Elisa: Enzyme-Linked Immunosorbent Assay
Elisa: Enzyme-Linked Immunosorbent Assay
Elisa: Enzyme-Linked Immunosorbent Assay
Based on antibodies
What is an antibody? What types of antibodies exist? What kinds of antibodies are used in our lab today?
-Hinge
-Fc= Fragment crystalline -Fab= Fragment antigen binding -F(ab)2= Protease digestion still useful to bind antigen
Polyclonal Antibodies
Monoclonal antibodies
Antibody-based assays
2. Indirect immunodetection
Secondary antibody conjugated with enzyme system
HRP HRP
HRP
HRP
Ag
Ag
Ag
Ag
HRP
HRP
HRP
HRP
HRP HRP
HRP
HRP
HRP Ag Ag
Ag
Ag
Streptavidin
2. Allow protein adsorption and block unoccupied sites with neutral protein
3. Add antibody solution (hybridoma supernatant) into each well
4. Add HRP or AP conjugated secondary antibody into each well and develop colorimetric reaction with appropriate substrate
5. Read absorbance in spectrophotometer with appropriate filter and quantitate relative antigen levels
Todays assay
2. Indirect immunodetection
Secondary antibody conjugated with enzyme system
HRP HRP
HRP
HRP
Ag
Ag
Ag
Ag
HRP
HRP
HRP
HRP
HRP HRP
HRP
HRP
HRP Ag Ag
Ag
Ag
Streptavidin
NPTII
Two neomycin phosphotransferase genes are used in selection of transformed organisms: the neomycin phosphotransferase I (nptI) gene and the neomycin phosphotransferase II (nptII) gene. The second one is the more widely used. It was initially isolated from the transposon Tn5 that was present in the bacterium strain Escherichia coli K12. The gene codes for the aminoglycoside 3'-phosphotransferase (denoted aph(3')-II or NPTII) enzyme, which inactivates by phosphorylation a range of aminoglycoside antibiotics such as:
* kanamycin * neomycin * geneticin (G418), and * paromomycin.