CN114317452B - Naphthylacetic acid monoclonal antibody, hybridoma cell strain and application - Google Patents
Naphthylacetic acid monoclonal antibody, hybridoma cell strain and application Download PDFInfo
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- CN114317452B CN114317452B CN202210088651.5A CN202210088651A CN114317452B CN 114317452 B CN114317452 B CN 114317452B CN 202210088651 A CN202210088651 A CN 202210088651A CN 114317452 B CN114317452 B CN 114317452B
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- monoclonal antibody
- naphthylacetic acid
- hybridoma cell
- acetic acid
- naphthalene acetic
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Abstract
The invention provides a naphthylacetic acid monoclonal antibody, a hybridoma cell strain and application thereof, belonging to the field of food safety immunodetection. The hybridoma cell lines were deposited with the accession numbers: CGMCC No.45024. The invention synthesizes the complete antigen of naphthylacetic acid, uses Freund's adjuvant to mix and emulsify, and injects to immunize BALB/c mice. Screening high potency, low IC 50 The mouse spleen cells are fused with mouse myeloma cells by a PEG method, and a selective culture medium is used for screening out hybridoma cells fused with two cells; and screening cells by an indirect competitive ELISA method and subcloning the cells for multiple times to obtain a monoclonal antibody hybridoma cell strain. The monoclonal antibody secreted by the cell strain has good detection sensitivity to naphthalene acetic acid, has 50% inhibition concentration IC50 of 6.77ng/mL to naphthalene acetic acid, is used for an immunodetection kit and a colloidal gold test strip, and provides a powerful detection means for detecting the addition of naphthalene acetic acid into plant-derived foods.
Description
Technical Field
The invention belongs to the field of food safety immunodetection, and particularly relates to a naphthylacetic acid monoclonal antibody, a hybridoma cell strain and application thereof.
Background
Naphthalene acetic acid is an organic compound that is insoluble in water, but slightly soluble in hot water, and readily decomposes in a humid environment. Has auxin activity and is absorbed by root, stem and leaf. Naphthalene acetic acid is widely used in the fields of agriculture, forestry, vegetables, flowers, fruit trees and the like, and is used for inducing adventitious root formation and improving tree cuttage survival rate; the fruit setting rate is improved, and fruit drop before picking is prevented. However, it is a toxic product and has very strong damaging effect on mucous membrane, upper respiratory tract, eye, skin and other tissues. The naphthylacetic acid has wide development prospect in crop production and plant cultivars, brings great economic and social benefits to human beings, and brings great attention to people.
Conventional detection methods of naphthalene acetic acid include high performance liquid chromatography tandem mass spectrometry, ultra-high performance liquid chromatography-tandem mass spectrometry, gas chromatography tandem mass spectrometry, and the like. These methods suffer from several drawbacks to varying degrees: time consuming, expensive instrumentation, and extensive sample pretreatment procedures, etc. Therefore, these methods are not suitable for the detection of naphthylacetic acid for high throughput analysis in the field. There is therefore a need for an analysis system, which means that these methods have limited application in field analysis.
The immunoassay method has the characteristics of low cost, high flux, high sensitivity, low relative requirements on technicians and the like, so that the method is suitable for rapid screening of a large number of samples.
Disclosure of Invention
In order to solve the problems in the related art, the invention provides a hybridoma cell strain secreting the naphthylacetic acid monoclonal antibody and application thereof, and the monoclonal antibody prepared by the cell strain has better affinity and detection sensitivity to naphthylacetic acid, can be used for establishing a naphthylacetic acid enzyme-linked immunosorbent assay method or establishing a colloidal gold immunochromatography test strip rapid detection method, and lays a foundation for research and development and popularization of an indirect competition ELISA kit and a colloidal gold test strip.
In one aspect, the invention provides a hybridoma cell strain secreting the naphthylacetic acid monoclonal antibody, which is preserved in China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) for 12 months and 15 days in 2021, wherein the preservation name is monoclonal cell strain HLH, the preservation number is CGMCC No.45024, and the preservation address is North Chen West Lu No. 1 and No. 3 in the Korean region of Beijing city.
The invention also provides a naphthylacetic acid monoclonal antibody which is secreted and produced by a hybridoma cell strain with the preservation number of CGMCC No.45024 and secreting the naphthylacetic acid monoclonal antibody.
In yet another aspect, a method for preparing a naphthylacetic acid monoclonal antibody is provided, comprising: taking BALB/c mice, injecting paraffin oil into the abdominal cavity, injecting hybridoma cell strain with the preservation number of CGMCC No.45024 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and preserving the obtained naphthylacetic acid monoclonal antibody at low temperature.
In yet another aspect, there is provided an application of the naphthalene acetic acid monoclonal antibody for detecting naphthalene acetic acid residues in food.
In yet another aspect, there is provided an application of a hybridoma cell strain secreting the naphthylacetic acid monoclonal antibody for preparing the naphthylacetic acid monoclonal antibody
In yet another aspect, an application of a hybridoma cell strain secreting the naphthylacetic acid monoclonal antibody is provided, which is applied to detecting naphthylacetic acid.
Further, the method is applied to detection of the naphthylacetic acid residues in foods.
In another aspect, a kit is provided comprising the naphthylacetic acid monoclonal antibody.
In yet another aspect, the kit is applied to the detection of naphthylacetic acid residues in food.
In yet another aspect, a colloidal gold test strip is provided, comprising the monoclonal antibody of naphthylacetic acid.
In yet another aspect, the colloidal gold test strip is applied to the detection of naphthylacetic acid residues in food.
The preparation method of the cell strain HLH provided by the invention comprises the following basic steps:
(1) Preparation and identification of immunogens: naphthalene acetic acid is used as a raw material, amino groups of an activated ester method protein carrier are connected, after the reaction is finished, a complete antigen and unconjugated small molecule hapten are separated through dialysis, and the complete antigen is identified through an ultraviolet absorption scanning method;
(2) Immunization of mice: BALB/c mice of 6-8 weeks of age were selected for immunization. After the immunogen and the Freund's adjuvant are completely emulsified, the mice are immunized by subcutaneous multipoint injection, the Freund's complete adjuvant is adopted for primary immunization, the Freund's incomplete adjuvant is adopted for boosting immunization, the immunization dose is half of the previous immunization dose during sprint immunization, and the mice are directly injected into the abdominal cavity after being uniformly mixed with normal saline; each immunization interval was three weeks. After the third immunization, blood sampling is carried out at intervals of one week to detect serum titer and inhibition;
(3) Cell fusion and cell strain establishment: fusing the spleen cells of the mice and myeloma cells of the mice by a polyethylene glycol (PEG 2000) method, culturing by a HAT culture medium, detecting positive cell holes by using an indirect ELISA (enzyme-linked immunosorbent assay), further measuring the inhibition effect of the positive cell holes by using an indirect competition ELISA method, subcloning the positive cell holes with the best inhibition for three times by using a limiting dilution method, and finally screening to obtain hybridoma cell strains HLH;
(4) Identification of hybridoma cell line properties: adopting enzyme-labeled secondary antibody suit determination for mouse monoclonal antibody Ig class/subclass identification; determination of IC50 values, cross-reactivity and affinity was by ELISA.
Compared with the prior art, the invention has at least the following beneficial effects:
the monoclonal antibody secreted by the hybridoma cell strain has good detection sensitivity to naphthylacetic acid; the method can realize the detection of the residual quantity of the naphthylacetic acid in the food, and provides a new means for establishing a rapid, simple, convenient, cheap, sensitive and specific naphthylacetic acid detection method; the method for synthesizing the naphthylacetic acid immunogen provided by the invention has the advantages that the synthesis steps are simplified and effective, and the thought and method for synthesizing the immunogen are provided for the research of people in future.
Preservation of biological materials
Hybridoma cell lines secreting naphthylacetic acid monoclonal antibodies are classified and named as follows: the monoclonal cell strain HLH has the following preservation units: the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) has a preservation address of: is No. 3 of North Chen Silu 1, the region of Chaoyang in Beijing, and the preservation number is: CGMCC No.45024, the preservation date is: 2021, 12, 15.
Drawings
The accompanying drawings are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate the invention and together with the description serve to explain, without limitation, the invention. In the drawings:
FIG. 1 (a) shows the chemical structural formula of a naphthaleneacetic acid hapten, and FIG. 1 (b) shows the chemical structural formula of a naphthaleneacetic acid hapten.
FIG. 2 is a standard inhibition curve for a naphthylacetic acid monoclonal antibody.
Detailed Description
The following examples of the present invention are merely further illustrative of the present invention and are not intended to limit the scope or spirit of the present invention. The invention is further illustrated by the following examples.
The following examples relate to the following media:
RPMI-1640 medium (mg/L): l-arginine 290, L-asparagine 50, L-aspartic acid 20, L-cystine dihydrochloride 65.15, L-glutamic acid 20, glycine 10, L-histidine 15, L-hydroxyproline 20, L-isoleucine 50, L-leucine 50, L-lysine hydrochloride 40, L-methionine 15, L-phenylalanine 15, L-proline 20, L-serine 30, L-threonine 20, L-tryptophan 5, L-tyrosine 23.19, L-valine 20, p-aminobenzoic acid 1, calcium nitrate 100, anhydrous magnesium sulfate 48.84, anhydrous sodium dihydrogen phosphate 676.13, potassium chloride 400, sodium chloride 6000, glucose 2000, reduced glutathione 1, phenol red 5, L-glutamine 300, biotin 0.2, D-calcium pantothenate 0.25, folic acid 1, i-inositol 35, nicotinamide 1, choline chloride 3, pyridoxine hydrochloride 1, riboflavin 0.2, thiamine hydrochloride 1, vitamin B12.005, sodium bicarbonate 2000.
The reagents involved in the following examples were as follows:
carbonate Buffer (CBS): weighing Na 2 CO 3 1.59g，NaHCO 3 2.93g, respectively dissolving in a small amount of double distilled water, mixing, adding double distilled water to about 800mL, mixing, adjusting pH to 9.6, adding double distilled water to 1000mL, and storing at 4deg.C for use.
Phosphate Buffer (PBS): 8.00g NaCl,0.2g KCl,0.2g KH 2 PO 4 ，2.9g Na 2 HPO 4 ·12 H 2 O is dissolved in 800mL of pure water, pH is regulated to 7.2-7.4 by NaOH or HCl, and volume is regulated to 1000mL;
PBST: PBS containing 0.05% Tween 20;
TMB color development liquid: and (3) solution A: na (Na) 2 HPO 4 .12H 2 18.43g of O, 9.33g of citric acid and pure water to 1000mL; and (2) liquid B: 60mg of TMB was dissolved in 100mL of ethylene glycol. A. The solution B is prepared from the following components in percentage by weight: 1 to obtain TMB color development liquid, and mixing immediately.
The detection method involved in the following examples is as follows:
the method for detecting the inhibition rate of the naphthylacetic acid comprises the following steps: the most appropriate antigen and antibody concentrations in ELISA experiments were selected by checkerboard method. The antigen was diluted to 0.01,0.03,0.1 and 0.3 μg/mL with Carbonate Buffer (CBS) and the antibody diluted to 0.03,0.1,0.3 and 1 μg/mL with antibody dilution. After selecting the optimal working point, the naphthylacetic acid standard was diluted to 7 concentrations (1.25,2.5,5, 10, 20, 40, 80 ng/mL) and finally mapped with OriginPro 9.0 (results shown in FIG. 2) according to the procedure of the IC-ELISA to obtain the standard inhibition curve of naphthylacetic acid, and IC was calculated 50 。
Example 1 preparation of an artificial naphthalene acetic antigen:
synthesis of naphthalene acetic acid complete antigen:
taking 3.7mg of naphthalene butyric acid, adding 4.2mg of EDC (1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride) and 2.3mg of NHS (N-hydroxysuccinimide), dissolving by using DMF (N, N-dimethylformamide), stirring at room temperature, and activating for 6 hours to obtain a naphthalene acetic acid hapten, wherein the chemical structure is shown in a figure 1 (a); another 18mg BSA (bovine serum albumin) was dissolved in 3mL, 0.05M, CB (carbonate buffer) solution at pH 9.6; the activating solution is added into BSA solution drop by drop, stirred at room temperature for reaction for 8 hours, then dialyzed for 3 days by 0.01MPBS, unreacted small molecule hapten is removed, and the complete naphthalene acetic acid antigen is obtained, and the chemical structure is shown in figure 1 (b), and is sub-packaged and stored at-20 ℃.
Example 2: preparation of Naphthylacetic acid monoclonal antibody secreting hybridoma cell strain
2.1 acquisition of animal immunization
Healthy Balb/C mice of 6-8 weeks of age were selected for immunization. After mixing and emulsifying the naphthylacetic acid immunogen and the equivalent Freund's adjuvant, BALB/c mice were subjected to subcutaneous multipoint injection immunization (except sprint immunization) on the back of the neck. The first immunization was performed with complete Freund's adjuvant at a dose of 100 ug/dose; multiple boosting with incomplete Freund's adjuvant and halving the dose to 50 ug/dose; the sprint immunity is directly diluted by normal saline without adjuvant, and then the dosage is halved to 25 ug/patient. One month is separated from the first immunization and the second immunization, 21 days is separated from the multiple boosting, and 18-21 days is separated from the final boosting. The immune effect of the mice is observed by an indirect competition enzyme-linked immunosorbent assay (ic-ELISA), namely the titer and inhibition of the serum of the mice are detected.
2.2 cell fusion and screening
After 3 days of impact immunization, cell fusion was performed according to the conventional PEG (polyethylene glycol, molecular weight 4000) method, specifically as follows:
a. taking out spleen of a mouse by aseptic operation, moderately grinding the spleen with a rubber head of a syringe, obtaining spleen cell suspension through a 200-mesh cell screen, collecting and centrifuging (1200 rpm,8 min), washing the spleen cells with RPMI-1640 medium for three times, diluting the spleen cells to a certain volume after the last centrifugation, and counting for later use;
b. collecting SP2/0 cells: 7-10 days before fusion, SP2/0 tumor cells were treated with 10% FBS (fetal bovine serum)RPMI-1640 medium in 5% CO 2 In an incubator. The number of SP2/0 tumor cells before fusion reaches 1 to 4 multiplied by 10 7 Ensuring SP2/0 tumor cells to be in logarithmic growth phase before fusion. During fusion, collecting tumor cells, suspending in RPMI-1640 basic culture solution, and performing cell count;
c. the fusion process was 7min. 1min, 1mL of PEG 1500 was added dropwise to the cells from slow to fast; and (2) standing for 2 min. Dripping 1mL of RPMI-1640 culture medium in the period of 1min for 3min and 4 min; dripping 2mL of RPMI-1640 culture medium in the period of 1min at the 5 th and 6 th min; at 7min, 1mL of RPMI-1640 medium was added dropwise every 10 s. Then, the mixture was incubated at 37℃for 5min. Centrifuging (800 rpm,8 min), discarding supernatant, re-suspending in RPMI-1640 screening medium containing 20% fetal bovine serum and 2% 50 XHAT, adding 200 μl/well to 96-well cell plate, and standing at 37deg.C and 5% CO 2 Culturing in an incubator.
2.3 cell Screen and cell line establishment
Half-changing the fused cells by HAT medium on the 3 rd day after cell fusion; full exchange with RPMI-1640 medium (HT medium) containing 20% fetal bovine serum, 1% 100×ht on day 5; cell supernatants were taken on day 7 for screening. Screening is carried out in two steps: the first step is to screen out positive cell holes by using an ic-ELISA method, and the second step is to select naphthalene acetic acid as a standard substance, and to measure the inhibition effect of positive cells by using the ic-ELISA method. Cell holes with better inhibition on the naphthylacetic acid standard are selected, subcloning is carried out by adopting a limiting dilution method, and detection is carried out by adopting the same method after seven days. And performing subcloning for three times according to the method to finally obtain the cell strain HLH secreting the naphthylacetic acid monoclonal antibody.
Example 3: preparation of Naphthylacetic acid monoclonal antibody
Taking 8-10 week-old BALB/c mice, and injecting paraffin oil into the abdominal cavity of each mouse by 1mL; intraperitoneal injection of 1X 10 per mouse after 7 days 6 Hybridoma cells, collecting ascites from day 7, purifying the ascites by octanoic acid-saturated ammonium sulfate method, and storing the obtained monoclonal antibody at-20deg.C.
Example 4: application of naphthylacetic acid monoclonal antibody
The monoclonal antibody prepared from hybridoma cell strain HLH through in vivo ascites is applied to a naphthylacetic acid ELISA additive recovery test, and the specific steps are as follows:
(1) Coating a 96-well ELISA plate with 0.1 mug/mL of naphthylacetic acid diluted by Carbonate Buffer (CBS) as a coating raw material, coating 100 mug/well by using PBST washing liquid for 2 hours at 37 ℃, washing the plate three times, 200 mug/well by using PBST washing liquid for 3 minutes, and drying;
(2) Blocking with CBS containing 0.2% gelatin, blocking at 37deg.C for 2 hr, washing the plate with PBST wash solution three times, 200 μl each time, 3min each time, and drying;
(3) 0,1.25,2.5,5, 10, 20, 40, 80. Mu.g/L of naphthylacetic acid standard solution were prepared with Phosphate Buffered Saline (PBS), respectively. Respectively adding the standard solution and the sample extracting solution to be detected into the sealed ELISA plate, wherein each hole is 50 mu L, each sample is repeated for 3 holes, and then 50 mu L of 1 is added into each hole: after 16000 diluted naphthylacetic acid monoclonal antibody reacts for half an hour at 37 ℃, washing the plate and beating;
(4) mu.L of PBS 1 with 0.1% gelatin was added to each well: after a reaction of 3000 dilution of HRP-marked goat anti-mouse IgG secondary antibody at 37 ℃ for half an hour, washing the plate and beating;
(5) 100 mu L of TMB developing solution is added into each hole, after developing for 15min at 37 ℃, 50 mu L of 2M H2SO4 stop solution is added into each hole, and the absorbance value is measured at 450 nm;
(6) And (3) adding and recycling and sample pretreatment: 1g of fresh cucumber is taken, washed, chopped and stirred in a masher to be evenly mixed. Three different doses of naphthalene acetic acid standard were added, 50ng, 100ng, 200ng, respectively. The mixture was placed in a 50mL centrifuge tube, 8mL of methanol solution was added, and the mixture was shaken on a vortex mixer for 10min, sonicated for 30min, and then placed in a centrifuge for centrifugation at 6000r/min for 10min. Constant volume to 10ml. Remove 1mL of supernatant and filter the membrane for detection. The recovery of the additives was 93.4%, 87.5% and 93.6% respectively by indirect competition ELISA.
Example 5 sensitivity and specificity
FIG. 2 is a standard inhibition curve of antibodies using an indirect competition ELISA method to determine the IC of monoclonal antibodies to naphthalene acetic acid 50 6.77ng/ml, and verified that it was against forchlorfenuron and the likeIC 50 And the cross-reactivity is shown in Table 1.
TABLE 1 IC of Naphthylacetic acid monoclonal antibody to Naphthylacetic acid, chlorpyrimide, abscisic acid, uniconazole 50 Cross-reactivity ratio
| IC 50 (ng/mL) | Cross reaction rate | |
| Naphthalene acetic acid | 6.77 | 100％ |
| Chloropyrrole (chlorfluazuron) | ＞500 | ＜1％ |
| Abscisic acid | ＞500 | ＜1％ |
| Enfazoles | ＞500 | ＜1％ |
As can be seen from the above examples, the synthetic procedure of the artificial antigen related to naphthylacetic acid of the present invention is simple and effective, and can be effectively used in immunoassay, providing a convenient way for subsequent research and analysis, providing monoclonal antibodies secreted by cell strain HLH, and having the effect of naphthylacetic acidBetter specificity and detection sensitivity (IC 50 6.77 ng/mL), can realize the detection of the residual quantity of the naphthylacetic acid in the equivalent food, and provides a new means for establishing a rapid, simple, convenient, low-cost, sensitive and specific naphthylacetic acid detection method.
Example 6: naphthalene acetic acid immunodetection kit
This example provides a naphthaleneacetic acid immunoassay kit comprising a naphthaleneacetic acid monoclonal antibody prepared in example 3, an enzyme-labeled plate, a naphthaleneacetic acid coated antigen, a naphthaleneacetic acid standard solution, an HRP-labeled goat anti-mouse IgG secondary antibody, and a TMB color-developing solution.
The principle of detecting the naphthylacetic acid by the naphthylacetic acid immunoassay kit is as follows: and detecting the content of naphthalene acetic acid in the sample to be detected by adopting an indirect competition ELISA method. And (3) coating a naphthylacetic acid coating antigen in the micropore of the ELISA plate in advance, adding a naphthylacetic acid standard solution or a sample to be detected, a naphthylacetic acid monoclonal antibody, an HRP-marked goat anti-mouse IgG secondary antibody and a TMB chromogenic solution, preparing a naphthylacetic acid standard inhibition curve, and determining the naphthylacetic acid content in the sample to be detected according to the naphthylacetic acid standard inhibition curve and the absorbance value of the sample to be detected. The detection of the naphthylacetic acid can be realized by adopting a method commonly used in the field for operation.
Example 7: naphthylacetic acid detection colloidal gold test strip
The embodiment provides a colloidal gold test strip, which comprises a sample pad, a colloidal gold binding pad, a nitrocellulose membrane and a water absorption pad, wherein the nitrocellulose membrane is sequentially provided with a detection line and a quality control line, and the colloidal gold binding pad is coated with the naphthylacetic acid monoclonal antibody prepared in the embodiment 5. The detection line is printed by a naphthylacetic acid coating antigen. The quality control line is printed by goat anti-mouse IgG secondary antibody. The colloidal gold test strip can be assembled in a manner commonly used in the art.
The principle of detecting the naphthylacetic acid by using the naphthylacetic acid detection colloidal gold test strip is as follows: and detecting whether the sample to be detected contains naphthylacetic acid by using an indirect competition method principle. If the sample to be detected contains naphthylacetic acid, the detection line does not develop color, and the quality control line develops color. If the sample to be detected does not contain naphthylacetic acid, the detection line and the quality control line are both developed. The detection of the naphthylacetic acid can be realized by adopting a method commonly used in the field for operation.
The foregoing description of the preferred embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify for specific embodiments and applications without departing from the true spirit and scope of the present invention, and therefore, all such modifications, equivalents, and improvements that fall within the true spirit and scope of the present invention should be considered to be within the scope of the following claims.
Claims (8)
1. Hybridoma cell lines secreting the naphthylacetic acid monoclonal antibodies are preserved in the China general microbiological culture Collection center (China Committee for culture Collection of microorganisms) on 12 months and 15 days of 2021, and have the preservation name of monoclonal cell lines HLH, the preservation number of CGMCC No.45024 and the preservation address of North Chen Xi Lu No. 1 and 3 in the Korea of Beijing city.
2. A naphthalene acetic acid monoclonal antibody which is secreted by the hybridoma cell line secreting naphthalene acetic acid monoclonal antibody with a preservation number of CGMCC No.45024 according to claim 1.
3. A method of producing a naphthylacetic acid monoclonal antibody according to claim 2, comprising: taking BALB/c mice, injecting paraffin oil into the abdominal cavity, injecting hybridoma cell strain with the preservation number of CGMCC No.45024 into the abdominal cavity, collecting ascites after injection, purifying the ascites, and preserving the obtained naphthylacetic acid monoclonal antibody at low temperature.
4. Use of a monoclonal antibody to naphthylacetic acid according to claim 2, for the detection of naphthylacetic acid residues in food products.
5. Use of a hybridoma cell line secreting a monoclonal antibody to naphthalene acetic acid according to claim 1 for the preparation of a monoclonal antibody to naphthalene acetic acid.
6. The use of a hybridoma cell line secreting a monoclonal antibody to naphthalene acetic acid according to claim 1, for the detection of naphthalene acetic acid residues in food products.
7. A kit comprising the naphthylacetic acid monoclonal antibody according to claim 2.
8. The use of the kit according to claim 7 for the detection of naphthylacetic acid residues in food products.
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| JPH05260989A (en) * | 1992-03-03 | 1993-10-12 | Natl Sci Council | Monoclonal antibody against indole-3-acetic acid and its production and use thereof |
| CN105131106A (en) * | 2015-07-27 | 2015-12-09 | 苏州博源医疗科技有限公司 | 5-hydroxyindoleacetic acid immunogen, antibody and detection reagent, and preparation methods thereof |
| CN108828219A (en) * | 2018-09-13 | 2018-11-16 | 苏州科铭生物技术有限公司 | Detect the ELISA adsorption analysis method of jasmine acid content and jasmonic and methyl jasmonate total content in plant |
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