5. Immunological applications Flashcards
Production of polyclonal antibodies (pAb)
- > antigen binds covalently to the affinity matrix
- > antigen specific antibodies bind. Non-specific are washed
- > antigen-specific antibodies are eluted.
Affinity purification:
involves the separation of molecules in solution (mobile phase) based on differences in binding interaction with a ligand that is immobilized to a stationary material (solid phase). A support or matrix in affinity purification is any material to which a biospecific ligand is covalently attached
Immunological Applications
ELISA (Enzyme-linked Immunosorbent Assay)
Immunofluorescence
Confocal Microscopy – gives depth
Immunohistochemistry
Immunoelectron microscopy
FACS (Fluorescence Activated Cell Sorting)
- ELISA (Enzyme-linked immunosorbent assay)
- > Anti - cancer antigen antibody coated plate (1st pair)
- > Add test sample containing cancer antigen
- > Add HPP - conjugated anti - cancer antigen antibody (2nd pair)
- > Add substrate
- > Develop colour and read on plate reader
The concentration o the cancer antigen is directly proportional to the colour intensity
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Cell selection (+ve or -ve)
Heterogenous population of lymphocytes is mixed with antibodies coupled to paragmetic particles or beads and poured over an iron wool mesh
When a magnetic field is applied the coupled clels stick to the iron wool; unlabeled cells are washed out.
The magnetic field is removed releasing the coupled cells
Visualising protein expression
Immunofluorescence
Confocal Microscopy
Immunohistochemistry
Immunoelectron microscopy
Excitation and emission wavelengths of some commonly used fluorochromes
R-phycoerythrin (PE)
-> excitation :
480 ; 565 nm
-> emission : 578 nm
Fluorescein
-> 495 nm#-> 519 nm
PerCP
- > 490 nm
- > 675 nm
Texas red
-> 589 nm
615 nm
Rhodamine
- > 550 nm
- > 573 nm
Green fluorescent protein (GFP) from jelly fish Aequorea victoria
380 -516 excitation + 440 - 529 emission -> GFP derives
540 - 590 -> mRFP1 derived
596 + 590 and 625 + 648 -> evolved by SHM
Flow cytometry or FACS (Fluorescent activated cell sorting)
Same fluorochromes but cells are in solution
Mixture of cells is labelled with fluorescent antibody
-> stream of fluid conrainign antibody labelled cells + Laser
-> green photomultiplier tube (PMT)
(CPU - Red PMT / side scatter / forward scatter)
Inreacellular flor cytometry
Detects proteins synthesised by T cells
- > Activated T cells are treated with an inhibitor which blocks protein export allowing cytokines to accumulate in the ER
- > The cell is fixed and permeabilized with mild detergents
- > Cytokine-specific antibodies penetrate the cell to bind the intracellular cytokine molecules
Key points of FACS
Most commonly used to measure cell surface molecules (intracellular possible too)
Multiple fluorochromes allow multi-molecules to be evaluated on individual cells, i.e. 12 -15
Cells can be sorted for future use
Used clinically for many leukaemias and lymphomas, and HIV
Typically 5 – 10,000 cells analysed in <10 sec