Lecture 15 – Immunological techniques in diagnostics and research

Question 1

7 IT (7)

Answer 1

Antibodies
Agglutination reactions
Flow cytometry
Confocal microscopy
IHC
ELISA
Western blotting

Question 2

Antibodies (6)

Answer 2

Produced by B cells.
Ab specific to TP (target protein) in mice.

1) TP in mice.
2) B cells + Tumour = Hybridoma.
3) Hybridoma produces Ab.
4) Ab cloned in IT.

Question 3

Agglutination reactions (4)

Answer 3

Diff blood types, Ab,Ag, Rh.

1) Mix sample blood with A, B/Rh /Ag.
2) Check to see if blood undergoes agglutination.
3) Aggultination = Ag present in blood sample.

Question 4

Flow cytometry (8)

Answer 4

Analysis of proteins ON cells that are suspended in fluid.
Involves FCA (Flurochrome-conjugated antibody).
1) FCA specific for TP are added to cells.
2) Cells chanelled past laser, excite flurochrome and emits light.
3) Emitted light is detected and plotted on a graph.

Amount of light emitted = Amount of Ab bound to protein = Amount of protein expressed = cell size/density.

Research = Cell sorting.
Diagnostic = CD4 in HIV.

Question 5

Confocal microscopy (5)

Answer 5

Analyse tissue sections/cells attached to microscopic slide.
Light emitted by FCA viewed under microscopic slide.
Collect 2D images, joins to make a 3D image.
It can visualise where TP is on cells.

Research - Identification and analysis of cells within tissues.

Question 6

IHC (7,2)

Answer 6

ImmunoHistoChemistry

Distribution/localisation of Ag using Ab-Ag interactions.

1) Thin sections of tissue.
2) Primary Ab recognise TP added to tissue.
3) Ab-Ag visualised using chromogenic detection.
4) Secondary Ab specific for primary Ab conjugated to HRP is added.
5) HRP catalyses conversion of DAB to produce brown precipitate at location of protein.
6) Brown ppt visualised with light microscope.

Diagostic use in cancer B-Raf gene = cell division.

Question 7

ELISA (3,4)

Answer 7

Quantifies amount of protein/antibody in liquid samples.
Applications
1. Detection of bacterial toxins - E.coli
2. Home pregnancy test - HCG in urine.

4 types
Direct/Indirect/Sandwich/Competitive.

Question 8

Western blotting (8)

Answer 8

Detects proteins
Southern - DNA Northern - RNA
1) Sample prep - Cells lysed
2) Electrophoresis - Lysates into a gel
3) Transfer to membrane - Fractionated proteins
4) Stain for protein of interest.

Application
Diagnostic - basically nonsense.
Research
- Cell signalling proteins
- Cancer drug mechanism of action.