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2. Immuno Assays Folkard Asch, 13.01.2000 - Slide 2 for Plant Hormone Analyses Immuno assays are based on the competition between a known amount of antigen and an unkown amount of sample antigen for binding sites on a limited amount of highly specific antibody.

3. Immuno Assays Folkard Asch, 13.01.2000 - Slide 3 for Plant Hormone Analyses Important pre-requisites: highly specific antibody a way to label the antibody without losing its specificity a process to separate the antibody bound antigen from the unbound antigen proper sample and purification techniques

4. Immuno Assays Folkard Asch, 13.01.2000 - Slide 4 for Plant Hormone Analyses we use two types of assays: Radio Immuno Assay (RIA) Abscisic Acid Enzyme Linked Immuno Sorbent Assay (ELISA) Absicic Acid Zeatin-riboside

5. Immuno Assays Folkard Asch, 13.01.2000 - Slide 5 for Plant Hormone Analyses RIA RIA combines a specific immuno reaction with the sensitivity of radio-isotope techniques.

6. Immuno Assays Folkard Asch, 13.01.2000 - Slide 6 for Plant Hormone Analyses RIA un-labelled antigen (ABA) competes with 3 H labelled antigen ( 3 HABA) for the binding sites of a monoclonal antibody (MAC250). Standard curves derived from antibody- bound labelled ABA (antigen) versus total supplied ABA (antigen) are used to determine the unkown amount of ABA (antigen) in a sample.

7. Immuno Assays Folkard Asch, 13.01.2000 - Slide 7 for Plant Hormone Analyses RIA to measure the bound labelled ABA liquid scinitillation is used. It uses the effect of matter fluorescence when bombarded with radioactive radiation to determine the amount of label in the solution which is equivalent to the ABA bound to the antibody

8. Immuno Assays Folkard Asch, 13.01.2000 - Slide 8 for Plant Hormone Analyses RIA Advantages: only one antibody needed monoclonal antibody commercially available easy to handle

9. Immuno Assays Folkard Asch, 13.01.2000 - Slide 9 for Plant Hormone Analyses RIA Dis-Advantages: relatively expensive, requires advanced equipment (temperature regulated centrifuge, scinitlation counter, radioactive handling permission) limited number of samples limited sensitivity for ABA

10. Immuno Assays Folkard Asch, 13.01.2000 - Slide 10 for Plant Hormone Analyses ELISA ELISA uses an indirect way to label the antibody. Antibody bounds to stationary antigen. A secondary enzym-marked antibody binds to the primary antibody. An ezymatic reaction on the secondary antibody creates a color reaction. The color intensity is equivalent to the amount of antibody bound to the antigen.

11. Immuno Assays Folkard Asch, 13.01.2000 - Slide 11 for Plant Hormone Analyses ELISA empty microtiter- plate-well add coating solution containing the antigen and the antigen carrier protein

12. Immuno Assays Folkard Asch, 13.01.2000 - Slide 12 for Plant Hormone Analyses ELISA Wash. Add sample solution containing the antigen and a limited amount of monoclonal anti- body

13. Immuno Assays Folkard Asch, 13.01.2000 - Slide 13 for Plant Hormone Analyses ELISA Wash after competition of the free antigen and the coating antigen for the anti-body. A relative amount of anti-body binds to the coating of the well Add secondary, enzyme-marked anti-body. The secondary, enzyme-marked anti-body binds to the primary anti-body.

14. Immuno Assays Folkard Asch, 13.01.2000 - Slide 14 for Plant Hormone Analyses ELISA Add the enzyme substrate. The enzyme, linked to the sencondary anti-body, produces a colored product. S P S P S P S P S S P The intensity of the color is relative to the amount of antigen in the sample. Measure with a photometer, i.e. an ELISA reader.

15. Immuno Assays Folkard Asch, 13.01.2000 - Slide 15 for Plant Hormone Analyses ELISA Advantages: low cost equipment requirements high sensitivity to ABA and CYT no radioactivity easy to set-up

16. Immuno Assays Folkard Asch, 13.01.2000 - Slide 16 for Plant Hormone Analyses ELISA Dis-Advantages: relatively difficult to handle ezyme reactions are not as precise as scintilation counting two antibodies required