Enzyme-linked immunosorbent assay for detecting of jasminic acid in plant and total content of jasmonic acid and methyl jasmonate
An enzyme-linked immunosorbent adsorption and methyl jasmonate technology, applied in the research field of analytical chemistry, can solve problems such as low content, limited sensitivity of analytical methods, interference from surrounding impurities, etc., and achieve high sensitivity, low test cost, and large test volume Effect
Inactive Publication Date: 2018-11-16
SUZHOU COMIN BIOTECH
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Problems solved by technology
However, the content of JAs in plants (µg ~ ng / g fresh weight) is very low and the surrounding impurities interfere a lot, which limits the sensitivity of its analytical method
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Embodiment
[0044] (1) Preparation of immunogen and coating antigen
[0045] 12.22 mg (0.0581 mmol) jasmonic acid (JA), 6.69 mg (0.0581 mmol) N-hydroxysuccinimide (NHS), 11.98 mg (0.0581 mmol) dicyclohexylcarbodiimide (DCC) were dissolved in 100 ~200 μL dimethylformamide (DMF), then add NHS and DCC solutions to the JA solution sequentially while stirring at room temperature, stir overnight at room temperature, centrifuge the mixture at 4°C for 10 minutes, and take the supernatant. Then slowly add the supernatant to 5 mL of 100 mg bovine serum albumin (BSA) (or ovalbumin (OVA) in NaHCO 3 Stir in the buffer solution for 1-10 hours, centrifuge, take the supernatant, dialyze for several days, freeze-dry the solution, and store it in the refrigerator at -20°C, where JA-BSA is the immunogen and JA-OVA is the coating antigen . For characterization results, see figure 1 .
[0046] (2) Preparation of monoclonal antibodies
[0047] Immunization: For the first immunization, dissolve the immunog...
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Abstract
The invention discloses an enzyme-linked immunosorbent assay (ELISA) for detecting the content of jasmonic acid (JA) in plant samples and the total content of jasmonic acid and methyl jasmonate (JAs),and is characterized by the use of hybridoma technology to successfully prepare a monoclonal antibody with high sensitivity and high specificity for methyl jasmonate (MeJA), and the establishment ofindirect competition of ELISA with methyl jasmonate (MeJA) as the detection target. The sensitivity (IC50 value) of the ELISA method for detecting methyl jasmonate (MeJA) is 2.02 ng / mL, and the detection limit (LOD value) is 0.20 ng / mL. The method for detecting the content of jasmonic acid phytohormone in plant samples established by the invention has the advantages of high sensitivity, high specificity, rapidity, small sample amount and high flux, and provides a powerful analytical test method for an in-depth study on the physiological effects of jasmonic acid plant hormones.
Description
1. Technical field [0001] The invention relates to an enzyme-linked immunosorbent assay method (ELISA) for detecting the content of jasmonic acid (JA) and the total content of jasmonic acid and methyl jasmonate (JAs) in plants, belonging to the research field of analytical chemistry. 2. Background technology [0002] Jasmonates (JAs), typically represented by jasmonic acid (JA) and methyl jasmonate (MeJA), are a new type of natural plant growth regulator with broad-spectrum physiological effects , widely present in a variety of higher plants. As natural endogenous plant hormones, both JA and MeJA can control signal transduction and regulate growth, and have various physiological effects that regulate plant growth and development at the molecular, cellular, organ and individual levels. Through self-regulation of jasmonate-like hormones (JAs)-responsive transcription factors, JAs play an important role in the accumulation of plant secondary metabolites, which are important re...
Claims
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IPC IPC(8): G01N33/577
CPCG01N33/577
Inventor 赵林川
Owner SUZHOU COMIN BIOTECH