CN110386979A - A kind of method preparing mulberry Ralstonia solanacearum antibody and its application in DAS-ELISA detection method - Google Patents
A kind of method preparing mulberry Ralstonia solanacearum antibody and its application in DAS-ELISA detection method Download PDFInfo
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Abstract
A kind of method preparing mulberry Ralstonia solanacearum antibody and its application in DAS-ELISA detection method.It is immune animal with BALA/c mouse, it is immune using Ralstonia solanacearum living strain, prepare the monoclonal antibody of anti-Ralstonia solanacearum and the DAS-ELISA detection method for constructing Ralstonia solanacearum.It the use of bacteria suspension concentration is 106‑108Mouse is immunized in the living body Ralstonia solanacearum bacteria suspension of CFU/mL, and filter out hybridoma, prepare mulberry Ralstonia solanacearum DAS-ELISA kit, wherein, the skimmed milk power of 0.1-10% only contains the medicament such as bovine serum albumin(BSA) of albumen as confining liquid, and the TMB developing solution time is 10-60min, and the antibody action time of horseradish peroxidase label is 10-60min, the antibody extension rate of horseradish peroxidase label is 1:100-1:5000, and capture antibody extension rate is 1:100-1:5000.This technology there is good detection sensitivity lowest detection to be limited to 3.13 × 10 mulberry Ralstonia solanacearum3CFU/mL is and easy to operate, it can be achieved that detection in 2.5h to mulberry Ralstonia solanacearum, and detection is quick.
Description
Technical field
The invention belongs to biotechnologys and field of immunology, and in particular to a kind of side for preparing mulberry Ralstonia solanacearum monoclonal antibody
Method and its application in DAS-ELISA detection method.
Background technique
Ralstonia solanacearum, i.e., green withered Raul Salmonella (Ralstonia solanacearum) are to cause vegetative bacteria wilting (green
Blight) one of main plant pathogen.Ralstonia solanacearum can infect 50 sections, more than 500 kinds of effects, be located at public in the world in plant disease
Recognize the second of most harmfulness pathogen seniority among brothers and sisters version.Just have nearly that 30 provinces have been reported that bacterial wilt disease in China, mainly
Endangering plant is numerous crops, the industrial crops such as mulberry tree, tobacco and the portions such as potato, tomato, peanut, eggplant, ginger, capsicum
Point rare traditional Chinese medicine causes significant damage for China's agricultural production and sericulture there development, if therefore can to Ralstonia solanacearum into
Row timely detects, and will strive for valuable time for guarantee agricultural production and sericulture there.
Currently, being mainly based upon the detection method of round pcr, for the detection mode of such pathogen with the spy of Ralstonia solanacearum
Specific nucleic acid sequences or Special Proteins establish the quantitative relation between target substance concentration and Ralstonia solanacearum strain concentration as target,
To carry out Ralstonia solanacearum strain quantity the detection of indirect with this.Such method needs use the complex instruments such as similar PCR instrument,
And complicated operation, more demanding to testing conditions, therefore, promotes be restricted in agricultural production, be unfavorable for existing
Field sample is detected, and detection timeliness is difficult to ensure.
The research work of such as Said (Microb Pathogenesis, 2018,115:216-221.) in the prior art
In, New Zealand White Rabbit is immunized using No. 3 physiological species 2 biochemical types in green withered Raul Bordetella, and prepared and exempted from this
Epidemic disease serum obtains rabbit source polyclonal antibody, for constructing the indirect ELISA detection side to the biochemical type of the physiological species 2 of Ralstonia solanacearum 3
Method, detection time is 3h and lowest detection is limited to 106CFU/mL.Antibody to animal immune and is prepared with good using living strain
Good feasibility, but the antibody as used in indirect ELISA method constructed in the research of Said etc. is polyclonal antibody,
There are problems that causing detection sensitivity to receive influence in terms of specificity.
Enzyme linked immunosorbent assay (ELISA) (Enzyme Linked Immunosorbent Assay, ELISA), comes across earliest
Engvall etc. (Immunochemistry, 1971,8 (9): 871-874.) is ground using standard measure measurement immunoglobulin
Study carefully, the main specific reaction combined using antibody antigen in immunology carries out specific recognition to target substance and enzyme is urged
Change reaction high efficiency by specific recognition signal by be catalyzed reaction shows, by naked eyes or spectrophotometer into
Row quantitative detecting analysis.Zhang etc. (Anal Chem, 2014,86 (2): 1115-1122.) is based on laccase according to ELISA principle
Design for detecting Escherichia coli O 157: the system of H7 has the stationary phase of capture antibody, rabbit using magnetic nano particle as load
Source polyclonal antibody captures Escherichia coli as capture antibody, for specificity, the source of mouse monoclonal antibody after biotin labeling
As labelled antibody, the H generated in glucose oxidase catalysis reaction is utilized2O2Under the catalysis of laccase with luminol generationization
Luminescence-producing reaction is learned, completes enrichment and detection signal amplification to Escherichia coli in the above manner.The system is to Escherichia coli
The lowest detection of O157:H7 is limited to 1.2 × 103CFU/mL, and detection time be less than 2h, be successfully used for spring, cider and
The sample detections such as skim milk.To sum up, based on immunology and the sensitivity with higher of serological ELISA detection technique and
Short detection time is expected to be applied in the sample of field in the detection and analysis of Ralstonia solanacearum.
In detection method building based on by ELISA principle, sensitivity of the selection of antigenic targets to detection method
With important influence.Two kinds of target antibodies are successively reported in terms of Ralstonia solanacearum targeting antibodies preparation.One kind is to Ralstonia solanacearum
Secretion has the antibody of specificity, such as monoclonal antibody for being used to detect Ralstonia solanacearum M2 bacteriocin early in report in 1991
(Science Bulletin, 1991, (14): 1098-1101.)；Brix leaf etc. (Chongqing Normal University, 2016.) selects the interior of mulberry Ralstonia solanacearum
Dextranase EGL is cut as target, prepares effect for mouse to be immunized using the EGL after prokaryotic expression as specific antigen
The EGL source of mouse antibody serum that valence is 20000, and detected with the Western blot of mulberry Ralstonia solanacearum viable bacteria in Soil of Mulberry Garden, show
It is capable of the endoglucanase of specific detection mulberry Ralstonia solanacearum.Another kind of is to have targeting ability to Ralstonia solanacearum epicyte protein
Antibody has good specific aim to Ralstonia solanacearum using the detection means that Ralstonia solanacearum secretion is target building, helps to reduce
False positive, but whether Ralstonia solanacearum is secreted in the soil and its secretory volume has otherness, thus cause to detect misalignment.
Summary of the invention
In view of the above technical problems, the present invention provides a kind of method for preparing mulberry Ralstonia solanacearum monoclonal antibody and its in DAS-
Application in ELISA detection method, i.e. invention are using bacterial strain surface antigen protein as target, by carrying out to BALA/c mouse
It is immune, the hybridoma that can secrete Ralstonia solanacearum resistance is filtered out, and isolate and purify out monoclonal antibody, it is withered for constructing Sang Qing
DAS-ELISA detection method, the detection method can realize the detection to Ralstonia solanacearum in 2.5h, and easy to operate, be convenient for field
Operation.
A kind of method preparing mulberry Ralstonia solanacearum monoclonal antibody and its application in DAS-ELISA detection method, with bacterium
Suspension concentration is 106-108The living body Ralstonia solanacearum bacteria suspension of CFU/mL takes and reaches after mouse immune processing as immunizing antigen
Mouse spleen cells and murine myeloma cell after more than 10000 potency carry out cell fusion, filter out secretory antibody albumen
Hybridoma, and to antibody protein carry out purification process to get mulberry Ralstonia solanacearum antibody 9G9,1G9 and 2H5.
Preferably, the Ralstonia solanacearum is ATCC-BAA1114, GIM1.76, GIM1.74, GIM1.71 or GIM1.73..
The above method prepares application of the mulberry Ralstonia solanacearum antibody in DAS-ELISA detection method.
The specific steps of application are such as: the mulberry Ralstonia solanacearum antibody that extension rate is 1:100-1:5000 is coated on enzyme mark in advance
On plate, uses the skimmed milk power of 0.1-10% or only contains the medicament of albumen as confining liquid Seal treatment is carried out to ELISA Plate,
After sample solution or standard items are added, the horseradish peroxidase labeling antibody that extension rate is 1:100-1:5000 is added, is made
Be 10-60 min with the time, the horseradish peroxidase labeling antibody be combined with horseradish peroxidase mulberry Ralstonia solanacearum it is anti-
Ralstonia solanacearum in body, sample or standard items is combined with the capture antibody on ELISA Plate, forms " capture antibody-Ralstonia solanacearum-enzyme mark
Antibody " compound forms visual yellow substance after TMB developing solution develops the color 10-60min, thus in judgement sample
Whether contain Ralstonia solanacearum, light absorption value is measured using microplate reader at 450 nm, setting Ralstonia solanacearum culture solution is negative control
(Negative, N), sample light absorption value are sample detection value (Positive, P)；When P/N >=2, it is determined as the positive；P/N is between 1-
When 2, it is determined as weakly positive；When P/N≤1, it is determined as feminine gender.
Preferably, described to be used for coated mulberry Ralstonia solanacearum antibody extension rate as 1:1000.
Preferably, the medicament is bovine serum albumin(BSA).
Preferably, the TMB developing solution time is 30min.
Preferably, the horseradish peroxidase mark labeling antibody action time is 60min.
Preferably, the horseradish peroxidase labeling antibody extension rate is 1:500.
Preferably, lowest detection is limited to 3.13 × 103CFU/mL。
The utility model has the advantages that
Compared with prior art, the invention discloses a kind of method for preparing mulberry Ralstonia solanacearum monoclonal antibody and its in DAS-
BALA/c mouse is immunized using living body Ralstonia solanacearum in application in ELISA detection method, and preparing monoclonal antibody can be significantly
The potency for improving Ralstonia solanacearum antibody overcomes the low problem of phytopathogen antibody mediated immunity specificity, and passes through cleaning and concentration
Control solves phytopathogen toxicity problem caused by immune animal, using a process for preparing Ralstonia solanacearum specificity it is single
Clonal antibody potency is 64000, is conducive to the sensitivity for improving DAS-ELISA detection method.By the optimization to testing conditions,
So that detection sensitivity is reduced to 3.13 × 103CFU/mL.In addition, using thallus directly as target in the present invention, detection
Accuracy is only related to Ralstonia solanacearum cell membrane surface target site, can exclude the influence of the specific growth conditions of Ralstonia solanacearum, can be more
Accurately detect Ralstonia solanacearum.Therefore, there is good operation possibility as target using Ralstonia solanacearum cell membrane surface protein, has
It hopes the detection effect improved in practical application to Ralstonia solanacearum, provides effective information for prevention bacterial wilt.
Specific embodiment
Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention
Rather than it limits the scope of the invention.In addition, it should also be understood that, after reading the content taught by the present invention, those skilled in the art
Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited
Range.
The preparation of Ralstonia solanacearum specific antibody in the present invention: bacteria suspension concentration 106-108The living body Ralstonia solanacearum of CFU/mL
(ATCC-BAA1114, GIM1.76, GIM1.74, GIM1.71 and GIM1.73) is used as immunizing antigen, according to patent of invention
CN102206274A " bacterial canker of tomato method for preparing monoclonal antibody and its kit and application method " preparation.
ATCC-BAA1114, purchase are received in Beijing North and create connection, and for green withered No. 1 microspecies of Raul Salmonella, host is tomato.
GIM1.76, purchase is in Guangdong common micro-organisms collection (GIMCC), for green withered No. 2 microspecies of Raul Salmonella, place
Main is horse-tail.
GIM1.74, purchase is in Guangdong common micro-organisms collection (GIMCC), for green withered No. 3 microspecies of Raul Salmonella, place
Main is the wooden potato.
GIM1.71, purchase is in Guangdong common micro-organisms collection (GIMCC), for green withered No. 4 microspecies of Raul Salmonella, place
Main is ginger.
GIM1.73, purchase is in Guangdong common micro-organisms collection (GIMCC), for green withered No. 5 microspecies of Raul Salmonella, place
Main is mulberry tree.
Embodiment 1
This example demonstrates that the monoclonal antibody that BALA/c mouse is prepared is immunized by living body Ralstonia solanacearum, it is right after purification
Ralstonia solanacearum has high-titer.It is immunized by the way of intraperitoneal injection, immune metering is 0.4mL, OD every time600=0.6, side is immunized
Case is as shown in the table.
It is measured using indirect elisa method, prepared tri- kinds of monoclonal antibodies of 1G9,2H5,9G9, Ralstonia solanacearum is selected
GIM1.73 biological strain, i.e. mulberry Ralstonia solanacearum, bacterial strain extension rate is set as 1000,2000,4000,8000,16000,
32000 and 64000, initial bacteria suspension concentration is 108CFU/mL。
Testing result is as follows:
Embodiment 1 the result shows that, three kinds of monoclonal antibodies have good specificity to mulberry Ralstonia solanacearum, and valence value is
64000, it help to obtain highly sensitive DAS-ELISA detection method.
Embodiment 2
The present embodiment 2 illustrates that mass fraction is 1% skimmed milk power as confining liquid, and DSA-ELISA detection method is to Sang Qing
The detection process of withered bacterium.
Using DAS-ELISA principle, the use of 9G9 is capture antibody and is coated on ELISA Plate in advance, 1G9 is anti-as enzyme mark
Body, setting bacterial strain extension rate are set as 1000,2000,4000,8000,16000,32000 and 64000, and initial bacteria suspension is dense
Degree is 108CFU/mL.Testing conditions: the skimmed milk power of 0.1-10% or the only medicament containing albumen (such as bovine serum albumin(BSA))
As confining liquid, the TMB developing solution time is 30min, and horseradish peroxidase labeling antibody action time is 60min, captures antibody
Extension rate is 1:100-1:5000, and horseradish peroxidase labeling antibody extension rate is 1:100-1:5000, in this embodiment
Selecting enzyme labelled antibody and capture antibody extension rate is all 1:1000.
Testing result is as follows:
Show to be coated with the enzyme of 9G9 monoclonal antibody in advance when using 1% skimmed milk power as confining liquid in embodiment 2
Target has good detection effect with mulberry Ralstonia solanacearum, and has lower negative background's value.When confining liquid concentration is
0.5%, when P/N >=2, corresponding bacterium solution is not do diluted original bacteria liquid；It is corresponding when confining liquid concentration is 3%, P/N >=2
Bacterium solution maximum dilution multiple be 2000；And when confining liquid concentration is 1%, P/N >=2, corresponding bacterium solution maximum dilution multiple
It is 8000.Therefore, 1% skimmed milk power can get highly sensitive detection method as confining liquid.
Embodiment 3
When the present embodiment 3 illustrates that TMB developing time is 30min, detection of the DSA-ELISA detection method to mulberry Ralstonia solanacearum
Journey.
Using DAS-ELISA principle, the use of 9G9 is capture antibody and is coated on ELISA Plate in advance, 1G9 is anti-as enzyme mark
Body, setting bacterial strain extension rate are set as 1000,2000,4000,8000,16000,32000 and 64000, and initial bacteria suspension is dense
Degree is 108CFU/mL.Testing conditions: 1% skimmed milk power as confining liquid, the TMB developing solution time be set as 10min,
20min and 30min, enzyme labelled antibody action time are 60min, and labeling antibody extension rate is 1000.
Testing result is as follows:
Show to be coated with the ELISA Plate of 9G9 monoclonal antibody in advance when using 30min as developing time in embodiment 3
There is good detection effect with mulberry Ralstonia solanacearum.When developing time is 10min, P/N >=2, corresponding bacterium solution extension rate is most
Greatly 16000；When developing time is 20min, P/N >=2, corresponding bacterium solution dilution times maximum number is 2000；And when colour developing
Between be 30min, P/N >=2 when, corresponding bacterium solution extension rate is up to 8000；But when using original bacteria liquid at the same time, developing time
P/S value for 30min detection group is 3.17 times that developing time is 10min group.In the actual operation process, 10min detection group
In repeatedly there is the incomplete phenomenon of developing the color, it is relatively low to may cause detected value.Therefore, it when 30min is as developing time, can get
Highly sensitive detection method.
Embodiment 4
This example demonstrates that when enzyme labelled antibody action time is 60min, inspection of the DSA-ELISA detection method to mulberry Ralstonia solanacearum
Survey process.
Using DAS-ELISA principle, the use of 9G9 is capture antibody and is coated on ELISA Plate in advance, 1G9 is anti-as enzyme mark
Body, setting bacterial strain extension rate are set as 1000,2000,4000,8000,16000,32000 and 64000, and initial bacteria suspension is dense
Degree is 108CFU/mL.Testing conditions: for 1% skimmed milk power as confining liquid, 30min is arranged in the TMB developing solution time, and enzyme mark is anti-
Body action time is set as 30min, 45min and 60min, and labeling antibody extension rate is 1000.
Testing result is as follows:
Show to be coated with 9G9 monoclonal antibody in advance when using 60min as enzyme labelled antibody action time in embodiment 4
ELISA Plate with mulberry Ralstonia solanacearum have good detection effect and negative control value it is lower.It is 30min, P/N >=2 when action time
When, corresponding bacterium solution extension rate is up to 1000；When action time is 45min, P/N >=2, corresponding bacterium solution dilution times is most
Big number is 8000；And when action time is 60min, P/N >=2, corresponding bacterium solution extension rate is up to 8000；But at the same time
When using original bacteria liquid, action time is that the P/S value of 60min detection group is 3.1 times that developing time is 45min group.Therefore,
When 60min is acted on as enzyme labelled antibody, highly sensitive detection method can get.
Embodiment 5
This example demonstrates that when enzyme labelled antibody extension rate is 1:500, the detection of DSA-ELISA detection method mulberry Ralstonia solanacearum
Process.
Using DAS-ELISA principle, the use of 9G9 is capture antibody and is coated on ELISA Plate in advance, 1G9 is anti-as enzyme mark
Body, setting bacterial strain extension rate are set as 1000,2000,4000,8000,16000,32000 and 64000, and initial bacteria suspension is dense
Degree is 108CFU/mL.Testing conditions: for 1% skimmed milk power as confining liquid, 30min is arranged in the TMB developing solution time, and enzyme mark is anti-
Body action time is 60min, and labeling antibody extension rate is set as 500,1000,2000 and 3000.
Testing result is as follows:
Show to be coated with the enzyme of 9G9 monoclonal antibody in advance when being 500 with enzyme labelled antibody extension rate in embodiment 5
Target has good detection effect with mulberry Ralstonia solanacearum.When enzyme labelled antibody extension rate is 500, P/N occurs with concentration gradient
Change of gradient and bacterium solution extension rate be 32000 when, P/N 3.99, correspond at this time Ralstonia solanacearum bacteria suspension concentration be 3.13
×103CFU/mL, detection time 2.5h.
In conclusion the present invention can be fast and convenient the detection for mulberry Ralstonia solanacearum, and have high sensitivity, can
Accurately to detect Ralstonia solanacearum.
Claims (10)
1. a kind of method for preparing mulberry Ralstonia solanacearum antibody, which is characterized in that with bacteria suspension concentration for 106-108The living body of CFU/mL
Ralstonia solanacearum bacteria suspension takes the Mouse spleen cells after reaching 10000 or more potency after mouse immune processing as immunizing antigen
Cell fusion is carried out with murine myeloma cell, filters out the hybridoma of secretory antibody albumen, and carry out to antibody protein
Purification process is to get mulberry Ralstonia solanacearum antibody 9G9,1G9 and 2H5.
2. preparing the method for mulberry Ralstonia solanacearum antibody according to claim 1, which is characterized in that the Ralstonia solanacearum is ATCC-
BAA1114, GIM1.76, GIM1.74, GIM1.71 or GIM1.73.
3. the application based on preparation mulberry Ralstonia solanacearum antibody described in claim 1 in DAS-ELISA detection method.
4. application according to claim 3, which is characterized in that resist the mulberry Ralstonia solanacearum that extension rate is 1:100-1:5000
Body is coated on ELISA Plate in advance, is used the skimmed milk power of 0.1-10% or is only contained the medicament of albumen as confining liquid to enzyme mark
Plate carries out Seal treatment, after sample solution or standard items are added, adds the horseradish peroxide that extension rate is 1:100-1:5000
Change hydrogen enzyme labelled antibody, action time 10-60min, the horseradish peroxidase labeling antibody is to be combined with horseradish hydrogen peroxide
The mulberry Ralstonia solanacearum antibody of enzyme, the Ralstonia solanacearum in sample or standard items are combined with the capture antibody on ELISA Plate, form that " capture is anti-
Body-Ralstonia solanacearum-enzyme labelled antibody " compound forms visual yellow substance after TMB developing solution develops the color 10-60min,
To whether contain Ralstonia solanacearum in judgement sample, light absorption value is measured using microplate reader at 450 nm, setting Ralstonia solanacearum culture solution is
Negative control (Negative, N), sample light absorption value are sample detection value (Positive, P)；When P/N >=2, it is determined as the positive；
When P/N is between 1-2, it is determined as weakly positive；When P/N≤1, it is determined as feminine gender.
5. application according to claim 4, which is characterized in that described to be for coated mulberry Ralstonia solanacearum antibody extension rate
1:1000。
6. application according to claim 4, which is characterized in that the medicament is bovine serum albumin(BSA).
7. application according to claim 4, which is characterized in that the TMB developing solution time is 30min.
8. application according to claim 4, which is characterized in that the horseradish peroxidase labeling antibody action time is
60min。
9. application according to claim 4, which is characterized in that the horseradish peroxidase labeling antibody extension rate is 1:
500。
10. application according to claim 4, which is characterized in that lowest detection is limited to 3.13 × 103CFU/mL。
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