Test 7 Flashcards
Describe single radial immunodiffusion test
Antibody is premixed in a gel. Antigen is put in holes in the gel, and diffuses out. A ring of precipitate is formed around the hole.
What is Ouchterlony’s technique?
Double immunodiffusion.
Result: precipitation line (qualitative)
Describe double immunodiffusion?
Radial diffusion of antigen and antibody from holes forming a line of precipitation
How are the results of double immunodiffusion interpreted?
reaction of identity; reaction of non-identity; reaction of partial identity
What is protein electrophoresis used for, and what medium is used?
screening of serum and other fluids for protein abnormalities, agarose gel
What are the protein fractions in protein electrophoresis?
Albumin
Alpha-1 globulins
Alpha-2 globulins
Beta-1 globulins
Beta-2 globulins
Gamma globulins
What is immunofixation?
Technique used to identify abnormal bands in electrophoretic pattern of serum proteins.
Detect monoclonal components.
Proteins are anchored in situ after electrophoresis by forming an insoluble complex with antibody.
What are the four stages of immunofixation?
- Separation of proteins by electrophoresis in agarose gel
- Fixation and immunoprecipitation of electrophoresed proteins
- Non-precipitating, soluble proteins are removed by blotting and washing
- Precipitated proteins are visualized by staining
Name the tracks of immunofixation:
electrophoretic pattern, gamma antiserum, alpha antiserum, mu antiserum, kappa, lambda
What is immunoelectrophoresis a combination of?
electrophoresis and gel diffusion
How are antigens separated in immunoelectrophoresis?
charge, size, conformation, composition, pH of buffer
Describe classical immunoelectrophoresis:
A gel is prepared with alternating wells and slots with antigen mixture placed in the wells and electrophoresis is performed to separate proteins in the sample.
Antibodies are added into the slots. Antigens and antibodies migrate towards each other and form arcs of precipitate.
How are proteins separated in classical immunoelectrophoresis?
positively charged proteins move to the negative electrode, and negatively charged proteins move to positive electrode
What is identified in classical immunoelectrophoresis?
serum proteins
What type of analysis is classical immunoelectrophoresis?
qualitative
Describe counter-immunoelectrophoresis
Antigens placed in one well, antibodies in the other. Electric current is used to drive the antigen towards the antibody.
What is rocket immunoelectrophoresis used for?
- quantitative
Determining levels of serum proteins
Describe rocket immunoelectrophoresis
Antigens are loaded into wells along the edge of a gel containing incorporated antibody.
Antigens are electrophoresed into the gel where they interact with antibody forming precipitation line.
Results: height of precipitation arc is directly proportional to the concentration of the antigen, compared to known concentrations.
What is the basic principle of enzyme immunoassays?
Enzyme of conjugate reacts with a colourless substrate to generate a colour reaction product
What is a chromogenic substrate?
A substrate used in enzyme immunoassays which produce colour
How are enzyme immunoassays divided?
Enzyme-linked immunosorbent assay (ELISA),
enzyme immunoblotting and
enzyme immunohistochemistry
What are the enzymes used for enzyme immunoassays?
Alkaline phosphatase and Horse Radish peroxidase
What system is used to increase sensitivity of enzyme-immunoassays?
Biotin-avidin systems.
Avidin can non-covalently bind up to four biotin molecules - used in amplification step.
Used in case of: low level of antigen or need for rapid detection
Steps of ELISA:
- binding of known component to solid surface
- blocking of the surface to prevent further non-specific binding
- incubation of the test sample with adsorbed antigen or antibody
- detection of antigen-antibody complexes with enzyme-linked antibody which produce a colour product from substrate to be detected
