RIA+ELIZA Flashcards
What is RIA?
Determine antibody levels by using an antigen that is labelled with a radioactive label and measuring the subsequent radio activity of the antibody compodent.
Uses of RIA
Drug tests
Hepatitis
Early cancer diagnosis
Growth hormone levels
Various neurotransmitters
Various proeins
How does RIA start (setting up the test)
Take a known quantity of antigen (control)
Make it radioactive
Mix it with a known quantity of the antibody we should be testing for
Radioacttive antigen will bind with the antibody
Stage 2 of RIA (Adding patient sample)
Take a sample from the patient
The unlabelleed patiet antigen competees with radioactive antigen in the control sample
The antigen and antibody binds
The higher concentration of unlabelled patient antigen displaces more radioactive antigen
*we can measure how much labelled antigen has been outcompeted
Stage 3 of RIA (Measuring what we have got)
The bound antigens are seperated from unbound radio active antigens
The radioactive o bound antigens are recorded/measures
Concentration of unlabelled antigen found by calibration curve
And a decrease activity
The activity will be in proportion to concentration of subtance being tested.
ELIZA
ELIZA is usually carried out on a multi-wel plate
Stage one of ELIZA
Antigens from the sample are attached to a surface
Time is given for the antigen to bind to the plate
A buffer solution (protien solution) is added
This blocks any unbound sights on the plate to prevent non-specific binding of the antibody
Stage 2 of ELIZA
A specific antibody is applied to the surface so that it can bind with the antigen
The antibody is linked to an enzyme
Any unbound antibodies are washed off
Stage 3 of ELIZA
A substance containing the enzymes substrate is added
The subsequent reaction produces a detectable signal most commonly a colour change
The higher the concentration of antibody, the stronger the colour change.
This can be detected by a spectometer.
