Lecture 15 - Immunoassays and their applications Flashcards
What is Ouchterlony Immunodiffusion?
A qualitative technique for detecting AB-antigen interactions in which antigens and ABs are placed in a gel of agar and allowed to diffuse towards one another
A positive action is indicated by the presence of precipitin lines (AB and antigen binding to form a complex comes out of solution as a precipitate)
In Ouchterlony Immunodiffusion, what happens if there is an excess of antibody?
No precipitation occurs
In Ouchterlony Immunodiffusion what happens of there is an excess of antigen?
No precipitation occurs due to steric hindrance.
How is an Ouchterlony Immunodiffusion set up?
- Petri dish taken containing agar gel
- Wells cut out with 1 central well and several wells surrounding it in a circle formation
- Add antibodies of interest to the inner well and antigen to the outer wells
In an Ouchterlony Immunodiffusion what result is indicative of identical antigenic determinants between 2 of the wells?
A smooth arc of precipitation is formed. The wells may not contain the same allergen but a smooth arc of precipitation shows they would both produce an immune response within the patient, and have common antigenic determinants
In an Ouchterlony Immunodiffusion what result is indicative of no common antigenic determinants between 2 of the wells?
Precipitin lines cross without any interaction, producing a cross swords reaction. This shows that they are unrelated.
In an Ouchterlony Immunodiffusion what result is indicative of identical antigenic determinants between 2 of the wells?
Precipitin lines fuse, but an additional spur forms towards the well containing one of the solutions, showing some common determinants but one of the wells has additional unique antigenic determinants.
What is western blotting?
A technique in which specific antibodies are used to identify particular antigens in mixtures of proteins that have been resolved and transferred to a membrane
What are the 4 stages involved in performing a western blot?
- Proteins are separated by an appropriate technique, usually SDS PAGE
- Transfer is carried out by electroblotting to a sheet of nitrocellulose or PVDF
- This is then incubated with labelled antibodies specific for the antigen of interest (Radiolabelled or enzyme linked ABs can be used.)
- Unbound antibodies are washed away, and the position of the labelled ABs is revealed using an appropriate technique such as chemical substrates or radiography
What are the stages in an SDS PAGE in 1D?
- Make polyachrilamide gel
- Take solution of antigen and mix it with limelight buffer
- Heating with mercaptoethanol allows disulphide bonds to break between protein subunits
- Use sodium didecylsulphate to give all the protein subunits a negative charge, with equal charge to mass ratio
- Run on the gel and separate according to molecular weight. High MW proteins found at the top of the gel and low MW proteins at the bottom.
On an SDS PAGE gel where are the high and low molecular weight proteins found respectively?
High mw proteins found at the top of the gel and low mw proteins found at the bottom
How are proteins separated on a 2D SDS-PAGE gel?
Similar principle to in 2D (negatively charged protein fragments migrate across gel toward positive charge) however:
Protein fragments first separated according to pH giving across the gel from lower to higher pH
Gel is then flipped and proteins separated according to molecular weight down the gel.
This gives spots on the gel rather than lines of separation
How are the proteins on the PAGE gel transferred to the western blot?
- Sandwich gel with nitrocellulose transfer membrane
- Expose membrane to electric current
- Proteins that have been separated within the gel migrate across onto nitrocellulose membrane, leaving a permanent record of the proteins
- Membrane can then be probed with AB of interest, with a colour change indicative of a positive result
What is immunofluorescence?
A qualitative technique in which ABs tagged with fluorescent agents are used to detect molecules of interest. It tells you the distribution of the antigen, and is particularly useful for when they are found in extracellular locations
What is the difference between direct and indirect immunofluorescence?
With direct immunofluorescence the antibody binds directly to the antigen of interest whereas with indirect, the primary antibody binds the antigen of interest, a secondary antibody conjugated to a fluorochrome then binds the Fc domain of the primary antibody in order to produce a more amplified signal.
