CN104316687B - A kind of DASELISA immunization detecting pseudomonas syringae spot pvs oryzae and oryzicola germ - Google Patents
A kind of DASELISA immunization detecting pseudomonas syringae spot pvs oryzae and oryzicola germ Download PDFInfo
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Abstract
Detect a DASELISA immunization for pseudomonas syringae spot pvs oryzae and oryzicola germ, belong to immunoassay field.The present invention's deactivation pseudomonas syringae spot pvs oryzae and oryzicola deactivation germ immunity 8 weeks age BALB/c mouse, through immunity, Fusion of Cells, screen 6 strains product specific monoclonal antibodies hybridoma cell strain.6 strain antibodies mark HRP and respectively with pseudomonas syringae spot pvs oryzae and oryzicola germ for object matches between two.With antibody 1D3(CGMCC? No. Q, No.9312 monoclonal cell strain) and antibody 6C6(CGMCC? No. R, No.9313 monoclonal cell strain) respectively as coated antibody and enzyme labelled antibody, establish the double antibody sandwich ELISA of pseudomonas syringae spot pvs oryzae and oryzicola germ, LOD is 1.5*10
5cfu/mL.It is good, highly sensitive that ELISA method of the present invention detects pseudomonas syringae spot pvs oryzae and oryzicola germ specificity, and cost is low, can realize pseudomonas syringae spot pvs oryzae and oryzicola germ high throughput assay.
Description
Technical field
The present invention relates to the double antibody sandwich method of a kind of quantitative detection pseudomonas syringae spot pvs oryzae and oryzicola germ, belong to immunoassay field.
Background technology
Pseudomonas syringae spot pvs oryzae and oryzicola (Pseudomonassyringaepv.Maculicola) is brassicaceous vegetable germ important in the world, mainly causes Cruciferae bacterial black rot.This germ belongs to prokaryotes bacterium circle, proteus door, pseudomonadaceae in classification.The suitableeest cultivation temperature of this bacterium is at 25-27 DEG C, and main host has the brassicaceous vegetables such as Chinese cabbage, radish, cauliflower.
Strengthen quarantine, it is the important measures controlling this germ that prevention infected seed is propagated at Pest-or disease-free area.Domestic and international report mainly, in pathogenesis, isolated strains somatotype etc., yet there are no the report of immune detection pseudomonas syringae spot pvs oryzae and oryzicola germ.
Summary of the invention
(1) technical matters that will solve
The object of the invention is to the double-antibody sandwich elisa detection method setting up a kind of pseudomonas syringae spot pvs oryzae and oryzicola germ, for special, sensitive, detect pseudomonas syringae spot pvs oryzae and oryzicola germ in batches, fast.
(2) technical scheme
Realize above-mentioned purpose, the present invention first apply deactivation pseudomonas syringae spot pvs oryzae and oryzicola germ immunity 8 weeks age BALB/c mouse, obtain after immunity, Fusion of Cells, screening 6 strains generation monoclonal antibody specifics hybridoma cell strain.6 strain antibodies mark horseradish peroxidase (HRP) and respectively with pseudomonas syringae spot pvs oryzae and oryzicola germ for object matches between two.Using antibody 1D3 and 6C6 as coated antibody and enzyme labelled antibody, establish the double-antibody sandwich elisa analytical approach of pseudomonas syringae spot pvs oryzae and oryzicola germ, LOD is 1.5*10
5cfu/mL.
A kind of DASELISA immunization detecting pseudomonas syringae spot pvs oryzae and oryzicola germ, it is the ELISA method based on monoclonal antibody, coated antibody is No. Q, 1D3 and CGMCCNo.9312 monoclonal cell strain, and detecting antibody is No. R, 6C6 and CGMCCNo.9313 monoclonal cell strain;
(1) preparation of pseudomonas syringae spot pvs oryzae and oryzicola germ monoclonal antibody:
With pseudomonas syringae spot pvs oryzae and oryzicola germ deactivation thalline (NCPPB1820, bacterial strain imports and exports inspection and quarantine bureau from Hunan Province) as immunogene, the BALB/c mouse in immune 8 week age; Obtain through hybridoma technology fusion, screening;
(2) the pairing screening of monoclonal antibody:
6 strain monoclonal antibodies after purifying are marked horseradish peroxidase HRP respectively, after the success of direct method identification marking, carries out sandwich method pairing; Pairing parameter is as follows: coated antibody 2 μ g/mL; Coating buffer is the carbonate buffer solution of 0.01M, pH9.6; Mark product concentration 1 × 10
8cfu/mL; The PBS of mark product dilution 0.01M, pH7.2; Enzyme labelled antibody dilutes 500 times of uses; With this understanding, Success in Experiment obtains the pairing of 6 couples of P/N value >5;
(3) foundation of sandwich method: select detectability stablize, sensitive pairing, namely with No. Q, 1D3 and CGMCCNo.9312 monoclonal cell strain for coated antibody, No. R, 6C6 and CGMCCNo.9313 monoclonal cell strain sets up sandwich method as enzyme labelled antibody; Design parameter is as follows:
Coated antibody 1D3 concentration: 5 μ g/mL;
Coating buffer: 0.01M, pH9.6 carbonate buffer solution;
Mark product dilution: 0.01M, pH7.2PBS+0.2%Tween;
Enzyme labelled antibody 6C6-HRP concentration: 2 μ g/mL;
Reaction time: coated antibody: close 37 DEG C, reaction 2h; Standard items: 37 DEG C, reaction 1h; Enzyme labelled antibody 37 DEG C, reaction 1h; Developing time 10min.
The LOD of the DASELISA immunization of described detection pseudomonas syringae spot pvs oryzae and oryzicola germ is 1.5 × 10
5cfu/mL.
The detection analysis principle of the inventive method is:
In ELISA Plate, bag is by appropriate capture antibody 1D3; Wash plate 3 times, wash away unconjugated antibody, add confining liquid 220 μ L and close unnecessary binding site on plate hole; Wash plate 3 times, add sample and contrast, hatch 1h for 37 DEG C; Wash plate 3 times, add enzyme labelled antibody 6C6-HRP, hatch 1h for 37 DEG C; Wash plate 4 times, add nitrite ion colour developing 12min.If sample has enough pseudomonas syringae spot pvs oryzae and oryzicola germs, so pseudomonas syringae spot pvs oryzae and oryzicola germ is captured antibody capture being combined with enzyme labelled antibody 6C6-HRP, and catalytic substrate produces absorption value (P/N >=2.1) at 450nm, and be judged as the positive; If sample is so captured without pseudomonas syringae spot pvs oryzae and oryzicola germ without pseudomonas syringae spot pvs oryzae and oryzicola germ or concentration too low (P/N≤2.1) or catches too little being not enough to of quantity cause enough signals, be judged as feminine gender.
(3) beneficial effect
Pseudomonas syringae spot pvs oryzae and oryzicola germ double-antibody sandwich detection method provided by the invention have employed the pseudomonas syringae spot pvs oryzae and oryzicola germ monoclonal antibody that physicochemical property height is homogeneous, specificity good, can prepare in a large number, the sandwich method set up is highly sensitive, good stability, cost are low, a large amount of sample can be detected simultaneously, be applicable to the testing requirement of pseudomonas syringae spot pvs oryzae and oryzicola germ, there are promotion and application and be worth.
Biological material specimens preservation: pseudomonas syringae spot pvs oryzae and oryzicola germ (NCPPB1820), imports and exports inspection and quarantine bureau from Hunan Province.No. Q, cell line, Classification And Nomenclature monoclonal cell strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number CGMCCNo.9312, preservation date on May 28th, 2014.No. R, cell line, Classification And Nomenclature monoclonal cell strain, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number CGMCCNo.9313, preservation date on May 28th, 2014.
Accompanying drawing explanation
The typical curve of Fig. 1 pseudomonas syringae spot pvs oryzae and oryzicola germ double antibody sandwich method.
Specific embodiments
Further illustrate the present invention by the following examples.
One, instrument:
TGL-40B table-type low-speed hydro-extractor, Anting Scientific Instrument Factory, Shanghai;
KFLOW water purification machine, Kai Folong company;
ZD – 9556 horizontal shaker, granary science and education equipment factory;
The 96 removable ELISA Plate in hole 8 × 12, Xiamen experiment equipment company limited of happy Jiamei;
MuLtiskaMks microplate reader, ThermoLabsystems company;
Adjustable pipettor, ThermoLabsystems company;
Turbine mixer, Shanghai Hu Xi instrumental analysis factory.
Two, reagent:
Tetramethyl benzidine (TMB), Shanghai Jingchun Industrial Co., Ltd.;
Other reagent are analytical reagent.
Three, step
1. the preparation of monoclonal antibody
A, animal used as test: select the BALB/c mouse in 58 week ages to carry out immunity;
B, antigen are prepared: by immunogene normal saline dilution, be made into 10
9the solution of cfu/mL;
C, emulsification: by above-mentioned immunogen solution and equivalent is complete or incomplete freund adjuvant mix and blend method by its emulsification, subcutaneous multi-point injection mouse after emulsification completely;
Immunization method: according to specific immune flow process immune mouse, 3 exempt from rear indirect competitive mensuration tires, and tires after reaching requirement, carries out spurt immunity; Punching is merged after exempting from posterior orbit blood sampling in 3 days;
D, blood sampling: within 1 week, carry out docking blood sampling after third time immunity, adopt indirect non-competing euzymelinked immunosorbent assay (ELISA) to measure antiserum titre;
E, fusion, screening: adopt hybridoma technology to merge, adopt indirect ELISA screening positive cell hole, adopt limiting dilution assay to carry out subclone to positive hole;
The purifying of f, antibody and preservation: adopt sad-saturated ammonium sulfate method purifying ascites, obtain monoclonal antibody after dialysis, adopt micro-UV process to measure its concentration, after packing, put into-20 DEG C of preservations.
2, ELISA course of reaction:
Antibody titer determination step:
A, coating antigen bag is buffered liquid makes serial dilution bag by 96 hole ELISA Plate, 100 μ L/ holes, in 4 DEG C of refrigerator overnight; Next day takes out ELISA Plate and is back to room temperature, and 200 μ LPBST solution are injected in every hole, and shaking table vibrates 3min, firmly gets rid of cleansing solution, thieving paper pats dry, and continues washing 2 times; Following washing methods is identical;
After b, fully washing, with Block buffer sealase target, 200 μ L/ holes, in 37 DEG C of incubation casees, after incubation 2h, taking-up is dried stand-by;
C, positive serum serial dilution correspondence is joined 7 ranks before ELISA Plate, eighth row adds negative serum, 100 μ L/ holes, 37 DEG C hatch 1h after washing, pat dry;
D, every hole add 100 μ L, the sheep anti-mouse igg of the HRP mark of 1:3000 dilution, 37 DEG C hatch 1h after washing, pat dry;
E, every hole add 100 μ L nitrite ions (TMB and substrate solution ratio are 1:5), and 37 DEG C, dark place reaction 15min, after taking out, every hole adds 100 μ L stop buffers (sulfuric acid of 2mol/L), measures light absorption value A by microplate reader
450.
Pseudomonas syringae spot pvs oryzae and oryzicola germ double antibody sandwich method determination step:
A, bag quilt: with the 1D3 coated elisa plate of 5 μ g/mL, 100 μ L/ holes, 4 DEG C are spent the night;
B, washing: use PBST detersive enzyme target three times, each 3min, 200 μ L/ holes, then dry ELISA Plate;
C, close: containing the CBS of 0.2% gelatin, 200 μ L/ holes, 37 DEG C of closed 2h;
D, washing: same to b;
E, sample: pseudomonas syringae spot pvs oryzae and oryzicola germ is diluted to 1 × 10 with PBST
8, 3.3 × 10
7, 1.1 × 10
7, 3.7 × 10
6, 1.23 × 10
6, 4.12 × 10
5, 1.37 × 10
5cfu/mL series concentration, separately establishes a PBST blank.Every hole adds 100 μ L samples, in 37 DEG C of incubation 1h;
F, washing: same to b;
G, add enzyme labelled antibody (6C6-HRP, 2 μ g/mL), 100 μ L/ holes, 37C reacts 1h;
H, washing: same to b;
I, colour developing: add substrate TMB100 μ L/ hole, colour developing 10min;
J, termination: add stop buffer 50 μ L/ hole;
K, mensuration: detect OD by microplate reader
450nm.
The mensuration of cross reaction:
By Burkholderia glumae (Burkholderiaglumae, NCPPB3591), xanthomonas oryzae pv. oryzicola (Xanthomonasoryzaepv.Oryzicola, NCPPB1585), P.stwartii subsp.stewartii (Pantoeastewartiisubsp.stewartii, NCPPB449), pseudomonas syringae cloves pvs oryzae and oryzicola (Pseudomonassyringaepv.syringae, NCPPB2844) is diluted to 10
8cfu/mL, 10
7cfu/mL, light absorption value is detected in the pseudomonas syringae spot pvs oryzae and oryzicola germ double-antibody sandwich law system set up, and establish blank well and pseudomonas syringae spot pvs oryzae and oryzicola germ positive control, each concentration is done 6 times and is measured mean value, does and repeats experiment for 3 times.
Test findings is as follows:
1, typical curve: the sensing range that the antigen that the present invention obtains detects is 10
6~ 10
8cfu/mL, specifically asks for an interview Figure of description Fig. 1.
2, LOD:LOD is that blank mean absorbance adds cell concentration corresponding to the standard deviation of the blank absorption value of 3 times, and pseudomonas syringae spot pvs oryzae and oryzicola germ double antibody sandwich method LOD is 1.5 × 10
5cfu/mL.
3, cross reacting rate (CR%)
Result: pseudomonas syringae spot pvs oryzae and oryzicola germ is normally developed the color, and 10
8other test strain of cfu/mL and following concentration does not all develop the color (OD<0.15).The pseudomonas syringae spot pvs oryzae and oryzicola germ double antibody sandwich ELISA and Burkholderia glumae set up are described, xanthomonas oryzae pv. oryzicola, P.stwartii subsp.stewartii, pseudomonas syringae cloves pvs oryzae and oryzicola no cross reaction, specificity is good.
Claims (2)
1. one kind is detected the DASELISA immunization of pseudomonas syringae spot pvs oryzae and oryzicola germ, it is characterized in that the ELISA method based on monoclonal antibody, coated antibody is No. Q, 1D3 and CGMCCNo.9312 monoclonal cell strain, and detecting antibody is No. R, 6C6 and CGMCCNo.9313 monoclonal cell strain;
(1) preparation of pseudomonas syringae spot pvs oryzae and oryzicola germ monoclonal antibody:
With pseudomonas syringae spot pvs oryzae and oryzicola germ deactivation thalline NCPPB1820 as immunogene, the BALB/c mouse in immune 8 week age; Obtain through hybridoma technology fusion, screening;
(2) the pairing screening of monoclonal antibody:
6 strain monoclonal antibodies after purifying are marked horseradish peroxidase HRP respectively, after the success of direct method identification marking, carries out sandwich method pairing; Pairing parameter is as follows: coated antibody 2 μ g/mL; Coating buffer is the carbonate buffer solution of 0.01M, pH9.6; Mark product concentration 1 × 10
8cfu/mL; The PBS of mark product dilution 0.01M, pH7.2; Detect antibody dilution 500 times use; With this understanding, Success in Experiment obtains the pairing of 6 couples of P/N value >5;
(3) foundation of sandwich method: select detectability stablize, sensitive pairing, namely with No. Q, 1D3 and CGMCCNo.9312 monoclonal cell strain for coated antibody, No. R, 6C6 and CGMCCNo.9313 monoclonal cell strain as detection antibody set up sandwich method; Design parameter is as follows:
Coated antibody 1D3 concentration: 5 μ g/mL;
Coating buffer: 0.01M, pH9.6 carbonate buffer solution;
Mark product dilution: 0.01M, pH7.2PBS+0.2%Tween;
Detect antibody 6C6-HRP concentration: 2 μ g/mL;
Reaction time: coated antibody: close 37 DEG C, reaction 2h; Standard items: 37 DEG C, reaction 1h; Detect antibody 37 DEG C, reaction 1h; Developing time 10min.
2. detect the DASELISA immunization of pseudomonas syringae spot pvs oryzae and oryzicola germ according to claim 1, it is characterized in that: the LOD of the DASELISA immunization of described detection pseudomonas syringae spot pvs oryzae and oryzicola germ is 1.5 × 10
5cfu/mL.
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| WO1996030053A1 (en) * | 1995-03-28 | 1996-10-03 | The General Hospital Corporation | Methods of screening compounds useful for prevention of infection or pathogenicity |
| CN101838703A (en) * | 2010-05-26 | 2010-09-22 | 中国农业科学院植物保护研究所 | Method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot |
| CN103713104A (en) * | 2013-12-26 | 2014-04-09 | 中华人民共和国淮安出入境检验检疫局 | Double-antibody sandwich method for detecting enterobacter sakazakii in food |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996030053A1 (en) * | 1995-03-28 | 1996-10-03 | The General Hospital Corporation | Methods of screening compounds useful for prevention of infection or pathogenicity |
| CN101838703A (en) * | 2010-05-26 | 2010-09-22 | 中国农业科学院植物保护研究所 | Method for quickly detecting pathogenic bacteria carried by plant seeds of bacterial black rot |
| CN103713104A (en) * | 2013-12-26 | 2014-04-09 | 中华人民共和国淮安出入境检验检疫局 | Double-antibody sandwich method for detecting enterobacter sakazakii in food |
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| Title |
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| Pseudomonas syringae pv. maculicola in Australia: pathogenic, phenotypic and genetic diversity;B. J. Peters et al.;《Plant Pathology》;20040204;第53卷(第1期);73-79 * |
| 丁香假单胞菌中六个致病变种的全细胞蛋白质电泳分析;黄琼等;《云南农业大学学报》;19961231;第11卷(第4期);197-202 * |
| 萝卜细菌性黑斑病在高山菜区发生规律观察;喻法金等;《湖北植保》;20070515(第3期);34-36 * |
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