.
2.6.2 indirect ELISA experiment
In order to screen hybridoma cell strain, the μ L of cell culture supernatant 100 additions are coated with detection antigen in taking title 2.5
ELISA Plate in, while set up positive, negative and blank, 37 DEG C of incubation 1h, washing；Add working concentration (1：3000 is dilute
Release) sheep anti mouse HRP-IgG, 100 μ L/ holes, 37 DEG C of incubation 1h, the same washing pats dry；Add the μ L/ of tmb substrate nitrite ion 100
Hole, 37 DEG C of 10 ~ 15 min of reaction；Add the μ L/ holes of terminate liquid 50, terminating reaction.Determine each hole OD450.Criterion：Hole to be checked
OD450The OD of/negative hole450It is the positive that inner cell strain in hole to be checked is judged to by >=2.1, can carry out next step experiment.
The cloning of 2.7 hybridomas
Cloning is carried out to the cell line of double test positive using limiting dilution assay.Mouse is prepared before clone to raise
Support cell.Living cells accurate counting in clone hole is treated, with the doubling dilution of HT culture mediums 10 into 20 cell/mL culture mediums, plus
Enter to being covered with 96 porocyte culture plates of feeder cells, every hole is had 1 cell in theory, be placed in 37 DEG C, 5%CO2Temperature
Cultivated in case.Detected when the 1/10 of cell growth to bottom hole area, strong positive hole is cloned according still further to same procedure,
So repeat 3 ~ 4 times, until positive rate reaches 100%, obtain the hybridoma cell strain of preliminary screening.By the hybridization of preliminary screening
Tumor cell strain Amplification Culture, freeze-stored cell, while retain supernatant, using indirect ELISA determine antibody titer, choose potency compared with
Some hybridoma cell strains high.
The screening of 2.8 hybridoma cell strains
Take Escherichia coli O 157:H7 surfaces lipopolysaccharides carries out SDS-PAGE electrophoresis, and if higher with potency in title 2.7
Dry hybridoma cell strain culture supernatant carries out immuning hybridization respectively, chooses the best clone of immune response effect anti-as secretion
Escherichia coli O 157:The hybridoma cell strain of H7 monoclonal antibodies, is named as 10G1A2.
The specificity of 2.9 monoclonal antibodies
Strain subject includesE. Coli O157:H7、EHEC O26:H11、EHEC O111、EPEC O127:H6、ETEC
O148:H28、E. coli O25、E. coli O55、E. coliO78、E. coli O103、E. coli O138、E. coli
O139、E. coli O141、E. coli O145、E. coli K88、E. coli K99、E. coli BL21、E. coli
Top10, staphylococcus aureus, bacillus dysenteriae, Klebsiella Pneumoniae, pseudomonas aeruginosa, Aeromonas hydrophila, secondary haemolysis arc
Bacterium, salmonella, Bacillus cereus, bacillus subtilis, citrobacter freundii, pasteurella multocida, pest of duck Richter scale
Bacillus, haemophilus parasuis, Bacillus acidi lactici, Brucella, Li bacillus, fowl mycobacterium, streptococcus, clostridieum welchii, meat poisoning
Clostridium, fowl Podbielniak bacterium.By each strain subject according to method coated elisa plate in title 2.6.1, indirect ELISA inspection is then carried out
Survey, investigate the specificity of hybridoma cell strain 10G1A2 nutrient solution supernatants.Result shows, hybridoma cell strain 10G1A2 nutrient solutions
Supernatant only withE. Coli O157:There is positive reaction in H7, other each bacterium are negative reaction（The OD in hole to be checked450/ negative hole
OD450>=2.1 are judged to the positive）.
3. the preparation of monoclonal antibody and property
The preparation of 3.1 monoclonal antibodies
Select female BAl BIc/c mouse more than 8 ~ 10 week old body weight 20g, intraperitoneal injection atoleine, 0.5mL/, abdomen after 1 week
Inject hybridoma cell strain 10G1A2 in chamber（2×106~5×106Cell/only）, ascites is extracted when animal belly is significantly increased,
3000r/min is centrifuged 10min, takes supernatant, -20 DEG C freeze it is standby.
Using HiTrapTMProtein G affinity columns（Sangon Biotech (Shanghai) Co., Ltd., article No.
SD6606）Ascites supernatant is purified.Agents useful for same is all chemical pure in purge process, and the solvent of all solution is all
Ionized water, with 0.45 μm of membrane filtration after preparing, 10000 r/min are centrifuged 3min to ascites supernatant before purification.The specific behaviour of purifying
Make step as follows：
(1) remove the stopper of chromatography column top, syringe and chromatographic column are connected together with connector.
(2) snap-off of chromatographic column afterbody is removed.
(3) 10ml binding buffer are drawn with syringe（Bioengineering (Shanghai) limited company, article No.
BSP048-2）, the injection of " drop to drop " formula enters chromatographic column, injection rate 1ml/min with air-prevention.
(4) ascites supernatant to be purified is drawn, is injected into chromatographic column.
(5) 10ml binding buffer are drawn in chromatographic column, foreign protein is eluted.
(6) with 5 ml elution buffer（0.1M citric acid solutions）Post is washed, prepares 1.5ml collecting pipes 5, often managed
The μ L of Tris-HCl buffer solutions 200 that concentration is 1M, pH9.0 are previously added, often pipe collects 1ml.
Ascites supernatant after purification, obtains purifying anti-Escherichia coli O 157:H7 monoclonal antibodies, through Nanodrop1000
（Thermo）Protein content is measured for 1.027mg/mL, -20 DEG C save backup, and with SDS-PAGE electrophoresis observation purification effects,
It can be seen that destination protein purity is very high, main stripe size is slightly over 200kDa, such as Fig. 1.
3.2 antibody titers are determined
Using the ELISA Plate for being coated with detection antigen（Prepared in title 2.6.1）, by hybridoma cell strain 10G1A2 cultures
Liquid supernatant ascites supernatant is cooked doubling dilution, and potency is detected using indirect ELISA method, and nutrient solution supernatant potency is 1:
2430；Titer of ascites is>1:81000.
3.3 subclass are determined
It is sub- according to Thermo scientific companies monoclonal antibody subgroup identification kit operational manual detection antibody
Class.The monoclonal antibody subclass that hybridoma cell strain 10G1A2 is produced is IgG.
The affinity costant of 3.4 monoclonal antibodies
The anti-Escherichia coli O 157 that hybridoma cell strain 10G1A2 is produced:The affinity costant Kd=5.69* of H7 monoclonal antibodies
10-8。
Embodiment 2 prepares anti-Escherichia coli O 157:The coated immunomagnetic beads of H7 monoclonal antibodies
1. 165 u L bead suspensions (5mg containing magnetic bead is purchased from Life technologies, article No. 1309004) are taken to put
In centrifuge tube, magnetic bead is precipitated with magnetic frame（Hereinafter referred to as magnetic force is precipitated）, concrete operation step is：Centrifuge tube is put in magnetic force
On frame, supernatant is discarded after supernatant liquid becomes clarification, retain the magnetic bead of bottom, remove magnetic frame；
2. 1mL magnetic bead coupling buffers, resuspended magnetic bead, magnetic force precipitation are added；
3. the anti-Escherichia coli O 157 of purifying prepared in 75.7 μ L magnetic beads coupling buffers, 50 μ L embodiments 1 is successively added:
H7 monoclonal antibodies（100ug containing antibody）Magnetic bead with after magnetic force precipitation in step 2, mixes, and adds 100 μ L treatment fluids, mixes
It is even, put after being incubated 18h on 37 DEG C of shaking tables, magnetic force precipitation；
4. the 1mL confining liquids resuspended magnetic beads of A are added, after being incubated 1h on 37 DEG C of shaking tables, magnetic force precipitation；
5. 1mL storing solutions are added, after vortex oscillation 5-10s, magnetic force precipitation；Repeat the operation once；Add 240uL deposits
Liquid is mixed（Overall solution volume 250uL）, obtain anti-Escherichia coli O 157:The coated immunomagnetic beads of H7 monoclonal antibodies, in 4 DEG C of ice
Case is preserved.
Magnetic bead coupling buffer：The borate buffer solution of 1mol/L, its pH is 7.4；
Treatment fluid：In the borate buffer solution of 1mol/L（PH is 7.4）The ammonium sulfate of the middle final concentration of 3mol/L of addition；
Confining liquid A：0.5g bovine serum albumin(BSA)s（BSA ）It is dissolved in the PBS of 100mL（0.02M, pH are 7.4）；
Storing solution：0.1g BSA are dissolved in 100mL PBSs（0.02M, pH are 7.4）；
PBS（0.02M, pH are 7.4）：Contain Na2HPO4 2.9g/L、KH2PO4 0.2g/L、NaCl 8.0g/L、
The aqueous solution of KCl 0.2g/L, its pH is 7.4.
Embodiment 3 utilizes anti-Escherichia coli O 157:The coated immunomagnetic beads detection Escherichia coli O 157 of H7 monoclonal antibodies:
H7
（1）According to SN/T0793-2010（Import and export meat, meat products and enterorrhagia Bacillus coil 0157 in other food:
H7 detection methods）Carry out the pre- increasing bacterium of testing sample：By in testing sample addition EC meat soups, 18- is cultivated under the conditions of 42 DEG C
24h, obtains enrichment liquid；
（2）Step is added in centrifuge tube（1）The enrichment liquid 20-30mL for obtaining, is subsequently adding anti-Escherichia coli O 157:H7
The coated immunomagnetic beads of monoclonal antibody（It is prepared by embodiment 2）50 μ L, 37 DEG C of incubation 1h；
（3）Washing magnetic bead：Centrifuge tube is put on magnetic frame and precipitates magnetic bead, supernatant discarded after 3min adds 30mL
PBS（0.02M, pH are 7.4）It is slow resuspended, magnetic bead is precipitated with magnetic frame, supernatant discarded.Such repeated washing 3 times；
(4) it is 50mmol/L glycine solutions, interruption vibration wash-out to 1mL concentration is added in the immunomagnetic beads after washing
Immunomagnetic beads 1h, centrifuge tube is put on magnetic frame, and supernatant is taken out when immunomagnetic beads is all adsorbed on tube wall, that is, wash
De- liquid.This eluent is Escherichia coli O 157:H7 pregnant solutions.From Escherichia coli O 157:Sample is directly extracted in H7 pregnant solutions
DNA, Escherichia coli O 157 is whether there is in then detecting testing sample with fluorescence quantitative PCR method:H7.
（5）Fluorescence quantitative PCR detection
Sample DNA is taken, TaKaRa PCR kit for fluorescence quantitative is used（DRR390A）, prepare reaction system as follows：
Premix Ex Taq (2 ×) 10ul, sense primer (10uM) 0.4ul, anti-sense primer (10uM) 0.4ul, Taqman probe
0.8ul、ROX Reference Dye Ⅱ（ROX reference dyes II, 50 ×）（）0.2ul, template（Sample DNA）2ul、dH2O
6.2ul。
Sense primer（SEQ ID NO:1）：5’-GCACTAAAAGCTTGGAGCAGTTC-3’.
Anti-sense primer（SEQ ID NO:2）：5’-AACAATGGGTCAGCGGTAAGGCTA-3’.
Taqman probes：5’-FAM-CGTTGGCGAGGACC-TAMRA-3’.FAM is reporter fluorescence group, and TAMRA is quenched
Fluorophor.The nucleotide sequence of Taqman probes such as SEQ ID NO:Shown in 3.
The 20ul reaction systems for preparing, are fitted into quantitative fluorescent PCR reaction tube, cover lid, centrifugation, confirm reaction tube
It is middle to react without being put into after drum in ABI7500 quantitative real time PCR Instruments.
Response procedures are as follows：The first step：95℃ 30s；Second step 95 DEG C of 5s, 60 DEG C of 34s, repeat 40 circulations；Reaction
During 60 DEG C collect fluorescence signals.Final result：It is the positive with CT value≤35, need to repeat experiment if CT values are equal to 35 tests
Card；CT values ＞ 35 is negative reaction.
Embodiment 4 utilizes anti-Escherichia coli O 157:The coated immunomagnetic beads detection Escherichia coli O 157 of H7 monoclonal antibodies:
The sensitivity of H7
Prepare Escherichia coli O 157:H7 bacterium solutions, 10 times of doubling dilutions are coated with sorbitol-MacConkey agar flat board（Reference is entered
Outlet meat, meat products and enterorrhagia Bacillus coil 0157 in other food:H7 detection methods）To determine clump count.
Choose 101、102、103、104、105CFU/ml dilutes bacterium solution, and Escherichia coli are carried out respectively using method in embodiment 3
O157:H7 detections, investigate the sensitivity of the inventive method.Result is 101CFU/ml-105CFU/ml respectively dilutes bacterium solution and detects
It is the positive.Therefore, within the inventive method Sensitivity is up to 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 anti-Escherichia coli O 157:The coated immunomagnetic beads detection Escherichia coli O 157 of H7 monoclonal antibodies:
The accuracy of H7
1. interference test：The bacteria suspension of each interference bacterial strain is prepared respectively（It is non-O157 in the title 2.9 of example 1 from bacterial strain:
H7 bacterial strains）(bacterial concentration is 10 to each 25ml5/ mL), add Escherichia coli O 157:H7 about 1-10, according to the behaviour in embodiment 3
Carried out as step.Result is the positive.
It is not added with Escherichia coli O 157:The bacteria suspension of each interference bacterial strain of H7（It is non-in the title 2.9 of example 1 from bacterial strain
O157:H7 bacterial strains）It is detection negative.
2. analog detection experiment：The random contact pollution modes of natural infection are simulated, random concentration is manually prepared（1CFU/
g-103CFU/g）Escherichia coli O 157:H7 pollutes 100 parts of sample.Respectively according to SN/T0793-2010, the inventive method（Embodiment
Method in 3）And fluorescence quantifying PCR method is detected.It was found that using SN/T0793-2010 methods（Import and export meat, meat products and
Enterorrhagia Bacillus coil 0157 in other food:H7 detection methods）Verification and measurement ratio be 57%, the inventive method recall rate is
98%, fluorescence quantifying PCR method recall rate is 69%.Show that the present invention, can by immunomagnetic beads and quantitative fluorescent PCR method for combined use
Effectively by the Escherichia coli O 157 in sample:H7 is enriched with, combined with fluorescent quantitative PCR method, substantially increases recall rate.This
The verification and measurement ratio of invention detection method is not only significantly higher than SN/T0793-2010 methods, and is significantly higher than quantitative fluorescent PCR side
Method.
SEQUENCE LISTING
<110>Propagation and Food Test Center of Jiangsu Entry-Exit Inspection and Quarantine Bureau
<120>Anti- Escherichia coli O 157:H7 monoclonal antibodies and its application
<130> 20141229
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> artificial
<220>
<223>Sense primer
<400> 1
gcactaaaag cttggagcag ttc 23
<210> 2
<211> 24
<212> DNA
<213> artificial
<220>
<223>Anti-sense primer
<400> 2
aacaatgggt cagcggtaag gcta 24
<210> 3
<211> 14
<212> DNA
<213> artificial
<220>
<223>The nucleotide sequence of Taqman probes
<400> 3
cgttggcgag gacc 14
Claims (6)
1. it is a kind of to produce anti-Escherichia coli O 157:The hybridoma cell strain 10G1A2 of H7 monoclonal antibodies, its preserving number is CGMCC
NO. 9999。
2. it is a kind of as described in claim 1 hybridoma cell strain 10G1A2 secrete anti-Escherichia coli O 157:H7 monoclonal antibodies.
3. anti-Escherichia coli O 157 described in claim 2:The coated immunomagnetic beads of H7 monoclonal antibodies.
4. immunomagnetic beads described in claim 3 is in the detection Escherichia coli O 157 for the purpose of non-diagnostic:Application in terms of H7.
5. apply according to claim 4, it is characterised in that using in immunomagnetic beads enrichment testing sample described in claim 3
Escherichia coli O 157:H7, obtains pregnant solution；Escherichia coli in the pregnant solution are detected using fluorescence quantifying PCR method
O157:H7。
6. apply according to claim 5, it is characterised in that the Escherichia coli O 157 in the enrichment testing sample:The side of H7
Method is：Adsorbed during immunomagnetic beads described in claim 3 is added into testing sample, the immunomagnetic beads precipitated with magnetic frame,
The immunomagnetic beads is eluted, eluent is taken as pregnant solution.
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