Which plant is transgenic? Determination using an enzyme-linked immunosorbent assay

transgenic plants / GMOs A transgenic plant has incorporated gene(s) from another species. Transgenics are useful for engineering a specific phenotype or studying a gene’s function.

transgenic selection: NPTII Selection of plants that incorporated the transgene can be performed by simultaneously introducing a drug resistance gene into the plant (just like we do with E.coli). nptII (most common) codes for the enzyme aminoglycoside 3'-phosphotransferase (NPTII) that phosphorylates (inactivates) aminoglycoside antibiotics including kanamycin, neomycin geneticin (G418) and paromomycin.

transgenic selection: kanamycin resistance due to NPTII kanamycin WT kanamycin Transgenic NPTII

Which plant is transgenic?

WT transgenic Need both WT and transgenic plants for experiment (today) so kanamycin selection is out. Perform test for transgene or transgene protein? ELISA is a fast way to test for the presence and amount of a protein such as NPTII.

ELISA understanding the acronym ELEnzyme-linked –An enzyme’s activity is used as a “reporter” to test for the presence/amount of a protein of interest. –The enzymatic reaction will produce a colored species.

ELISA understanding the acronym ELEnzyme-linked ISImmunosorbent –An antibody or antigen (“immuno”) is adsorbed (“sorbent”) onto the polystyrene wells in which we conduct the test. –One antibody is already adsorbed added to the wells and a second antibody with our enzyme will be added in the type of ELISA we will perform today.

ELISA understanding the acronym ELEnzyme-linked ISImmunosorbent AAssay –We will could determine the amount of NPTII (quantitative assay). –We will use NPTII concentration standards to construct a standard curve.

ELISA: uses antibodies What is an antibody? What types of antibodies exist? What kinds of antibodies are used in our lab today?

What is an antibody? Protein secreted by B-cells that specifically bind a foreign substance (antigen) Immunoglobulin domains Complementarity-determining Regions (CDRs) Fab= Fragment antigen binding Hinge Fc= Fragment crystalline F(ab)’2= Protease digestion still useful to bind antigen

producing polyclonal antibodies

producing monoclonal antibodies 1 2 3

Antibody-based assays

Ag Types of immunodetection systems 1. Direct immunodetection Primary antibody conjugated with enzyme system 2. Indirect immunodetection Secondary antibody conjugated with enzyme system 3. Sandwich indirect immunodetection Antigen applied in soluble form 4. Indirect immunodetection with biotin linkers Biotinylated primary antibodies Ag HRP Ag HRP Streptavidin HRP streptavidin HRP horseradish peroxidase antigen Ag

Simple ELISA protocol (For screening monoclonal antibodies) 1. Coat antigen onto microplate 2. Allow protein adsorption and block unoccupied sites with neutral protein 3. Add antibody solution into each well 4. Add enzyme conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate 5. Read absorbance in spectrophotometer

Today’s assay

Sandwich indirect immunodetection Antigen applied in soluble form HRP Substrate

Sandwich ELISA protocol 1. Coat primary antibody onto microplate. 1a. Allow antibody adsorption and block unoccupied sites with neutral protein (BSA). 2. Add antigen sample to be detected into each well. Incubate 30 min at 37 0 C. 4. Develop colorimetric reaction with appropriate substrate. Incubate 15 min at room temperature. 5.Stop reaction with 3M H 2 SO 4. Read absorbance in ELISA spectrophotometer and quantitate relative antigen levels. 3. Add second primary antibody against antigen and HRP-conjugated secondary antibody (antibody mix) into each well. Incubate 30 min at 37 0 C.

What you will do today (1): –Collect and weigh tissue sample plant A, B and C. –Repeat for second and third plants. –Add 400 μL PEBX1 buffer to each microcentrifuge tube and grind plant tissue using pestle. –Add 100 μL of PEBX1 to wells A and B. –Add 100 μL of one sample to wells C and D. –Add 100 μL of second sample to wells E and F. –Add 100 μL of third sample to wells G and H. –HANDLE THE WELLS LIKE A CUVETTE (read at bottom) A B C D E F G H

What you will do today (2): Incubate 30 minutes at 37 °C. –Place wells in a zip-close bag to prevent them from drying out in the oven. Wash wells 3 times with PBST Add antibody-enzyme conjugate (MRS-2 Ab) –Note the dilution factor for the conjugate Incubate 30 minutes at 37 °C. –Place wells in a zip-close bag to prevent them from drying out in the oven. Wash 4 times with PBST.

What you will do today (3): Add substrate and allow blue color to develop. Stop the enzymatic reaction with 3M H 2 SO 4. Presence of NPTII will result in color change from blue to yellow.

How we will detect: read absorbance at 450 nm

Data Absorbance 450 nm NPTII Std WELLWELL A B C D E F G H

Data analysis (due 4/23/12) Total amount of plant tissue (mg)A450 NPTII concentration (ng/mL) NPTII amount (ng protein /mg tissue)Transgenic?A 450 NPTII concentration (ng/mL) Your Sample Your sampleStandard WELLWELL A BlankN/A 0 2 B BlankN/A 0 1 C Plant __ 0.5 D Plant __ Average Yes/No 0.25 E Plant __ F Plant __ Average Yes/No G Plant __ H Plant __ Average Yes/No