CN104745537B - A kind of monoclonal antibody of pseudomonas syringae pv.tomato and its application - Google Patents
A kind of monoclonal antibody of pseudomonas syringae pv.tomato and its application Download PDFInfo
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- CN104745537B CN104745537B CN201510098828.XA CN201510098828A CN104745537B CN 104745537 B CN104745537 B CN 104745537B CN 201510098828 A CN201510098828 A CN 201510098828A CN 104745537 B CN104745537 B CN 104745537B
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Abstract
Monoclonal antibody and its application the invention discloses a kind of pseudomonas syringae pv.tomato.The present invention provides the monoclonal antibody hybridoma cell strain Pst10 for producing pseudomonas syringae pv.tomato, its preserving number is CGMCC No.10119.Additionally provide the monoclonal antibody produced by above-mentioned No. 10 secretions of hybridoma cell strain.The monoclonal antibody of pseudomonas syringae pv.tomato provided by the present invention it is specific good, potency is high, reacts with 3 different strains of pseudomonas syringae pv.tomato, has preferable broad spectrum activity.Applied in pseudomonas syringae pv.tomato serodiagnosis reagent, specificity is good, avoids cross reaction.The monoclonal antibody for the pseudomonas syringae pv.tomato that the present invention develops can be used for the immunological diagnostic reagent for developing pseudomonas syringae pv.tomato, such as enzyme-linked diagnostic reagent, colloid gold immune test paper bar, antibody chip, biology sensor etc..
Description
Technical field
The present invention relates to biological technical field, more particularly to a kind of monoclonal antibody of pseudomonas syringae pv.tomato and its
Using.
Background technology
Pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato, Pst) is China's Import quarantine
Property harmful organism, is Gram-negative bacteria, thalline rod-short is straight or slightly curved, unicellular, and size is 1.5-4 μm of 0.1-1 μ ms.
The germ is mainly caused harm leaf, stem, flower, petiole and the fruit of tomato.The disease from 1933 first report since, the U.S., Britain,
26 countries such as the former Soviet Union, Canada, France, South Africa, Australia and China Jilin, Liaoning, Heilungkiang, Hebei, Tianjin,
Report on the ground such as Gansu, Xinjiang, Fujian, Guangxi, Taiwan.Germ can be overwintering on seed, invalid body, soil and weeds,
Can be with seed long-distance communications.The disease has the trend risen year by year, and serious threat is formed to the tomato production in China.
The quick detect and diagnose of bacteriosis is the basis of tomato health production on tomato, using ELISA exempting from as representative
Epidemiology detection method is most important a kind of detection method, is widely used in the detection of phytobacterial disease, and high specificity, sensitive
Spend the basis that high monoclonal antibody is various immunological detection methods.
The content of the invention
A purpose of the invention is to provide a kind of monoclonal antibody hybridoma cell strain of pseudomonas syringae pv.tomato.
Hybridoma cell strain Pst 10 provided by the invention, its preserving number are CGMCC No.10119.
The monoclonal antibody produced by above-mentioned No. 10 secretions of hybridoma cell strain Pst is also the scope of protection of the invention.
It is a further object to provide a kind of enzyme labelled antibody.
Enzyme labelled antibody provided by the invention, the enzyme mark to be formed after above-mentioned monoclonal antibody is marked with marker enzyme resist
Body, the marker enzyme are specially alkaline phosphatase.
Above-mentioned monoclonal antibody or above-mentioned enzyme labelled antibody are in pseudomonas syringae pv.tomato detection kit is prepared
Application be also the scope of protection of the invention.
Above-mentioned monoclonal antibody answering in the colloidal gold colloidal gold detection test paper strip for preparing detection pseudomonas syringae pv.tomato
With being also the scope of protection of the invention.
The examination for being used to detect pseudomonas syringae pv.tomato containing above-mentioned monoclonal antibody or above-mentioned enzyme labelled antibody
Agent box is also the scope of protection of the invention.
It is also for the colloidal gold colloidal gold detection test paper strip for detecting pseudomonas syringae pv.tomato containing above-mentioned monoclonal antibody
The scope of protection of the invention.
In kit provided by the present invention, the coated antibody of independent packaging is further included；Wherein, coated antibody is thin for tomato
The polyclonal antibody of bacterium property leaf spot fungi；In the present invention, the pseudomonas syringae pv.tomato coated antibody is specially ACD
The product of Diagnostics companies, its catalog number are B264-C2.
At least one of in kit provided by the present invention, following 1)-the 5 of independent packaging is further included) reagent：
1) it is coated with buffer solution：Sodium carbonate 1.59g, sodium acid carbonate 2.93g and sodium azide 0.20g are dissolved in water, determined with water
Hold to 1L；The pH value for being coated with buffer solution is 9.6.
2) Sample extraction buffer solution：Sodium sulfite 1.3g and polyvinylpyrrolidone 20g are dissolved in lavation buffer solution,
1L is settled to lavation buffer solution.
3) lavation buffer solution：By sodium chloride 8.0g, potassium dihydrogen phosphate 0.2g, disodium hydrogen phosphate 1.15g, potassium chloride 0.2g,
Polysorbas20 0.5mL, sodium azide 0.2g is soluble in water, and 1L is settled to water；The pH value of the lavation buffer solution is 7.4.
4) enzyme labelled antibody dilution buffer：It is slow that polyvinylpyrrolidone 20g and bovine serum albumin(BSA) 2g are dissolved in washing
In fliud flushing, 1L is settled to lavation buffer solution.
5) substrate solution：Diethanol amine 97mL and 4-NPP (now with the current) 1g is soluble in water, adjust
PH value is settled to 1L to 9.8, with water.
Application of the mentioned reagent box in qualitatively or quantitatively detection pseudomonas syringae pv.tomato, falls within the guarantor of the present invention
Protect scope.
In the present invention, above all of pseudomonas syringae pv.tomato can be specially pseudomonas syringae pv.tomato
(Pseudomonas syringae pv.tomato)ATCC BAA-871。
It is demonstrated experimentally that the monoclonal antibody of pseudomonas syringae pv.tomato provided by the present invention specific good, potency
Height, reacts with 3 different strains of pseudomonas syringae pv.tomato, has preferable broad spectrum activity.Applied to tomato bacterium
Property leaf spot fungi immunological diagnostic reagent in, specificity it is good, avoid cross reaction.The tomato bacterial leaf that the present invention develops
The monoclonal antibody of pinta bacterium can be used for the immunological diagnostic reagent for developing pseudomonas syringae pv.tomato, and such as enzyme-linked diagnosis tries
Agent, colloid gold immune test paper bar, antibody chip, biology sensor etc..
Join the biomaterial (strain) of evidence：Pst 10
Scientific description：Secrete the cell line of pseudomonas syringae pv.tomato monoclonal antibody
Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism is referred to as：CGMCC
Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Preservation date：On December 11st, 2014
Collection is registered on the books numbering：CGMCC No.10119
Brief description of the drawings
Fig. 1 is monoclonal antibody SDS-PAGE electrophoresis
Fig. 2 determines for monoclonal antibody affinity costant
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, is commercially available unless otherwise specified.
Freund's complete adjuvant：Sigma companies, catalogue numbering is F5881.
Incomplete Freund's adjuvant：Sigma companies, catalogue numbering is F5506.
It is coated with buffer solution：Sodium carbonate 1.59g, sodium acid carbonate 2.93g and sodium azide 0.20g are dissolved in water, with water constant volume
To 1L；The pH value for being coated with buffer solution is 9.6.
Lavation buffer solution：By sodium chloride 8.0g, potassium dihydrogen phosphate 0.2g, disodium hydrogen phosphate 1.15g, potassium chloride 0.2g, spits
Temperature -200.5mL, sodium azide 0.2g is soluble in water, and 1L is settled to water；The pH value of lavation buffer solution is 7.4.
Sample extraction buffer solution：Sodium sulfite 1.3g and polyvinylpyrrolidone 20g are dissolved in PBST, delayed with PBST
Fliud flushing is settled to 1L.
Enzyme labelled antibody dilution buffer：Polyvinylpyrrolidone 20g and bovine serum albumin(BSA) 2g are dissolved in washing buffer
In liquid, 1L is settled to lavation buffer solution.
Alkaline phosphate ester enzyme substrate solution：Diethanol amine 97mL and 4-NPP (now with the current) 1g are dissolved in
In water, pH value is adjusted to 9.8, is then settled to 1L with water.
The nitrite ion of horseradish peroxidase reaction：1%A liquid+10%B liquid (A liquid：1% 3,3', 5,5'- tetramethyl join
Aniline (Sigma, T8768-5G) is dissolved in dimethyl sulfoxide (DMSO), (Sigma, D2650)；B liquid：0.1% is added in citrate buffer solution
H2O2)。
The secondary antibody of horseradish peroxidase-labeled：Goat anti-mouse IgG/HRP (has purchased from Beijing Zhong Shan Golden Bridge biotechnology
Limit company, cat. no ZB2305).
Embodiment 1, the acquisition of mouse hybridoma cell strain and the preparation of pseudomonas syringae pv.tomato monoclonal antibody
Pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato)：Compiled quoted from ATCC, its ATCC
Number it is BAA-871.
Balb/c mouse：6-8 week old female mices, purchased from the Chinese military medicine academy of sciences.
SP2/0 myeloma cell：This laboratory preserves.
First, animal immune
1st, Bacteria Culture
By the bacterium of pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato) ATCC BAA-871
Strain, lining NA culture mediums, (yeast extract 1.0g, beef extract 3.0g, peptone 5.0g, glucose 10.0g, agar 15.0g are double
Water 1000mL is steamed, adjusts pH to 7.2,121 DEG C of moist heat sterilization, 15min) on tablet, 28 DEG C of culture 2-3d, use brine
Flat-plate bacterial colony is into 2mL centrifuge tubes, brine 2 times, degree of thickening to 108CFU/mL, is bacteria suspension, for mouse immune
Experiment.
2nd, mouse immune
It is immune for the first time：Take the bacterium of isometric Freund's complete adjuvant and the pseudomonas syringae pv.tomato of step 1 acquisition
Suspension, Balb/c mouse are subcutaneously injected after emulsifying completely.
Second immune：Second is carried out after 2 weeks immune for the first time to be immunized, and takes isometric incomplete Freund's adjuvant and step
The bacteria suspension of rapid 1 pseudomonas syringae pv.tomato obtained, is subcutaneously injected after emulsification, and every mouse dosage is same to be exempted from for the first time
Epidemic disease.
Third and fourth time immune：After second 2 weeks immune, carry out third time and be immunized, after third time is 2 weeks immune, carry out the 4th
It is secondary immune.Method and dosage are the same as second.
Booster immunization：1 week tail vein blood carries out titration after the 4th is immunized, and enzyme-linked survey potency reaches 104During the above
Balb/c mouse booster immunizations are directly injected intraperitoneally in the bacteria suspension of the pseudomonas syringae pv.tomato obtained with step 1, and dosage is
108CFU pseudomonas syringae pv.tomatos.
2nd, mouse hybridoma cell strain obtains
Cell fusion assay is carried out when 80 is small after booster immunization.The splenocyte of immune mouse and SP2/0 myeloma is thin
Born of the same parents carry out cell fusion, and (splenocyte and the quantitative proportion of SP2/0 myeloma cell are 9:1).Screened and secreted using limiting dilution assay
The mouse hybridoma cell strain of monoclonal antibody；It is 10 with concentration8The bacteria suspension bag of the pseudomonas syringae pv.tomato of CFU/mL
By ELISA Plate, it is measured using indirect ELISA, screening secretory antibody potency is high and thin with monoclonal that immune bacterium reacts
Born of the same parents' strain.The cell limiting dilution assay in positive hole is cloned 3 times, makes positive rate up to 100%.
The step of above-mentioned indirect elisa method screening positive cell, is specific as follows：
1) it is coated with：100 μ L concentration 10 are added in 96 hole elisa Plates8The pseudomonas syringae pv.tomato ATCC of CFU/mL
The bacteria suspension (solvent is coating buffer solution) of BAA-871,4 DEG C of overnight incubations, are washed 3 times with cleaning solution.
2) close：With the phosphate buffer sealing plate containing 2% balf serum albumin (BSA), 37 DEG C of incubation 2h, with washing
Liquid washs 3 times.
3) sample to be tested is added：100 μ L Hybridoma Cell Culture liquid to be measured is added in ELISA Plate, while sets feminine gender right
It is 100 μ L/ according to (SP2/0 culture supernatants), blank control (PBS), positive control (positive serum PBS dilutes 200 times)
Hole, is incubated 1h, board-washing 3 times under the conditions of 37 DEG C.
4) ELIAS secondary antibody is added：Goat anti-mouse HRP ELIAS secondary antibodies (are purchased from the limited public affairs of Beijing Zhong Shan Golden Bridge biotechnology
Department, cat. no ZB2305) with enzyme labelled antibody dilution buffer 20000 times are diluted, add 100 μ L per hole, under the conditions of 37 DEG C
It is incubated 1h, board-washing 3 times.
5) develop the color：The nitrite ion that horseradish peroxidase reacts is added in ELISA Plate hole, per 100 μ L of hole, during colour developing
Between for 5min or so.
6) reaction is terminated：The 2M sulfuric acid terminate liquids of 50 μ L are added per hole, terminate reaction.
7) reading：With 450nm, the dual wavelength measure of 630nm respectively treats gaging hole OD values, with (thin with SP2/0 with negative control hole
Born of the same parents' culture supernatant replaces the control of sample to be tested) ratio (P/N) of OD values is limited more than 2.1, and it is positive or definite as being judged as
The critical point of potency.ELISA result judgement methods：If P/N>2.1, then it is determined as positive cell.
Screened again through indirect elisa method, 3 subclonings obtain 1 plant can stably excreting tomato bacterial leaf spot
The mouse hybridoma cell strain of the monoclonal antibody of bacterium, is named as Pst 10.The mouse hybridoma cell strain is in 2014
December 11 was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC, address：Beijing
The institute 3 of city Chaoyang District North Star West Road 1, Institute of Microorganism, Academia Sinica, postcode 100101), deposit number CGMCC
No.10119, scientific description：Secrete the cell line of pseudomonas syringae pv.tomato monoclonal antibody.
3rd, cell cryopreservation and recovery
No. 10 CGMCC No.10119 of mouse hybridoma cell strain Pst are made 1 × 10 with frozen stock solution6The cell of a/mL
Suspension, preserves for a long time in liquid nitrogen.During recovery, cryopreservation tube is taken out, 37 DEG C of water-bath middling speeds is immediately placed in and melts, centrifugation removes frozen stock solution
Culture culture in glassware is moved into afterwards.
4th, preparation, purifying and the hypotype identification of monoclonal antibody
1st, increment culture prepares monoclonal antibody and titration
The preparation method of cell culture medium：Calf serum is added into DMEM culture mediums and (is purchased from GIBCOBRL, catalogue
Number it is 26170-043), final concentration of 20% (volumn concentration) of calf serum, pH 7.4.
No. 10 CGMCC No.10119 of mouse hybridoma cell strain Pst are placed in above-mentioned cell culture medium, 37 DEG C of trainings
Support, during which observe daily, and timely amplification cultivation.Titration, concrete operations are carried out to culture supernatant using indirect elisa method
Referring to " indirect elisa method screening positive cell " in step 2, potency is with P/N>The maximum dilution multiple table of 2.1 culture supernatant
Show.Experiment is in triplicate.
The results show that the antibody titer of No. 10 CGMCC No.10119 cells and supernatants of mouse hybridoma cell strain Pst
For 1/1600.
2nd, the purifying of monoclonal antibody
The cell culture fluid obtained with G-protein column (GE Products, its catalog number are 17-0404-01) to step 1
Antibody, which is purified, in supernatant (uses the HiTrap Protein G antibody purification columns of 1mL.The 20mM of 10mL is first used, pH7.0's
Sodium phosphate combination buffer, purification column is washed by the speed of 1mL/min.The monoclonal antibody supernatant of 2mL is delayed with syringe
In slow push-in purification column, pillar, then glycine-salt of the 0.1M with 2-5mL, pH 2.7 are washed with the combination buffer of 5-10mL
Sour elution buffer elution, by antibody collection, in advance, added with the centrifuge tube of the 1M Tris-HCl of 60 μ L, often pipe is collected
0.5mL, surveys OD280nmValue determines the concentration of antibody) monoclonal antibody that obtains after purification (is pseudomonas syringae pv.tomato
Monoclonal antibody) -20 DEG C preserve.
3rd, the identification of monoclonal antibody Ig types
Step 2 is obtained into the monoclonal antibody solution of No. 10 CGMCC No.10119 secretions of mouse hybridoma cell strain Pst
With Southern Biotech companies monoclonal antibody grouping reagents (product identification is respectively 1020-05,1070-05,1080-05,
1090-05,1100-05,1040-05,1050-05,1060-05) detection monoclonal antibody Ig types, concrete operations are referring to examination
Agent box specification.
As a result such as table 1, the monoclonal antibody of No. 10 CGMCC No.10119 secretions of display mouse hybridoma cell strain Pst
Immunoglobulin subclass be IgG1 types.
Table 1 is cell strain of monoclonal antibody subgroup identification
| Cell line is numbered | M | G1 | G2a | G2b | G3 | A | K | λ |
| 2 | 0.028 | 0.385 | 0.033 | 0.036 | 0.037 | 0.049 | 0.179 | 0.058 |
| 8 | 0.957 | 0.06 | 0.064 | 0.05 | 0.023 | -0.006 | 1.192 | 0.018 |
| 10 | 0.006 | 0.468 | 0.022 | 0.022 | 0.012 | 0.001 | 0.234 | 0.043 |
| 12 | 0.705 | 0.036 | 0.03 | 0.024 | 0.021 | 0.021 | 0.699 | 0.038 |
| 13 | 0.644 | 0.03 | 0.032 | 0.03 | 0.018 | 0.012 | 1.023 | 0.065 |
| 15 | 1.126 | 0.096 | 0.153 | 0.051 | 0.039 | 0.022 | 0.976 | 0.095 |
| 16 | 0.57 | 0.019 | 0.03 | 0.024 | 0.007 | -0.001 | 0.663 | 0.013 |
| Negative control | 0.038 | 0.035 | 0.039 | 0.028 | 0.017 | 0.012 | 0.022 | 0.034 |
The detection for the pseudomonas syringae pv.tomato monoclonal antibody that embodiment 2, embodiment 1 obtain
First, the specific detection of the monoclonal antibody for the pseudomonas syringae pv.tomato that embodiment 1 obtains
1st, strains tested
In pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato) ATCC BAA-871 and table 2
Control bacterium.
The monoclonal antibody specificity detection control bacterium used of 2 pseudomonas syringae pv.tomato of table
2nd, the specific detection of monoclonal antibody
(1) preparation of alkaline phosphatase mark pseudomonas syringae pv.tomato monoclonal antibody
100 μ L alkaline phosphatases (Sigma, P6774-10KU) are taken to be added in the 0.01M PBS, pH7.2 of 300 μ L,
The glutaraldehyde (Alfa Aesar, A17876) for adding 10 μ L 25% mixes, and (25 DEG C) of room temperature is incubated activation 1h, adds
The pseudomonas syringae pv.tomato monoclonal antibody for the purifying that 0.5mg embodiments 1 obtain, reacts at room temperature 2h, that is, obtains alkaline phosphorus
The monoclonal antibody of acid esters enzyme mark, is enzyme labelled antibody.
(2) specific detection
Using DAS-ELISA methods, the monoclonal antibody difference for the pseudomonas syringae pv.tomato that detection embodiment 1 obtains
There is no cross reaction with compareing bacterium.Concretely comprise the following steps：
1. it is coated with：ACD companies (are purchased from, its catalog number is using the coated antibody of pseudomonas syringae pv.tomato
B264-C2) according to concentration (1:200) coated elisa plate, 100 μ L/ holes, solvent are coating buffer solution；37 DEG C of incubation 2h, are used
Cleaning solution washes ELISA Plate.
2. plus for examination bacterium：It is separately added into (dense with the diluted pseudomonas syringae pv.tomato of Sample extraction buffer solution, control bacterium
Spend for 108CFU/mL) bacterium solution or it is enzyme-linked use positive control, 100 μ L/ holes, 4 DEG C of refrigerator overnights, ELISA Plate is washed with cleaning solution.
3. enzyme mark monoclonal antibody：Will be according to volume ratio 1:The alkaline phosphatase mark that the step of 200 dilution (1) is prepared
Monoclonal antibody be added in ELISA Plate, 100 μ L/ holes, 37 DEG C incubation 4h, wash ELISA Plate with cleaning solution.
4. develop the color：Substrate pNPP is added in substrate solution by the concentration of 1mg/mL, is added to after mixing in enzyme mark hole,
100 μ L/ holes, 37 DEG C of avoid light places, 30min, 60min, 90min or the 405nm readings for reacting the longer time.
In triplicate, results are averaged for experiment.
It the results are shown in Table 3, it can be seen that the OD of pseudomonas syringae pv.tomato405nmIt is worth for 1.719, and remaining control bacterium
OD405nmValue is respectively less than 0.1.As it can be seen that the monoclonal antibody specificity for the pseudomonas syringae pv.tomato that embodiment 1 obtains is good, with
Experiment control bacterium used does not have cross reaction.
The specificity of 3 monoclonal antibody of table
2nd, the monoclonal antibody that embodiment 1 obtains and the reaction of pseudomonas syringae pv.tomato different strains
1st, the preparation of pseudomonas syringae pv.tomato bacterial strain
With pseudomonas syringae pv.tomato (Pseudomonas syringae pv.tomato) ATCC BAA- of culture
871st, NCPPB1106 and LX11 (China Inst. of Quarantine Inspection Sciences plant quarantine research institute QB0127) is material, for test
With.
2nd, the reaction of monoclonal antibody and pseudomonas syringae pv.tomato different strains
Using DAS-ELISA methods, the pseudomonas syringae pv.tomato monoclonal after purification that detection embodiment 1 obtains resists
The reaction for the pseudomonas syringae pv.tomato different strains that body is obtained with step 1.Concrete operations, only will step referring to step 12 (2)
The pseudomonas syringae pv.tomato different strains that step 1 acquisition is changed to for examination bacterium of rapid 2. middle addition, its dosage are 108CFU/
ML, per 100 μ L of hole.
In triplicate, results are averaged for experiment.
4 are the results are shown in Table, from table 4, it can be seen that the OD of pseudomonas syringae pv.tomato different strains405nmValue is in 0.980-
Between 1.720, strong positive reaction is presented.As it can be seen that embodiment 1 obtain pseudomonas syringae pv.tomato monoclonal antibody with kind
The reaction of eggplant bacterial leaf spot different strains is positive, and has preferable broad spectrum activity.
The reaction of 4 monoclonal antibody of table and pseudomonas syringae pv.tomato different strains
| The bacterial strain of pseudomonas syringae pv.tomato | OD405nmValue |
| ATCC BAA-871 | 1.719 |
| NCPPB 1106 | 1.013 |
| LX-11 | 0.979 |
The purity and affinity costant of the monoclonal antibody for the pseudomonas syringae pv.tomato that embodiment 3, embodiment 1 obtain
First, the purity of the monoclonal antibody for the pseudomonas syringae pv.tomato that embodiment 1 obtains
1.SDS-PAGE detects monoclonal antibody purity
Using SDS-PAGE methods, concretely comprise the following steps：
(1) glue is matched somebody with somebody：Configure PAGE concentration glue and 10% separation gel.
(2) loading：Example 1 obtains monoclonal antibody after purification and after isometric sample-loading buffer boils 5min, and 4
DEG C preserve.Loading cumulative volume is no more than 25ul/ holes.Add molecular weight marker proteins Pierce companies (article No. at the same time：26616)
Marker。
(3) glue is run：Concentrate glue voltage is 80V or so, after bromophenol blue indicator goes to concentration glue-separation gel intersection, is adjusted
Electrophoresis apparatus voltage is saved to 100V.Stop electrophoresis when bromophenol blue reaches glue bottom.
(4) dye and decolourize：0.125% coomassie brilliant blue staining liquid is added, boiling (2-3min) is heated to and shakes 1h.Return
Dyeing liquor is received, is decolourized overnight with destainer (5% methanol, 10% glacial acetic acid).
The results are shown in Figure 1, it can be seen that the monoclonal antibody (# for the pseudomonas syringae pv.tomato that embodiment 1 obtains
10) heavy chain molecule amount is about 55kD, and light chain molecule amount is about 25kD.No other miscellaneous bands occur, and illustrate the purity of monoclonal antibody
It is higher.
2.ELISA detects the affinity costant of monoclonal antibody
Specific assay method：
(1) it is coated with：100 μ L concentration 10 are added in ELISA Plate8Pseudomonas syringae pv.tomato (the ATCC of CFU/mL
BAA-871 bacteria suspension (solvent is coating buffer solution)), 4 DEG C of overnight incubations, are washed 3 times with cleaning solution.
(2) close：With the phosphate buffer sealing plate containing 2% balf serum albumin (BSA), 37 DEG C of incubation 2h, with washing
Liquid is washed to wash 3 times.
(3) test antibodies are added：Add the pseudomonas syringae pv.tomato of the purifying of the preparation of embodiment 1 of 2 times of gradient dilutions
Monoclonal antibody, 100 μ L/ holes, 37 DEG C of incubators, 1h；Wash liquid is used afterwards 3 times.
(4) ELIAS secondary antibody is added：Goat anti-mouse HRP ELIAS secondary antibodies (are purchased from the limited public affairs of Beijing Zhong Shan Golden Bridge biotechnology
Department, cat. no ZB2305) with enzyme labelled antibody dilution buffer 20000 times are diluted, add 100 μ L per hole, under the conditions of 37 DEG C
It is incubated 1h, board-washing 3 times.
(5) develop the color：The nitrite ion that horseradish peroxidase reacts is added in ELISA Plate hole, per 100 μ L of hole, during colour developing
Between for 5min or so.
(6) reaction is terminated：The 2M sulfuric acid terminate liquids of 50 μ L are added per hole, terminate reaction.
(7) reading：With 450nm, the dual wavelength measure of 630nm respectively treats gaging hole OD values, with the ratio with negative control hole OD values
Value (P/N) is limited more than 2.1, as the critical point for being judged as positive or definite potency.ELISA result judgement methods：If P/N>
2.1, then it is determined as the positive.
As a result such as table 5.
5 monoclonal antibody affinity costant of table measures
According to the measurement result of table 5, Fig. 2 is drawn out, according to affinity costant ≈ 150000 × A/ antibody concentrations, in A representatives
The corresponding antibody extension rate of the 1/2OD values of platform.Draw No. 10 CGMCC No.10119 of mouse hybridoma cell strain Pst
The affinity costant of the monoclonal antibody of secretion is 9.65E+08.
The characteristic for summarizing monoclonal antibody prepared by above-described embodiment 1 is as shown in table 6：
The fundamental characteristics of 6 monoclonal antibody of table
Claims (8)
1. hybridoma cell strain Pst 10, its preserving number is CGMCC No.10119.
2. the monoclonal antibody produced as No. 10 secretions of hybridoma cell strain Pst described in claim 1.
3. a kind of enzyme labelled antibody, for the enzyme labelled antibody formed after the monoclonal antibody described in claim 2 is marked with marker enzyme.
4. enzyme labelled antibody according to claim 3, it is characterised in that：The marker enzyme is alkaline phosphatase.
5. the enzyme labelled antibody described in monoclonal antibody or claim 3 or 4 described in claim 2 is preparing tomato bacterial leaf
Application in pinta bacterium detection kit.
6. the monoclonal antibody described in claim 2 is preparing the colloidal gold colloidal gold detection test paper strip of detection pseudomonas syringae pv.tomato
In application.
It is 7. thin for detecting tomato containing the enzyme labelled antibody described in the monoclonal antibody or claim 3 described in claim 2
The kit of bacterium property leaf spot fungi.
8. the colloidal gold detection examination for being used to detect pseudomonas syringae pv.tomato containing the monoclonal antibody described in claim 2
Paper slip.
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| CN102586470A (en) * | 2012-04-14 | 2012-07-18 | 黑龙江八一农垦大学 | Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit |
| CN103025160A (en) * | 2010-07-28 | 2013-04-03 | 日本农药株式会社 | Sigatoka disease control method |
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| CN102586470A (en) * | 2012-04-14 | 2012-07-18 | 黑龙江八一农垦大学 | Polymerase chain reaction (PCR) detection kit for maize curvularia leaf spot and detection method for PCR detection kit |
Non-Patent Citations (1)
| Title |
|---|
| Production of monoclonal antibodies to Pseudomonas syringae pv. phaseolicola and Xanthomonas campestris pv.Phaseoli;W.C.Wong;《Letters in Applied Microbiology》;19901231;第10卷;第243页左栏，表1 * |
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