W4L8 - Advanced Antibody Based Immunoassays

Question 1

Enzyme Linked ImmunoSorbent Assay (ELISA)

Answer 1

Used in routine diagnostic labs for detection of antibody or antigen
Qualitative or quantitative
Variety of formats

Question 2

Qualitative vs Quantitative ELISA

Answer 2

Qualitative
- to test if something is present or not
Quantitative
- to test how much is present

Question 3

Competitive Assays

Answer 3

Variation of ELISA and RIA
Known amount of labelled antigen compete with antigen in sample for binding onto antibody
The more antigen in sample the more antibody sights taken up
Concentration of sample antigen is inversely proportional to signal

Question 4

Immuno PCR

Answer 4

Tag secondary antibody with DNA fragment
Use specific primers to detect DNA by PCR
Up to 1000 times more sensitive than ELISA

Question 5

Immunofluorescence

Answer 5

Fluorescent compounds emit light of certain wavelength when excited by light or shorter wavelength
These are used to label antibodies for the detection of antigen
Glass slides with cells or tissue as target
Visualised by fluorescent microscopy
Direct method to detect antigen or Ag-AB complexes formed in vivo

Question 6

Immunohistochemistry

Answer 6

Similar to ELISA but on a tissue section
Similar to immunofluorescence except no fluorescent dye
Use of antibodies labelled with an enzyme on tissue sections as an alternative to fluorescent dye
- enzyme acts on a substrate to produce colour

Question 7

Antigen Retrieval

Answer 7

Frozen sections used for IF and IHC because the antigen are in tact
Antigen retrieval needed to unmask epitopes

Question 8

Heat Induced Epitope Retrieval (HIER)

Answer 8

Placing slide with tissue fixed on surface into a buffer and applying heat
- water bath
- pressure cooker
- steamer
- microwave oven
Slides must remain hydrated

Question 9

Antigen Retrieval Steps

Answer 9

Before Antigen Retrieval
- chemicals involved in tissue fixation create aldehyde cross links between proteins
- antibody is unable to bind to antigen of interest
Citrate buffer + heat + time
After Antigen Retrieval
- citrate buffer unmasks antigens by breaking cross links allowing the antibody to bind to the antigen of interest

Question 10

Flow Cytometry

Answer 10

Cells from blood are labelled with monoclonal antibody specific for surface marker
Cells injected into sheath fluid as it passes in single file through the flow cell
Passes through laser light
Measurement of:
- forward scatter (size of cell)
- side scatter (granularity of cell)
- fluorescence detection (identify surface marker)

Question 11

Bead Technology

Answer 11

Polystyrene beads coloured different shades of red
Each bead of specific colour coated with different antibody
Reacted with sample
Antigen will react with specific bead
Second antibody labelled with green
Interrogated through a flow cytometry system
Each bead can be identified and positive reaction identified by green

Question 12

Western Blot

Answer 12

Used to identify specific protein

Used to place specific proteins onto a matrix which is then used for detection of antibodies

Question 13

SDS-PAGE

Answer 13

Sodium dodecyl sulfate - polyacrylamide gel electrophoresis
SDS
- anionic detergent
- breaks secondary and tertiary structure of protein
- applies negative charge to protein
- boiling or use of 2-ME breaks disulphide bonds
- separation based on size
PAGE
- when polymerises forms a matrix with pores
- larger proteins will be hindered more than small proteins
- Identify protein specific molecular weight