6. Antibodies and biotechnology Flashcards
difference between monoclonal and polyclonal Ab?
monoclonal - Ab produced which can only recognise 1 type of Ab
polyclonal - many Ab produced - many epitopes recognised
what cells allow proliferation of cells?
tumour/myeloma cells
Advantage of polyclonal Ab
good precipitation and agglutination
advantages of monoclonal Ab - 2
- pure sample - specific binding
2. no batch variation
disadvantages of polyclonal Ab - 3
- not pure = non-specific binding
- limited serum supply = batch variation
- ethics - animals
disadvantages of monoclonal Ab -2
- POOR agglutination and precipitation
2. costly - labour intensive
4 types of blotting techniques - what do each of them detect?
western = proteins eastern = carbs/lipids Southern = DNA northern = RNA
what 3 things does western blotting tell us?
- presence/absence of protein
- drug effect
- subcellular distribution of proteins, glycosylation and size
method used for extracting pure sample of epitope
immunoaffinity production
2 types of immunoaffinity production
- affinity chromatography
2. immunoprecipitation
3 commonly used proteins for purification in immunoprecipitation
- 6 x histidine
- Gluthione-S-transferase
- Maltose binding protein
what does immunohistochemistry detect?
distribution of antigens in tissue
what does immunocytochemistry detect?
specific ag in whole cells
in IHC - what is used to preserve the cells?
paraffin wax
2 uses of ICC in clinical practice?
a) cancer diagnosis
b) change in disease with drug
what does ELISA stand for?
enzyme-linked immunosorbent assay
4 types of ELISA
- direct
- indirect
- sandwich
- competitive
purpose of competitive elisa?
test Ag concentration
which ELISA start with an Ab coated well?
indirect
which ELISA only uses 1 AB?
direct
how to detect outcome in ELISA?
add substrate which will react with enzyme on conjugated Ab - changing colour of solution
what method is used in diagnosing blood disorders?
flow cytometry
FACS
fluorescence-activated cell sorting
in FACS - what does forward scatter determine?
size and volume of cell
in FACS - what does side scatter determine?
granularity, nucleus structure, vesicles inside of cell
which cells have large forward and side scatter?
monocytes and granulocytes
3 problems of lab methods
- non-specific binding
2, tissues are auto-fluorescent - Ab need to be optimised
solution to non-specific binding
use positive and negative controls
solution to auto-fluorescent tissues
enzymatic markers
solution to Ab optimisation
alter conc to allow max binding
2 enzyme markers
- horse-radish peroxidase
2. alkaline phosphatase
2 fluorochromes
- fluorescein isiothiocyanate
2, Alexa 488
how can Ab be altered so that they don’t cause an autoimmune response in humans?
changing constant region of Ab to human
name 8 diseases Ab can be used as target therapy
- cancer
- transplant rejection
- crohn’s disease
- multiple sclerosis
- inflammatory lesions
- bacterial infections
- sepsis
- viral infections
example of Ab treatment used to combat HER2 on cancer cells
Herceptin
2 mechanisms of Herceptin
- binds to EGF on tumour cell - stops proliferation
2. binds to Fc domain - NK cell apoptosis
4 ways Ab can be used against cancer
- naked
- attach to toxin
- attach radioactive substance
- attach enzyme which activates drug
hormone which is elevated in pregnancy?
hCG
