Determination of plant hormones by enzyme-linked immunosorbent assay
Determination of plant hormones by enzyme-linked immunosorbent assay
Plant growth and development, gene expression, and response to environmental stimuli are governed by hormone balance in the body. However, due to the extremely low content of endogenous hormones, it is difficult to determine them accurately and rapidly in the laboratory. Immunoassay of plant hormones (planl hormone) has greatly facilitated the study of plant hormones. The purpose of this experiment is to master the principle and operation of enzyme-linked immunosorbent assay (ELISA) for the determination of plant hormone content, and to familiarize with the use of enzyme-linked immunoassay detector.
Principle
1. In ELISA, the detection of antigen-antibody reactions relies on enzyme markers, commonly used enzymes are horseradish peroxidase and alkaline phosphatase. The enzymes can be used to label hormone molecules directly, called enzyme-labeled phytohormones, or they can be labeled on a secondary antibody (an antibody that recognizes the Fc fragment of an anti-hormone antibody or the Staphylococcus aureus A protein), which is called an enzyme-labeled secondary antibody. These two types of markers are used in solid-phase antibody-based and solid-phase antigen-based ELISA, respectively .
(1) Solid-phase antibody-based ELISA: An anti-hormone monoclonal antibody (MAb) is conjugated to a rabbit anti-mouse 1 g antibody (RAMIG) that has been adsorbed on a solid-phase carrier, and then hormone standards or samples to be tested are added so that they bind to the solid-phase MAb, and then horseradish peroxidase (HRP) is added to label the hormone (the enzyme-labeled hormone). By determining the amount of enzyme-labeled hormone bound, the amount of hormone in the unknown sample can be converted.
(2) Solid-phase antigen-based ELISA: The "hormone-protein" complex (the protein should be different from the carrier protein in the immunogen) is encapsulated in a solid-phase carrier, and the hormone to be tested (sample or standard) is added to react with the anti-IAA polyclonal antibody (PAb) to carry out a competitive reaction, and then the HRP-labeled goat anti-rabbit Ig antibody ( HRP-GARIG) is allowed to bind to the sample. HRP-GARIG) reacts with the PAb bound to the solid phase, and the amount of enzyme bound to the solid phase is determined. The amount of hormone in the unknown sample was converted.
Operation method
Determination of plant hormones by enzyme-linked immunosorbent assay
Principle
1. In ELISA, the detection of antigen-antibody reactions relies on enzyme markers, commonly used enzymes are horseradish peroxidase and alkaline phosphatase. The enzymes can be used to label hormone molecules directly, called enzyme-labeled phytohormones, or they can be labeled on a secondary antibody (an antibody that recognizes the Fc fragment of an anti-hormone antibody or the S. aureus A protein), called an enzyme-labeled secondary antibody. These two types of markers are used in solid-phase antibody-type and solid-phase antigen-type ELISA, respectively.(1) Solid-phase antibody-type ELISA: Anti-hormone monoclonal antibody (MAb) is conjugated to rabbit anti-mouse 1 g antibody (RAMIG) which has been adsorbed on solid-phase carrier, and then hormone standards or samples to be tested are added to make it bind to solidified MAb, and then horseradish peroxidase (HRP)-labelled Horseradish peroxidase (HRP)-labeled hormone (enzyme-labeled hormone). By determining the amount of labeled hormone bound, the amount of hormone in the unknown sample can be converted. (2) Solid-phase antigen-based ELISA: The "hormone-protein" complex (the protein should be different from the carrier protein in the immunogen) is encapsulated in a solid-phase carrier, and the hormone to be tested (sample or standard) is added to the anti-IAA polyclonal antibody (PAb) for a competitive reaction, and then an HRP-labeled goat anti-rabbit Ig antibody ( HRP-GARIG) is added to the solid-phase carrier for a competitive reaction. HRP-GARIG) to react with PAb bound to the solid phase, and the amount of hormone in the unknown sample was converted by measuring the amount of enzyme bound to the solid phase.
Materials and Instruments
Material: Move The basic process of enzyme-linked immunosorbent assay (ELISA) for the determination of phytohormone content can be divided into the following steps: (i) Solid-phase antibody-based ELISA 1. Select the optimum working concentration of each reactant by titration using the square array method. 2. 2. 100 μL of RAMIG solution is encapsulated in microtiter wells of a polystyrene reaction plate at 4 °C for 12 h . 3. The solution in the wells is discarded. 3. Discard the solution in the wells, wash 3 times with washing buffer and shake dry. 4. Add 100 μl of anti-hormone MAb solution at 37 °C for 70 min. 5. Discard the solution in the wells. 5. Discard the solution in the wells, wash three times with washing buffer and shake dry. 6. 6. Add 50 μL of the sample solution to be tested to each well, 3 replicates of each sample, 15-20 °C, 20 min . 7. Add 50 μL of HRP-labeled hormone at 37 °C for 60 min. 8. Discard the solution in the wells, wash three times with washing buffer, and shake dry. 9. Add 100 μL of OPD substrate solution and develop the color at 37 °C for 15~20 min, then add 50 μL of 3mol-L-1H2SO4 to terminate the reaction. Determine the A490 value of each well at 490 nm by ELISA, and find out the average value of duplicate wells for each sample. 10. 10. Use the master solution of hormone standard and the reference series solution. The same method on the same microtiter plate. (ii) Solid phase antigen type ELISA 1. Select the optimal working concentration of each reactant by titration using the square array method. 2. 100 "Hormone-protein" complexes are coated in microtiter plates at 37 °C for 12 h. 3. The wells are discarded. 3. Discard the solution in the wells, wash 3 times with washing buffer and shake dry. 4. 4. Add 100 μL of 1% Sequestered Protein Solution to the plate at 37 °C for 30 min. 5. 5. Discard the solution in the wells, wash three times with washing buffer and shake dry. 6. 6. Add 50 μL of sample solution and 50 μL of anti-hormone PAb solution to each well simultaneously, and zero the wells with normal rabbit serum solution (non-specific adsorption wells) as a blank, 37 °C, 60 min. 7. Discard the solution in the wells, wash 3 times with washing buffer and shake dry. 8. Add 100 μL of HRP-GARIG solution at 37 °C for 60 min. 9. Discard the solution in the wells, wash 3 times with washing buffer, and shake dry. 10. The subsequent color development, suspension and colorimetric procedures are as above. 11. Use the hormone standard mother solution and the reference series solution in the same microtiter plate with the same method as above. (iii) Calculation of results 1. Factorized antibody ELISA. (1) The wells with the hormone mother liquor (non-specific adsorption wells) are used as blanks for zeroing, the wells with hormone-free PBS are used as B0 wells, and the wells with the hormone standard reference solution are used as B0 wells. The wells to which the hormone standard reference series solution is added are called Bi wells . Take the common logarithm of the amount of standard hormone substance lg n as the horizontal coordinate (x), and the corresponding ln [ Bj/ ( Bo - Bj )] as the vertical coordinate (y), a straight line y = a + bx can be obtained. (2) Substitute the human pour value of the sample well into the above equation, convert the amount of hormone in the sample to be measured, multiply by the dilution factor, divide by the weight of the sample, that is, the hormone content per gram of sample. 2. Solid phase antigen ELISA . Take the wells adding hormone-free phosphate buffer as Bo wells, and take the wells adding hormone standard reference series solution as Bj wells, and calculate the standard curve and analyze the results as above. Caveat 1. note: the principle and use of the enzyme immunoassay. 2. it is important to know the specificity of the antibodies used. 2. It is important to know the specificity of the antibodies used. Antibodies that have not undergone a rigorous cross-reactivity test should not be used in the immunoassay, as this may lead to erroneous results. For more product details, please visit Aladdin Scientific website.
Tissues or organs of higher plants, fungi, algae, etc.
Reagents:
(1) Washing buffer: 0.01 mol - L
-1
pH 7.4 phosphate buffer (PBS) containing 0.055% Tween-20;
(2) RAMIG solution or "hormone-protein" complex;
(3) 1% blocking protein (this protein should be different from the carrier protein in the immunogen);
(4) Anti-hormone MAb or PAb;
(5) Hormone standard master mix and reference series solution (double or 4-fold series dilution);
(6) HRP-labeled hormone or HRP-GARIG;
(7) o-phenylenediamine (OPD) matrix solution: weigh 5 mg OPD dissolved in 5 mL of 0.01 mol - L
-1
pH 5.0 phosphate buffer, add 30% BAX 12.5 μL before use.
Equipments: enzyme immunoassay, high-speed freezing centrifuge, thermostat, continuous injector, vortexer, 96-well microtiter plate, centrifuge.
96-well microtiter plate, centrifuge tubes, mortar or homogenizer, test tubes, etc.