CN114942331A - Enzyme linked immunosorbent assay kit for detecting phytophthora parasitica and application thereof - Google Patents
Enzyme linked immunosorbent assay kit for detecting phytophthora parasitica and application thereof Download PDFInfo
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- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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Abstract
The invention discloses an enzyme-linked immunoassay kit for detecting tobacco black shank bacteria and application thereof. It includes: obtaining tobacco black shank bacterium hypha and spores through culture; immunizing a female BALB/c mouse with 6 weeks of age by hypha and spores, fusing splenocytes of the immunized mouse with myeloma cells SP2/0, obtaining a specific monoclonal antibody cell strain by screening, and obtaining a specific monoclonal antibody by purifying in a mode of preparing ascites; the HRP coupled antibody is used, and the paired antibody is screened by a double-antibody sandwich ELISA method, so that the tobacco phytophthora parasitica can be quantitatively detected; and producing a kit, and detecting the actual sample. The method can be used for qualitatively and quantitatively detecting the tobacco phytophthora parasitica in the tobacco, and has the advantages of strong specificity, high sensitivity, simplicity, rapidness and the like.
Description
Technical Field
The invention relates to the technical field of agricultural biology, and relates to preparation and application of an enzyme-linked immunosorbent quantitative detection kit for phytophthora parasitica.
Background
Tobacco black shank (Phopthhro nicotinae Breda de Hann) was first found in Java in 1896, is one of the devastating diseases of tobacco worldwide and can occur from seedling stage to adult stage. The tobacco black shank is also called black stalk crazy, aconite and the like, is a relatively serious soil-borne fungal disease caused by Phytophthora nicotianae (Phytophthora nicotianae), and due to the reasons of wide distribution of the tobacco black shank, long field latency and the like, tobacco plants are damaged by germs and then have the phenomena of withered and yellow leaves, rotting leaves, browning stems and even plant death, and the like.
The traditional method for detecting the phytophthora parasitica is mainly used for detecting the form and physiological and biochemical characteristics of pathogenic bacteria. With the development of molecular biology, molecular biology identification based on PCR is widely used. However, both the traditional method and the molecular biological identification have high requirements on professional literacy of operators, cannot be widely popularized, and cannot carry out on-site rapid detection on the tobacco phytophthora parasitica.
Enzyme-linked immunoassay (elisa kit detection method) is an immunological detection method developed in the late 60 s of the 20 th century, and is the most widely applied labeling immunoassay at present. In 1966, Nakene and Pierce published both "enzyme-labeled antibodies: the application in preparation and antigen positioning, firstly, the basic principle of enzyme-linked immunosorbent assay (ELISA kit detection method) is provided. Engval and Perlmann published article "determination of IgG content by ELISA KIT method" in 1971, which makes enzyme-linked immunosorbent assay (ELISA KIT detection) a practical method for detecting trace substances in liquid samples. At present, enzyme-linked immunosorbent assay (ELISA kit) detection technology is widely used in immunology, microbiology, parasitology and the like. Due to the advantages of high sensitivity, stable reagent, simple equipment, safe operation and the like, the method is also applied to the field of food in recent years to detect the physiologically active substances in certain foods.
Disclosure of Invention
In order to rapidly screen and detect the tobacco phytophthora parasitica, the invention provides preparation and application of an enzyme-linked immunosorbent quantitative detection kit of the tobacco phytophthora parasitica.
In order to achieve the purpose, the invention adopts the following technical scheme:
the first scheme comprises the following steps: culturing the tobacco black shank bacterium to obtain tobacco black shank bacterium hypha and spores; inducing and immunizing a New Zealand white rabbit by using the hypha spores, obtaining rabbit serum, and purifying to obtain a rabbit polyclonal antibody; inducing and immunizing a female BALB/c mouse with the age of 6 weeks by using the hypha spores, fusing splenocytes of the immunized mouse with myeloma cells SP2/0, obtaining a specific monoclonal antibody cell strain by screening, and obtaining a specific monoclonal antibody by purifying in a mode of preparing ascites; screening paired antibodies by using an ELISA method of double antibody sandwich; an ELISA kit system is obtained through optimization, and an ELISA method of double antibody sandwich is established; producing a kit and detecting the actual sample.
Scheme II: the invention provides a kit for detecting phytophthora parasitica, which comprises the monoclonal antibody and the polyclonal antibody of the first aspect.
Preferably, the kit comprises the following components:
(1) scheme one monoclonal antibody pre-coated enzyme label plate
(2) A sample diluent which is a phosphate buffer solution;
(3) the washing solution is phosphate buffer solution containing Tween-20;
(4) reaction terminating solution of H 2 SO 4 A solution;
(5) an enzyme-labeled antibody which is a polyclonal antibody labeled with horseradish peroxidase (HRP) and described in the first aspect;
(6) the color developing agent is TMB color developing agent;
(7) tobacco phytophthora parasitica standard.
The coated antibody and the enzyme-labeled antibody are respectively the monoclonal antibody and the polyclonal antibody in the first scheme, and in the component (1), the preparation process of the antibody pre-coated enzyme-labeled plate is as follows:
(1) diluting the antibody to a certain concentration, adding the antibody to a 96-well enzyme label plate, reacting at 37 ℃ for 3h, wherein each well is 100 ul;
(2) spin-drying the plate hole solution, adding a washing solution, soaking for 5min, spin-drying the plate hole solution, and patting dry on absorbent paper;
(3) adding the confining liquid to a 96-well enzyme label plate, reacting for 2h at 37 ℃ with each well being 150 ul;
(4) drying the plate hole solution by spin drying, patting the plate hole solution on absorbent paper, and drying the plate hole solution by a freeze dryer;
(5) packaging in aluminum foil bags by a vacuum packaging machine.
In the preparation process of the antibody pre-coated ELISA plate, the coating buffer solution is carbonate buffer solution; further preferably, the carbonate buffer is Na 2 CO 3 -NaHCO 3 Buffer at 0.1M pH 9.6.
The antibody is the first monoclonal antibody or the second monoclonal antibody of the monoclonal antibody pair described in scheme two.
The coating concentration of the antibody is 1.8-2.2 mu g/ml, preferably 1.9-2.1 mu g/ml, and more preferably 2 mu g/ml.
The blocking solution is a carbonate buffer solution containing BSA.
In the component (5), the enzyme-labeled antibody is a second monoclonal antibody or a first monoclonal antibody of a second monoclonal antibody pair and is labeled by HRP;
the concentration of the enzyme-labeled antibody is 8-12 mug/ml, preferably 9-11 mug/ml, and more preferably 10 mug/ml;
the incubation temperature after adding the antibody is 22-26 ℃, preferably 25 ℃, and the incubation time is 40-50 minutes, preferably 45 minutes.
The tobacco black shank bacterium standard substance in the component (7) is hypha obtained by culturing tobacco black shank bacterium.
The sulfuric acid in the component (4) is 1.8-2.2M, preferably 1.9-2.1M, and more preferably 2M.
The third scheme is as follows: the invention provides a method for detecting tobacco phytophthora parasitica, which adopts a kit in scheme II and comprises the following steps:
(1) extracting protein from a sample to be detected to obtain a protein extracting solution;
(2) and (3) detecting the protein extracting solution by using the kit of the fourth aspect, wherein the main processes comprise sample adding, incubation, washing, enzyme adding, incubation, washing, color development, termination and reading.
(3) And calculating the concentration of the tobacco black shank bacteria in the sample to be detected through a concentration-absorbance standard curve prepared by the tobacco black shank bacteria standard product.
The invention has the beneficial effects that: the antibody provided by the invention has strong specificity, high sensitivity and high titer, can be used for accurately and quantitatively detecting the tobacco phytophthora parasitica in tobacco specifically, has accurate and reliable identification result and high sensitivity, lays a foundation for the culture, popularization and application of tobacco crops, is simple and quick in detection method, and provides powerful guarantee for the field detection of large-scale breeding progeny selection. The antibody of the present invention can also be used in WB, ELISA, IP and other experiments.
Drawings
FIG. 1 is a standard curve for the detection of tobacco phytophthora parasitica using the method of the present invention.
Detailed Description
The invention is described in more detail below in an exemplary manner.
The reagents, antibodies, plasmids and instruments related to the embodiment of the invention are shown in the following table 1:
TABLE 1 Main reagents, antibodies, plasmids and instruments
Example 1 culture of tobacco Heiyiphora nicotianae
(1) Isolation and harvesting of pathogens
Respectively collecting diseased plants from Yibin Xinghun county, Yun Lian county and Davidia county tobacco fields, cumulatively collecting 83 diseased plants, successfully separating 70 tobacco phytophthora nicotianae by using a tissue isolation method, and identifying the tobacco phytophthora nicotianae (phytophthora nicotianae) through morphological characteristics and molecules.
(2) Cultivation of germs and acquisition of hypha spores
Inoculating the activated phytophthora nicotianae mycelia on a 15mL oat culture medium plate, and culturing at 28 ℃ for 10-12 days. The culture medium full of mycelia was divided into 2mm × 3mm rectangular pieces, and the pieces were picked into 10 sterile petri dishes containing 30mL of Peyer's solution. Culturing in 26 deg.C incubator in dark, and sucking 10 μ L of culture solution with pipette at the edge of hypha block (white floccule has grown) after 3 d. The culture dish which has induced a large amount of sporangia is placed in a refrigerator at 4 ℃ for 1h, and then is continuously placed in an incubator at 26 ℃ for dark culture for 24 h. Filtering with 2 layers of gauze, sucking 10 mu L of filtrate by using a pipette, respectively dripping the filtrate on a blood counting chamber, and calculating the content of zoospores in each treatment.
Example 2 antibody preparation
(1) Preparation of polyclonal antibodies
1. Preparing immunogen: mixing hypha spore with equal volume of Freund's adjuvant and Youlong adjuvant, and emulsifying to obtain water-in-oil emulsion for immunizing rabbit.
2. Immunization strategy: 2 New Zealand white rabbits were immunized with hyphal spores, subcutaneously 3 times at 4-week intervals, and finally subjected to indirect ELISA.
The indirect ELISA method comprises the following steps:
1) the hypha spores were diluted to 1ug/ml with 0.1mol/L carbonate buffer at pH9.6, and then added to a 96-well microplate at 100 ul/well and reacted at 37 ℃ for 3 hours or left overnight at 4 ℃.
2) Spin off the plate well, add 250ul wash buffer, stand for 30s, spin off the plate well and repeat 3 times.
3) Adding 100ul of detection sample into each well, adding positive control (positive rabbit serum obtained in step (2)), negative control (rabbit serum before immunization) and blank control (no rabbit serum) at 37 deg.C, reacting for 45min,
4) repeating the step 2);
5) adding HRP labeled goat anti-rabbit enzyme labeled secondary antibody, reacting at 37 deg.C for 45min with each well at 100 ul.
6) Repeating the step 2);
7) adding color developing agent, 100ul per well, and reacting for 15min at room temperature in dark.
8) Stop solution was added at 100ul per well and OD was read at wavelength 450 using a microplate reader, and the antiserum titers are as in table 2 below:
TABLE 2 antiserum titers
3. Polyclonal antibody purification: centrifuging rabbit serum for 15min (4000rpm, room temperature), collecting supernatant, adding saturated ammonium sulfate dropwise slowly under stirring at 4 deg.C to half saturation, stirring for 30min, centrifuging for 30min (13000rpm, 4 deg.C), and discarding supernatant; the pellet was dissolved in an appropriate amount of PBS (0.01M, pH 7.4); slowly adding saturated ammonium sulfate dropwise to 33% under stirring at 4 deg.C, stirring for 30min, centrifuging for 30min (13000rpm, 4 deg.C), and removing supernatant; the precipitate was dissolved in an appropriate amount of PBS (0.01M, pH7.4), dialyzed overnight at 4 ℃ and assayed for antibody content, and frozen at-20 ℃ for future use. Ammonium sulfate precipitation, purifying with ProteinA column, passing 5ml ultrapure water through the new column, and balancing the column with 5ml 0.4M PB buffer (pH 7.0); the antibody passes through the column slowly in the process, so that the antibody protein is better combined on the combining site; the column was equilibrated with 10ml of 0.4M PB buffer (pH 7.0); 5ml of 0.1M glycine-hydrochloric acid buffer (pH 3.0) elute the antibody at the binding site, and 1M Tris-HCl (pH 8.0) is added to neutralize the glycine, keeping the pH at neutrality suitable for antibody preservation.
(2) Preparation of monoclonal antibody
1. Preparing immunogen: mixing hypha spore with equal volume of Freund's adjuvant and Youlong adjuvant, and emulsifying to obtain water-in-oil emulsion for immunizing mouse.
2. Immunization strategy: the hypha spore is used for immunizing 4 Balb/c mice, subcutaneous immunization is carried out for 3 times, the interval is 4 weeks, and finally indirect ELISA detection is carried out.
The indirect ELISA method comprises the following steps:
1) the hypha spores were diluted to 1ug/ml with 0.1mol/L carbonate buffer at pH9.6, and then added to a 96-well microplate at 100 ul/well for reaction at 37 ℃ for 3 hours or left overnight at 4 ℃.
2) Spin off the plate well, add 250ul wash buffer, stand for 30s, spin off the plate well and repeat 3 times.
3) Adding 100ul of detection sample into each well, adding positive control (positive mouse serum obtained in step (2)), negative control (mouse serum before immunization) and blank control (no mouse serum) at 37 deg.C, reacting for 45min,
4) repeating the step 2);
5) adding HRP labeled goat anti-mouse enzyme labeled secondary antibody, reacting at 37 deg.C for 45min with each well at 100 ul.
6) Repeating the step 2);
7) adding color developing agent, 100ul per well, and reacting for 15min at room temperature in dark.
8) Stop solution was added at 100ul per well and OD was read at wavelength 450 using a microplate reader, and the antiserum titers are as in table 3 below:
TABLE 3 antiserum titres
3. Cell fusion: two weeks after the last immunization, the antigen was intraperitoneally injected for booster immunization, and three days later, cell fusion was performed. Killing mouse neck, soaking in 70% ethanol for 30min for sterilization, cutting abdominal cavity on an ultra-clean bench, taking out spleen, grinding, sieving with 80 mesh sieve to obtain spleen cell, adding SP2/0 myeloma cell, performing cell fusion under the action of PEG4000,
4. fusion screening: and (3) paving the fused cells into a 96-well plate, culturing by using HAT culture solution, changing the culture solution after three days, and culturing by using HT culture solution. After 10 days, cell culture supernatants were taken for detection.
5. Cloning and establishing strains: cloning positive holes by using a limiting dilution method, detecting after 10 days, and cloning positive clones by continuing the limiting dilution method until all the obtained clones are positive, thereby establishing a positive cell strain. Finally, 5 positive cell strains are obtained.
6. And (3) amplification culture: and (4) carrying out expanded culture on the established monoclonal cells, and freezing and storing.
(3) Preparation and purification of ascites
1. Preparing ascites: injecting mineral oil into the abdominal cavity of the mouse one week ahead of time, injecting a certain amount of cells into the abdominal cavity of the mouse, collecting ascites after about 10 days, and centrifuging at 4000rpm to obtain supernatant, namely monoclonal antibody ascites.
2. Monoclonal antibody purification: centrifuging ascites for 15min (4000rpm, room temperature), collecting supernatant, adding saturated ammonium sulfate dropwise slowly at 4 deg.C under stirring to half saturation, stirring for 30min, centrifuging for 30min (13000rpm, 4 deg.C), and discarding supernatant; the pellet was dissolved in an appropriate amount of PBS (0.01M, pH 7.4); slowly adding saturated ammonium sulfate dropwise to 33% under stirring at 4 deg.C, stirring for 30min, centrifuging for 30min (13000rpm, 4 deg.C), and removing supernatant; the pellet was dissolved in PBS (0.01M, pH7.4), dialyzed overnight at 4 ℃ to determine the antibody content, and frozen at-20 ℃ for further use. Ammonium sulfate precipitation, purifying with Protein G column, passing 5ml ultrapure water through the new column, and balancing the purified column with 5ml 0.4M PB buffer solution (pH 7.0); the antibody passes through the column slowly in the process, so that the antibody protein is better combined on the binding site; the column was equilibrated with 10ml of 0.4M PB buffer (pH 7.0); the antibody on the binding site was eluted with 5ml of 0.1M glycine-hydrochloric acid buffer (pH 2.7), and glycine was neutralized by adding 1M Tris-HCl (pH 8.0) to maintain the pH at neutrality suitable for antibody preservation.
(4) Antibody screening
1. Potency assay
The titer of the purified antibody was measured by indirect ELISA method, and the results are shown in the following Table 4:
2. subtype detection
The subtype detection of the antibody is carried out by using a mouse antibody subtype detection kit, and the results are shown in the following table 4:
TABLE 4 antibody subtype detection
3. Antibody pairing
And screening the prepared antibodies by using a double-antibody sandwich ELISA method to screen out antibody pairs which can be paired.
The ELISA method for the double antibody sandwich was as follows:
a. diluting the coated antibody to 10ug/ml with CB, each well is 100ul, and coating is carried out at 37 ℃ for 3 h;
b. washing the plate for 3-5 times, patting to dry, adding positive standard/negative control/positive sample/negative sample, reacting at 25 deg.C for 45min, and adding 100ul per well.
c. Washing the plate for 3-5 times, drying, adding enzyme-labeled antibody, reacting at 25 deg.C for 45min at 100ul per well.
d. Washing the plate for 3-5 times, patting to dry, adding color developing agent, and reacting at 25 deg.C in dark for 15 min.
e. 100ul of stop solution was added and OD450 was read, and the results are shown in Table 5:
TABLE 5 antibody pairing
The FL3601-16 has the best matching effect, so the kit is prepared by using the FL3601-16 and rabbit polyclonal antibody.
Example 3 assembly of the kit of the invention:
1. preparation of buffer solution
(1) Coating buffer solution, 0.1M carbonate buffer solution CB, and the pH value is 9.6;
(2) diluent, 0.01mM phosphate buffer PBS, pH 7.4;
(3) washing solution, PBS containing 0.2% Tween-20;
(4) blocking solution, CB with 1% BSA;
(5) stop solution, 2M H 2 SO 4 ；
2. Preparation of ELISA plates
(1) Coating quilt
And (3) transferring a certain amount of the tobacco phytophthora parasitica monoclonal antibody into coating liquid with the required volume to ensure that the concentration of the monoclonal antibody is about 2 mu g/mL, and preparing the coating liquid. The coating solution was added to wells of an ELISA plate at a volume of 100. mu.L per well and allowed to stand at 4 ℃ overnight (care was taken to prevent evaporation of water).
(2) Sealing of
A blocking working solution was prepared by adding a predetermined amount of BSA, sodium salicylate, and sucrose to CB buffer (pH9.6) so that the concentrations of BSA, sodium salicylate, and sucrose were 1%, 0.05%, and 5%, respectively. The ELISA plate is blotted and washed twice with a plate washer, and the plate bar is patted dry on clean absorbent paper. Add 150. mu.L of blocking solution to the wells and bake at 37 ℃ for 3 hours.
(3) Pumping seal
Removing the sealing liquid, drying the batten on clean absorbent paper, putting the batten into a freeze vacuum drier, pumping for 3 hours, and carrying out vacuum heat sealing.
3. Preparation of tobacco black shank bacterium standard freeze-dried powder
Culturing the obtained tobacco black shank bacterium hyphae, diluting to 16 mu g/mL with the mother solution concentration of 3mg/mL, adding 100 mu L of 16 mu g/mL standard substance into a 3mL brown glass bottle, and performing overnight pumping drying by a freeze dryer to obtain the standard substance freeze-dried powder.
4. Preparation of enzyme-labeled antibody working solution
Preparation of enzyme-labeled antibody: and (3) coupling the purified tobacco phytophthora parasitica polyclonal antibody with HRP to obtain an enzyme-labeled antibody. The preparation method of the horseradish peroxidase (HRP) -labeled tobacco phytophthora parasitica polyclonal antibody comprises the following steps:
(1)5mg of HRP is dissolved in pure water, sodium periodate is added, and the reaction is carried out for 30 minutes at room temperature;
(2) 5mg of antibody was dialyzed against coupling buffer overnight;
(3) adding HRP into the antibody solution, and reacting at room temperature for 2 hours;
(4) adding sodium borohydride to seal the reaction site;
(5) the mixture was dialyzed overnight against phosphate buffer, and the mixture was stored at-20 ℃ with the addition of an equal amount of glycerol.
Adding a certain amount of enzyme-labeled antibody into the diluent, fully and uniformly mixing to ensure that the final concentration is 2 mu g/mL, and storing at the temperature of 2-8 ℃ in a dark place;
5. kit assembly, see table 6:
TABLE 6 kit Assembly
| Name (R) | Number of |
| Precoated |
1 block (A) |
| Standard substance freeze-dried |
2 bottle |
| Enzyme-labeled |
1 |
| Sample diluent | |
| 2 bottle | |
| |
1 |
| Terminating agent | |
| 1 | |
| Cleaning solution | |
| 1 | |
| Description | |
| 1 part of |
Example 4 detection of tobacco Heiyiphora nicotianae spiked samples Using the kit of the invention
(1) Preparing standard and adding standard sample
1) Preparing a standard substance:
dissolving the tobacco black shank bacterium standard substance by using 2mL of sample extracting solution, wherein the concentration of the solution is 8 ppm;
taking 8ppm of the tobacco phytophthora parasitica black shank standard substance, adding 300 mu L of the sample extracting solution into 300 mu L of the tobacco phytophthora parasitica black shank standard substance, wherein the concentration of the solution is 4 ppm;
taking 4ppm of a tobacco phytophthora parasitica reference substance, adding 300 mu L of a sample extracting solution into 300 mu L of the tobacco phytophthora parasitica reference substance, wherein the concentration of the solution is 2 ppm;
taking 2ppm of a tobacco phytophthora parasitica reference substance, adding 300 mu L of a sample extracting solution into 300 mu L of the tobacco phytophthora parasitica reference substance, wherein the concentration of the solution is 1 ppm; taking 1ppm of the tobacco phytophthora parasitica standard, adding 300 μ L of the sample extracting solution into 300 μ L of the tobacco phytophthora parasitica standard, wherein the concentration of the solution is 0.5 ppm.
2) Processing of the labeled sample:
the sample pretreatment method comprises the following steps: weighing 1g of negative sample, mashing the negative sample, putting the mashed negative sample into a 15mL centrifuge tube, adding 10mL of extracting solution, uniformly mixing the extracting solution by oscillation for 5 minutes, centrifuging the extracting solution at 4000rpm for 3 minutes, and taking supernatant as the extracting solution of the negative sample.
And adding a certain amount of high-concentration hypha solution into the negative sample extracting solution to prepare a positive labeled sample for detection.
(2) Adding standard or unknown concentration samples:
adding 100 mu L of sample diluent (blank control)/standard substance/positive labeled sample into corresponding micropores, gently shaking and uniformly mixing, and reacting for 45min at 25 ℃ in a dark environment.
(3) Washing the plate:
spin-drying the liquid in the pores, washing with 250 μ L of washing solution/pore for 3 times, each time at an interval of 1min, and drying with absorbent paper.
(4) Adding an enzyme-labeled antibody:
adding 100 mu L of enzyme-labeled antibody into the mixture per hole, slightly oscillating the mixture and uniformly mixing the mixture, reacting the mixture for 45min in a dark environment at the temperature of 25 ℃, taking out the mixture and repeating the plate washing step 3.
(5) Color development:
adding 100 mul/hole color developing agent, and reacting for 15min in a dark environment at 25 ℃.
(6) Termination and measurement:
add 100. mu.L of stop solution/well, mix by gentle shaking, set the microplate reader to 450nm, determine the OD value of each well (preferably using double wavelength 450/630nm to detect, read the data within 5 min). If no enzyme marker is present, the determination can be carried out by visual inspection without adding stop solution.
(7) And (3) calculating:
drawing a standard curve according to the measured OD value of the standard substance by using statistical drawing software; and calculating the concentration of the tobacco phytophthora parasitica in the sample according to the standard curve and the OD value of the sample.
(8) The results are shown in Table 7.
TABLE 7 test results for spiked samples
As can be seen from the results in Table 7, the percent recovery of the phytophthora parasitica detected by using the kit and the method of the present invention is between 85% and 120%, which indicates that: the method for detecting the phytophthora parasitica in the plants has better accuracy.
Example 5 detection of tobacco Heiyiphora nicotianae in plants Using the kit of the present invention
And (3) sensitivity test: 5 parts of positive serum are respectively diluted by 1:10, 1:100, 1:1000, 1:10000, 1:100000 and 1:1000000, then detection is carried out by using optimized conditions, and the maximum serum dilution with positive detection result is searched as the result of the sensitivity test. The result shows that the antibody can still be detected after being diluted by 10000 times, and the sensitivity is as high as 0.1-1mg/L
TABLE 8 tobacco Heiguobacter nigrum antibody pairing sensitivity assay
| |
1 | 2 | 3 | 4 | 5 |
| CK | 0.0898 | 0.0885 | 0.0963 | 0.0792 | 0.0947 |
| 10 | 1.4729 | 1.4471 | 1.8346 | 1.3849 | 1.9021 |
| 100 | 0.6215 | 0.6227 | 0.8908 | 0.5533 | 0.8261 |
| 1000 | 0.3196 | 0.3202 | 0.6916 | 0.4275 | 0.6418 |
| 10000 | 0.1932 | 0.1867 | 0.2756 | 0.2259 | 0.2459 |
| 100000 | 0.0939 | 0.0908 | 0.1221 | 0.1048 | 0.1076 |
| 1000000 | 0.0855 | 0.0827 | 0.0941 | 0.0808 | 0.0929 |
In conclusion, the monoclonal antibody provided by the invention has the advantages of strong specificity, high sensitivity and high titer, can be used for specifically and quantitatively detecting the phytophthora parasitica in plants accurately, has accurate and reliable identification result and high sensitivity, is simple and quick in detection method, and provides powerful guarantee for large-scale on-site detection.
The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above, i.e. it does not mean that the present invention must be implemented in dependence on the above. It will be apparent to those skilled in the art that any modification of the present invention, equivalent substitutions of selected materials and additions of auxiliary components, selection of specific modes and the like, which are within the scope and disclosure of the present invention, are contemplated by the present invention.
Claims (8)
1. An enzyme linked immunosorbent assay kit for detecting phytophthora parasitica, which is characterized in that:
the kit comprises a pre-coated enzyme label plate and an enzyme-labeled antibody, wherein the pre-coated enzyme label plate is an enzyme label plate coated by a monoclonal antibody for resisting tobacco black shank bacteria, and the enzyme-labeled antibody is a polyclonal antibody for resisting tobacco black shank bacteria and labeled by horse radish peroxidase.
2. The ELISA kit for detecting phytophthora parasitica according to claim 1, wherein the ELISA kit comprises:
the preparation method of the monoclonal antibody comprises the following steps: the preparation method comprises the steps of immunizing a BALB/c mouse by using tobacco phytophthora parasitica mycelium, preparing a monoclonal antibody hybridoma cell strain through cell fusion and cloning screening, preparing ascites, and purifying the ascites to obtain the monoclonal antibody hybridoma cell strain.
3. The ELISA kit for detecting phytophthora parasitica according to claim 1, wherein the ELISA kit comprises:
the preparation method of the polyclonal antibody comprises the following steps: the rabbit serum is obtained by culturing tobacco phytophthora parasitica to obtain hypha and spores, immunizing rabbits with the hypha and the spores, and purifying after obtaining the rabbit serum.
4. The ELISA kit for detecting phytophthora parasitica according to claim 1, wherein the ELISA kit comprises:
the concentration of the monoclonal antibody coating of the anti-phytophthora parasitica is 2 mug/mL, and the concentration of the enzyme-labeled antibody is 1 mug/mL.
5. The ELISA kit for detecting phytophthora parasitica according to claim 1, wherein the ELISA kit comprises:
the kit also comprises standard substance freeze-dried powder, sample diluent, a color developing agent, a stop solution and a washing solution.
6. The ELISA kit for detecting phytophthora parasitica according to claim 5, wherein the ELISA kit comprises:
the standard freeze-dried powder is tobacco black shank bacterium hyphae;
the sample diluent is PBS buffer solution with 0.01mM and pH value of 7.4;
the solvent of the washing solution is 0.01mM PBS buffer solution with the pH value of 7.4, and contains 0.2 percent of Tween-20 by volume percentage concentration;
the stop solution is 2M sulfuric acid aqueous solution;
the color developing agent comprises a horseradish peroxidase catalytic substrate A solution and a horseradish peroxidase catalytic substrate B solution; the horseradish peroxidase catalytic substrate A solution is H with the volume concentration of 3% 2 O 2 An aqueous solution; the preparation method of the horseradish peroxidase catalytic substrate B liquid comprises the following steps: 1 mL of a 10 mg/mL solution of 3,3',5,5' -tetramethylbenzidine was added to 100 mL of a phosphate buffer solution of 0.1mol/L, pH of 6.0 to prepare a mixture.
7. An application of an enzyme linked immunosorbent assay kit in detecting tobacco phytophthora parasitica.
8. The use of the enzyme linked immunosorbent assay kit of claim 7 for detecting phytophthora parasitica, wherein the enzyme linked immunosorbent assay kit comprises the following components in percentage by weight:
the application method comprises the following steps:
(1) extracting protein from a sample to be detected to obtain a protein extracting solution;
(2) detecting the protein extracting solution by using the kit, wherein the main process comprises the steps of sample adding, incubation, washing, enzyme adding, incubation, washing, color development, termination and reading;
(3) and calculating the concentration of the tobacco black shank bacteria in the sample to be detected through a concentration-absorbance standard curve prepared by the tobacco black shank bacteria standard product.
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| CN101041853A (en) * | 2006-12-26 | 2007-09-26 | 重庆大学 | Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco black shank bacterium |
| WO2017107974A1 (en) * | 2015-12-23 | 2017-06-29 | 中国人民解放军第二军医大学 | Detection test kit for serum psmd4 proteins and detection method and application thereof |
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|---|---|---|---|---|
| CN101041853A (en) * | 2006-12-26 | 2007-09-26 | 重庆大学 | Primer, probe and real-time fluorescent PCR reagent case for detecting tobacco black shank bacterium |
| WO2017107974A1 (en) * | 2015-12-23 | 2017-06-29 | 中国人民解放军第二军医大学 | Detection test kit for serum psmd4 proteins and detection method and application thereof |
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| PEKAROVA-KYNEROVA, BLANKA等: "Preparation and use of monoclonal antibodies to detect: Phytophthora nicotianae var. nicotianae", 《PLANT PROTECTION SCIENCE》, vol. 35, no. 02, 30 June 1999 (1999-06-30), pages 41 - 46 * |
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