Immunology & antibodies

Question 1

antibody dependant cell mediated cytotoxicity

Answer 1

antibodies attached to target cells cause destruction by eosinophilis and nk cells

Question 2

activation and cell lysis

Answer 2

cause inflamation and cell lysis

Question 3

antibodies in situ

Answer 3

created against a single antigen by injecting sample(proteins, polysaccharides, nucleic acids, haptens) into a lab animal

Question 4

steps in antibody production

Answer 4

blood collected from (immunized) animals
cell spun out to obtain serum
Affinity chromatography column produced(binds Fc region)
plasma poured through column to bind Fc region(tail of antibody)
elution by low pH buffer(then neutralized in fraction tube)
test by SDS-page to determine peak fraction

Question 5

problems with making antibodies

Answer 5

• in one organism there may be cross reactive or non-specific antibodies in the same isolation
• new antibodies can contaminate your animal extractions if they get exposed to pathogens
• animals die

Question 6

hybridoma

Answer 6

B-cells fused with Cancerous B cells and metabolicly and immunologicallly selected for

Question 7

Elisa uses IgG’s to

Answer 7

determine how much of a protein is present in a mixture

Question 8

agglutination tests use IgG’s to

Answer 8

determine the identity of a pathogen

Question 9

ouchterlony tests use IgG’s to

Answer 9

determine the antigens/antibodies in a sample

Question 10

fluorescent staining use IgG’s to

Answer 10

localize proteins in a cell

Question 11

Isotope labelling

Answer 11

• 125I or 35S

- As the isotope decays electrons expose X-ray film layered over the blot

Question 12

Enzyme labelling

Answer 12

Common enzymes(HRP, AP) catalyze a reaction to give coloured substance or chemiluminescence

Question 13

Fluorescent labelling

Answer 13

Dye attached that absorbs one colour of light and gives off another

Question 14

Western blot applications

Answer 14

detection of a protein of interest
expression profiling
verying transgenic organsims
detect infections

Question 15

RPE65

Answer 15

gene involved in recycling vitamin A(without it you’re blind)

Question 16

Steps in elisa test

Answer 16

samples placed in wells
blocking proteins added(washed away)
detection antibodies added(washed away)
secondary enzyme w/ linked enzyme added(washed away)
chromogenic reagent added

Question 17

fluorescence microscopy

Answer 17

cells fixed in place on slides with paraformaldehyde or methanol/acetone
permeabilized(cell membrane broken) with dilute detergent
cells blocked(flooded with blocking reagent)
primary antibody added
secondary fluorescent antibody added

Question 18

standard fluorescent microscopy

Answer 18

whole image illuminated, cheap, fuzzy photodamage

Question 19

confocal fluorescent microscopy

Answer 19

whole image illuminated, out of focus light blocked, good resolution, photodamage

Question 20

two photon fluorescent microscopy

Answer 20

less energetic light, 2 photons must hit dye to exite it

lower photodamage, better tissue penetration, costliest, somewhat inferior resolution to confocal