5. Immunological applications Flashcards

1
Q

Production of polyclonal antibodies (pAb)

A
  • > 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

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2
Q

Immunological Applications

A

ELISA (Enzyme-linked Immunosorbent Assay)

Immunofluorescence

Confocal Microscopy – gives depth

Immunohistochemistry

Immunoelectron microscopy

FACS (Fluorescence Activated Cell Sorting)

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3
Q
  1. ELISA (Enzyme-linked immunosorbent assay)
A
  • > 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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4
Q

2.

A

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5
Q

3.

A

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6
Q

4.

A

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7
Q

5.

A

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8
Q

6.

A

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9
Q

Cell selection (+ve or -ve)

A

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

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10
Q

Visualising protein expression

A

Immunofluorescence

Confocal Microscopy

Immunohistochemistry

Immunoelectron microscopy

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11
Q

Excitation and emission wavelengths of some commonly used fluorochromes

A

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

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12
Q

Flow cytometry or FACS (Fluorescent activated cell sorting)

A

Same fluorochromes but cells are in solution

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13
Q

Mixture of cells is labelled with fluorescent antibody

A

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

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14
Q

Inreacellular flor cytometry

A

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
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15
Q

Key points of FACS

A

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

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