lecture 18-20 Flashcards
What are the five varieties of antibodies?
– IgG (main)
– IgM (first)
– IgA (secreted)
– IgD (dispensible) – IgE (allergies)
Test #1 – does the B- cell make host binding antibodies?
If yes, kill cell (clonal deletion)
Test #2 – does the B- cell make useful antigen binding antibodies?
If yes, stimulate cell to divide
Protective mechanisms of binding antibodies to antigens
- aggulation
- opsonization
- neutralization
- activation of complement
- antibody-dependant cell-mediated cytotoxicity
Immune reactions are polyclonal:
several B-cells each make a different antibody that recognizes the antigen
monoclonal cells
are single B-cells made immortal so a single epitope is recognized and available for a long time
western blot
determine quantity and band identity on a gel
ELISA
to determine how much protein is present in a mixture
aggulation test
to determine identity of a pathogen
Ouchterlony Diffusion Assay
to determine the antigens/antibodies in a sample
fluorescent staining
localize proteins in a cell
antibody labels
isotope, enzyme, fluorescent
Western Blot
– Protein gel transferred to membrane, blocked, Ab
bound, washed, exposed/detected
– Allow for both the size of the target protein and abundance of target protein to be quantified
standard fluorescente microscopy
wholeimageisilluminatedandout-of- focus light makes the in-focus image fuzzy
– Pro: cheap
– Cons:fuzziness & photodamage
confocal
wholeimageisilluminatedandout-of- focus light is blocked
– Pro: very good resolution – Con: photodamage
two photon
lessenergeticlightisused(infrared) and 2 photons must hit the dye simultaneously to excite it
– Pro: lower photodamage, better tissue penetration
– Con: costliest, somewhat inferior resolution to confocal
