W4L7 - Basic Antibody Based Immunoassays

Question 1

Cross Linking

Answer 1

When several antibodies bind to one antigen at different epitopes it can cause cross linking
If antigen is small (soluble antigens) the cross linking forms aggregates which precipitate
If antigen is large (e.g. cells) cross linking may cause agglutination

Question 2

Agglutination Assays

Answer 2

Direct Agglutination
- RBC, bacteria, fungi agglutinated by serum antibody
- determine if specific antibodies in serum
Passive (indirect) Agglutination
- uses inert particles or cells with no foreign antigenic markers
- coated with a known soluble antigen and mixed with serum
Haemagglutination Inhibition
- serum containing suspected antibody is treated with antigen to remove the antibody before agglutination reaction
- if antibody is present then agglutination is inhibited

Question 3

Latex Particle Agglutination

Answer 3

Reaction in a positive test for antibodies
- when particles coated with antigens, agglutination indicates presence of antibodies
Reaction in a positive test for antigens
- when particles coated with monoclonal antibodies, agglutination indicates presence of antigens

Question 4

Precipitation Assays

Answer 4

Small soluble antigens
Cross linking of antigen-antibody complexes causes aggregations to form
Produces visible precipitate

Question 5

Single Radial Immunodiffusion (Mancini plates)

Answer 5

Gel is made with antibody incorporated in agar
Hole made in gel and antigen to be measured is added
Antigen diffuses through gel and contacts antibody
Antigen diffuses through gel and contacts antibody
Zone of precipitation is formed
Diameter of zone is proportional to concentration of anitgen

Question 6

Ouchterlony Gels

Answer 6

Two adjacent wells in gel
One well has antigen the other antibody
Both diffuse through the gel
As antigen and antibody diffuse toward each other they form antigen-antibody complexes
This forms a line of precipitation between the wells

Question 7

Immunofixation Electrophoresis (IFE)

Answer 7

Immunoprecipitation Assay
Used to detect/characterise paraprotein in serum to test for Multiple Myeloma
Electrophoresis of serum in agarose gel is followed by over laying with specific antisera
Antigen-antibody complex precipitates in gel

Question 8

Nephelometry

Answer 8

Antigen mixed with specific antibody in a tube causes turbidity as aggregates form
Can be measured by passing light through tube and detecting scattering of light
Rate of increase in light scatter is proportional to concentration of antigen
Can be used to determine concentrations of Ig, complement, CRP

Question 9

Labelled Antibodies

Answer 9

Antibodies can be labelled or conjugated to:
Fluorescent dyes
- commonly Fluorescein isothiocyanate (FITC)
Enzyme
- usually horse radish peroxidase or alkaline phosphatase
Radio isotope
- I125
Others
- streptavidin, biotin, gold
When antibodies are labelled they are termed conjugated antibodies or conjugates