5. Immunological applications

Question 1

Production of polyclonal antibodies (pAb)

Answer 1

• > 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

Question 2

Immunological Applications

Answer 2

ELISA (Enzyme-linked Immunosorbent Assay)

Immunofluorescence

Confocal Microscopy – gives depth

Immunohistochemistry

Immunoelectron microscopy

FACS (Fluorescence Activated Cell Sorting)

Question 3

1. ELISA (Enzyme-linked immunosorbent assay)

Answer 3

• > 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

Question 4

2.

Answer 4

-

Question 5

3.

Answer 5

-

Question 6

4.

Answer 6

-

Question 7

5.

Answer 7

-

Question 8

6.

Answer 8

-

Question 9

Cell selection (+ve or -ve)

Answer 9

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

Question 10

Visualising protein expression

Answer 10

Immunofluorescence

Confocal Microscopy

Immunohistochemistry

Immunoelectron microscopy

Question 11

Excitation and emission wavelengths of some commonly used fluorochromes

Answer 11

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

Question 12

Flow cytometry or FACS (Fluorescent activated cell sorting)

Answer 12

Same fluorochromes but cells are in solution

Question 13

Mixture of cells is labelled with fluorescent antibody

Answer 13

-> stream of fluid conrainign antibody labelled cells + Laser
-> green photomultiplier tube (PMT)
(CPU - Red PMT / side scatter / forward scatter)

Question 14

Inreacellular flor cytometry

Answer 14

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

Question 15

Key points of FACS

Answer 15

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