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

First, the principle

Plant Hormone Immunoassay

Plant hormone immunoassays are commonly based on two main techniques: radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). While RIA is highly sensitive, it requires specialized equipment and handling, making it less practical for routine use. As a result, ELISA has become the preferred method due to its simplicity, safety, and cost-effectiveness.

ELISA works by detecting antigen-antibody interactions using an enzyme label. The most frequently used enzymes are horseradish peroxidase (HRP) and alkaline phosphatase (AP). These enzymes can either be directly conjugated to the hormone molecule, known as enzyme-labeled hormones, or attached to secondary antibodies that recognize the primary antibody’s Fc region or other binding sites, such as S. aureus A protein. These labeled reagents are used in different types of ELISA, depending on whether the solid-phase component is an antibody or an antigen.

A. Solid Phase Antibody ELISA: In this method, an anti-hormone monoclonal antibody (Mab) is first immobilized on a solid support, often a polystyrene microplate. This Mab is then combined with a rabbit anti-mouse IgG (RAMIG) that has been pre-coated onto the plate. When a sample containing the hormone is added, the Mab binds to the hormone. An HRP-labeled version of the hormone is also introduced, allowing for detection through enzymatic colorimetric reactions. By measuring the amount of bound enzyme, the concentration of the hormone in the unknown sample can be determined.

B. Solid Phase Antigen ELISA: In this approach, a hormone-protein complex—where the protein is distinct from the carrier used in immunization—is coated onto the solid phase. The sample or standard solution containing the hormone is then mixed with an anti-hormone polyclonal antibody (Pab). This creates a competitive reaction between the free hormone and the immobilized hormone. After incubation, an HRP-labeled goat anti-rabbit IgG (HRP-GARIG) is added, which binds to the Pab attached to the solid phase. The amount of enzyme bound to the plate reflects the hormone concentration in the sample, which can be quantified by measuring the optical density at a specific wavelength.

Second, materials, equipment and reagents

(1) Materials: Tissues or organs from higher plants, fungi, or algae may be used as sources of plant hormones.

(2) Instruments and equipment: 1. ELISA reader; 2. High-speed refrigerated centrifuge; 3. Incubator; 4. Automatic pipette; 5. Vortex mixer; 6. 96-well microplate; 7. Centrifuge tubes; 8. Mortar or homogenizer; 9. Test tubes.

(3) Reagents:

1. Washing buffer: 0.01 mol/L phosphate buffer (PBS), pH 7.4, containing 0.05% Tween-20.

2. RAMIG solution or "hormone-protein" complex.

3. 0.1% blocking protein (should be different from the carrier protein used in immunization).

4. Anti-hormone monoclonal antibody (Mab) or polyclonal antibody (Pab).

5. Hormone standard stock solution and reference series (diluted in double or quadruple serial dilutions).

6. HRP-labeled hormone or HRP-conjugated goat anti-rabbit IgG (HRP-GARIG).

7. O-phenylenediamine (OPD) substrate solution: Dissolve 5 mg OPD in 12.5 ml 0.01 mol/L PBS, pH 5.0, and add 12.5 μl of 30% H₂O₂ just before use.

Third, the experimental steps

Solid Phase Antibody ELISA

1. Perform a titration using a square matrix method to determine the optimal working concentrations of all reagents.

2. Coat 100 μL of RAMIG solution into each well of a polystyrene microplate and incubate overnight at 4°C in a humidified chamber.

3. Remove the solution, wash the wells three times with washing buffer, and allow them to air dry.

4. Add 100 μL of anti-hormone Mab solution and incubate at 37°C for 70 minutes.