Determination of phytohormone by enzyme-linked immunosorbent assay (ELISA)

First, the principle

Plant hormone

(planthormone) Immunoassay mainly includes radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). The latter type is often used because of the insufficiency of operation of the former. In ELISA, the detection of antigen-antibody reaction is achieved by means of an enzyme label, and the commonly used enzymes are horseradish peroxidase and alkaline phosphatase. The enzyme can directly label the hormone molecule, which is called the enzyme standard plant hormone, and can also be labeled with the second antibody (the antibody recognizing the anti-hormone antibody Fc fragment or the S. aureus A protein), which is called the enzyme-labeled secondary antibody. These two types of markers are used for solid phase antibody type and solid phase antigen type ELISA, respectively. A. Solid phase antibody type ELISA: an anti-hormone monoclonal antibody (Mab) is combined with a rabbit anti-mouse Ig antibody (RAMIG) that has been adsorbed on a solid phase carrier, and then added to a hormone standard or a sample to be tested, thereby The solidified Mab is combined with horseradish peroxidase (HRP) labeled hormone (enzyme-labeled hormone). By measuring the amount of bound enzyme hormone, the amount of hormone in the unknown sample can be converted. B. Solid phase antigen type ELISA: The "hormone-protein" complex (the protein should be different from the carrier protein in the immunogen) is coated on the solid phase carrier, and the hormone to be tested (sample or standard) and anti-IAA are added. The antibody (Pab) was cloned and subjected to a competition reaction. Then, HRP-labeled goat anti-rabbit Ig antibody (HRP-GARIG) was reacted with Pab bound to the solid phase, and the amount of the hormone in the unknown sample was converted by measuring the amount of the enzyme bound to the solid phase.

Second, materials, equipment and reagents

(1) Materials: tissues or organs such as higher plants, fungi, algae.

(2) Instruments and equipment: 1. Enzyme-linked immunosorbent detector; 2. High-speed refrigerated centrifuge; 3. Thermostat; 4. Continuous injector; 5. Vortex; 6. 96-well microplate; 7. Centrifugation Tube; 8. mortar or homogenizer; 9. test tube.

(3) Reagents 1. Washing buffer: 0.01 mol/L pH 7.4 phosphate buffer (PBS), containing 0.05% Tween-0; 2. RAMIG solution, or "hormone-protein" complex; 3. 0.1% closed Protein (this protein should be different from the carrier protein in the immunogen); 4. Anti-hormone Mab, or Pab; 5. Hormone standard mother liquor, and reference series solution (diluted in double or quadruple series); 6. HRP Labeling hormone, or HRP-GARIG; 7. O-phenylenediamine (OPD) substrate solution: 5 mg of OPD was dissolved in 12.5 ml of 0.01 mol/L pH 5.0 PBS, and 30% H2O 212.5 μl was added before use.

Third, the experimental steps

Solid phase antibody type elisa.

1. Select the optimum working concentration of each reactant by titration with square matrix method;

2. 100 μl of RAMIG solution coated with polystyrene reaction plate micropores, 4 ° C wet box, 12h;

3. Discard the solution in the well, wash it 3 times with washing buffer, and dry it;

4. Add 100 μl anti-hormone Mab solution, 37 ° C for 70 min;