ELISA Class

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

TransgenicNPTII

Which plant is transgenic?

Which plant is transgenic?

WT WT 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.

ELISAunderstanding the acronym

• EL Enzyme-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.

ELISAunderstanding the acronym

• EL Enzyme-linked• IS Immunosorbent

– 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.

ELISAunderstanding the acronym

• EL Enzyme-linked• IS Immunosorbent• A Assay

– 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 antibodies1 2

3

Antibody-based assays

Ag 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 Ag AgAg

HRPHRP

HRPHRP HRPHRP

HRPHRP

Ag Ag

HRP

Streptavidin

HRP

HRP HRP

HRP

streptavidin

HRPhorseradishperoxidase

antigenAg

Today’s assay

Sandwich indirect immunodetection Antigen applied in soluble form

HRPHRP HRPHRPSubstrate

Substrate

Substrate

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 370 C.

4. Develop colorimetric reaction with appropriate substrate. Incubate15 min at room temperature.

5. Stop reaction with 3M H2SO4. Read absorbance in ELISA spectrophotometerand 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 370 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 H2SO4.• 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

1 2 3 4 5 6 7 8 9 10 11 NPTII Std

WELL

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

SampleYour

Sample Your Sample Your Sample Your sample Standard Standard

WELL

A Blank N/A  0 N/A N/A 2B Blank N/A  0 N/A N/A 1C Plant __   0.5D Plant __  Average Average Yes/No 0.25E Plant __   0.125F Plant __  Average Average Yes/No 0.0625G Plant __   0.03125H Plant __  Average Average Yes/No 0.015625