Antibody practical

Why can antibodies be used to identify proteins

They are highly specific about which antigen they bind to so can be used to distinguish different proteins

What are the different methods of identifying proteins with antigens

• Radial immunodiffusion

• Double immunodiffusion

• Immunoelectrophoresis

What is immunological testing used for

• Serum immunoglobulin concentrations

• Detecting specific antibodies

• Distinguish between antigens

How many binding sites do antigens have

Several sites

How many binding sites do antibodies have

At least two

What does the size of immune complexes depend on

• Relative amounts of antigen or antibody

• Number of binding sites on the antigen

• Class of antibody

What do immune complex formations look like

What is single radial immunodiffusion

• Gel contains antibodies which will bind target molecules

• The further the antigen diffuses from the well the more dilute it becomes

• Higher concentrations of antigen will reach the zone of equivalence further from the well

• Diameter of preciptin ring is proportional to Ag concentration

What happens in double immunodiffusion

• Hot agar solution poured onto plate

• Cools and sets

• Holes punched

• Ag and Ab added to well

• Ags and Abs diffuse into gel setting up a concentration gradient

• Ags and Abs combine forming large aggregates at approximately equimolar concentrations

• Precipitation of large Ag:Ab aggregates form the preciptin line

What is immunoelectrophoresis

• Technique used to identify any myelomas by demonstrating monoclonal immunoglobulins in sera

• These are detected using antisera to specific IgG, IgA and IgM heaby chains and light chains

• Deficiencies can also be identified as can acute bacterial infections

What is myeloma

• A blood cancer arising from plasma cells

• Tend to create huge amounts of a monoclonal antibody

• Can identify this excess production in sera of patients as identical antibodies with only 1 kind of heavy chain and 1 light chain

What happens in immunoelectrophoresis

1. Patient or Normal serum goes into central well

2. Electrophoresis seperates proteins

3. Immunoglobulins detect by antisera in troughs