Immunoassay Flashcards
Ouchterlony Double Diffusion Assay
technique used to detect Ag or Ab by precipitant formation
agar plate with two wells
looking for precipitant line between the two wells
Radial Immunodiffusion (RID)
Antibody in gel at constant concentration
Antigen in the well
circular pattern shows the zone of precipitance
size of circle related to concentration of antigen in well
also called single diffusion
Rocket Immunodiffusion
Speed up radial diffusion method
apply electrical current to drive diffusion
height of rocket proportional to concentration
Counter Immunoelectrophoresis (CIE)
Not used much any more
was used to detect antigens in css
drive contents of two wells together
Immunoelectrophoresis (IEP)
- Electrophoresis of a sample in a gell
- application of an antibody in well parallel to Gell
- double immunodiffusion to look for precipitant band
- don’t do this much anymore, difficult to read
Immunofixation Electrophoresis (IFE)
This is done now
Serup electropheresed in six lanes
anti sera then added to wells laid over lanes
IgG, IgA, IgM, kappa, lambda
Immunotyping
Latest method to prove band of restricted mobility is a M-Spike
Identifies monoclonal immunoglobulins via subtraction
Capillary electrophorus with antibodies lining the capillary column.
not as sensitive as IFE
Technique to measure Ag in solution
Nephelometry & Turbidimetry
Good for mg/dl concentrations antigens
Turbidimetry - photomultiplier tube opposite light source. more antigen means less light.
Nephelometry - photomultiplier tube at angle from source. more antigen means more light. linear, so good at low and high concentrations better than turbidimetry.
How to measure analyses in very low concentrations?
- Competitive immunoassay
- Radioimmunoassay (RIA)
- Noncompetitive immunoassay
- Immunometric assay
Competitive Immunoassay
- Kit includes Antibody and labeled-Ag (limited amounts)
- Serum is added: competition is usually between:
- labeled-Ag (kit)
- unlabeled-Ag (pt-serum)
- for binding to Ab
- Good for small antigens
Non-competitive immunoassay
- aka immunometric assay
- usually a double antibody assay (“sandwich”)
- 2 different antibodies that bind to the same analyte
- bind to 2 different epitopes
- one Ab is bound to a solid phase
- captures antigen
- second antibody: free provides signal
- sensitive method
- couple types of signal Ab
- immune-radiometric (IRMA) readioactive
- Enzyme (IEMA)
- Immuno-chemiluminescent assay (ICMA)
- Rare earth element - expose to electrical field, light up. can do it over and over. very high sensitive (electrochemiluminescencm)
Competitive vs. Non-competitive
- non-competitive is linear relationship between concentration and signal.
- lower-limit of detection
- higher upper linear limit
- competitive is inverse relationship between concentration and signal
- signal is asymptotic on low and high ends
- good for small sized antigens
Homogeneous Assay
- Don’t have to separate different antigens to measure one
- Florecense polarization assay
- antibody bound antigen spins more slowly, so that it has not rotated out of polarization when signal released.
Elisa
- Enzyme-linked immunosorbant assay
- coat a well with antigen OR antibody
- add pt serum
- wash
- add detection antibody
- add substrate, get product
EMIT assay
- Enzyme-Multiplied Immune Test
- Measure small drugs
- steric hindrance from antibody in kit, prevents product
- add drug from patient, antibody is competed off, less steric hindrance, then get product
