pre-IC4 Flashcards
What is an immunoassay?
Assays that employ antibodies to detect and
quantify a specific analyte (usually a biomolecule)
Key component in immunoassays
Antibodies against the biomolecule of interest; Polyclonal or Monoclonal antibodies may be used; usually raised in an animal
What are the 2 means of detection of Ag-Ab binding in immunoassays?
- Using a marker (radioimmunoassays/ enzyme immunoassays)
2. Agglutination/ haemagglutination
What is agglutination, and how is this observed?
Clumping of red blood cells (bound to antibodies); visibly observed as cloudiness/ turbidity/ precipitation
What is agglutination, and how is this observed?
Clumping of red blood cells (bound to antibodies); visibly observed as cloudiness/ turbidity/ precipitation
Solid phase enzyme immunoassay: What does a low absorbance mean?
Greater amount of antigen present in sample, lesser enzyme-linked antigens bound to solid phase, lesser enzyme activity thus lesser product formed.
What does ELISA stand for?
Enzyme-linked immunosorbent assay
What can be found on the solid surface of ELISA?
Can be antibody/ antigen depending on analyte to be determined in sample
What does indirect ELISA detect in a sample?
Indirect ELISA employs antigen to detect the presence of a specific antibody in the sample.
What does sandwich ELISA detect in a sample?
Sandwich ELISA employs antibodies to detect the presence of a particular antigen in the sample.
What happens in competitive ELISA?
Enzyme-linked antigen (AKA inhibitor antigens) competes with antigen in sample for antibody binding.
Advantages of ELISA
- Ease of use (usually only UV spectrophotometer needed)
2. Safe and specific (depending on specificity of Ab used)
Disadvantages of ELISA
- False positive (inadequate washing, use of polyclonal antibodies, inadequate blocking, cross reactivity of secondary Ab)
- False negative (antibodies and conjugated enzymes are denatured)
How does haemagglutination assay detect enveloped viruses in samples?
Viral particles interact with red blood cells through a viral surface glycoprotein called hemagglutinin. Presence of virus causes clumping of red blood cells (hemagglutination), forming a lattice instead of a nice full red dot.
When does clumping (agglutination) occur?
It happens as long as long antigen and/or antibody is particulate in nature (i.e. semi-solid or solid by being conjugated to a solid particle).
How can agglutination be measured quantitatively?
Agglutination is a visible change in turbidity, which can be detected by measuring the UV absorbance.
How does pregnancy test direct/ passive agglutination work?
hCG present in urine binds to anti-hCG Ab on latex particles, showing cloudiness.
What is passive agglutination?
Only works with particulate Ag. RBCs coated with a soluble Ag is used to test for Ab.
What is haemagglutination inhibition?
Soluble Ag in sample inhibits agglutination of semi-solid Ag-coated RBCs by Ab.
For haemagglutination inhibition, what would a positive test result show?
No agglutination as the Ag in sample successfully competed with other particulate for binding to Ab. Thus lower turbidity, and low absorbance.
