Immunoassays & Microscopy Flashcards
RIA uses labels known as ___. What are the two types?
Radiolabels - iodine 125
Competitive RIA = antigen is labeled; tracer molecule
Non-competitive = antibody is labeled
RIA - drawback
Hazardous, short half-life, expensive to dispose
Common enzyme labels (3)
Horseradish peroxidase
Alkaline phosphatase
Glucose-6-PD
How enzyme labels are measured
Enzyme reacts with substrate to produce a color change
-darker color = higher intensity
Common enzyme substrate
Orthophenylene diamine (OPD)
Common fluorescent labels and the colors they emit (2)
FITC = green
Rhodamine = orange
Common chemiluminescent labels (3)
Acridinium esters
Luminol
Nitrophenyl oxalates
Describe a competitive immunoassay
Labeled antigens are mixed with a patient’s antigens and competes for binding sites on a limited amount of antibodies. Measure bound labeled signal. Higher signals indicate more labeled antigens have bound to the antibodies
- lower signal = higher concentration of patient antibodies bound
- higher signal = lower concentration of patient antibodies bound
- inversely proportional
Describe a non-competitive immunoassay
“Sandwich” assays
- antigen or antibody sandwiched in the middle
- concentration directly proportional to signal
Difference between competitive vs non-competitive immunoassays as they relate to the signal given. Which one is more sensitive and specific?
Competitive = signal inversely proportional to concentration
Non-competitive = signal proportional to concentration
-more sensitive and specific
Homogeneous vs heterogeneous immunoassay. How are they different? Which one is more sensitive?
Homogeneous - requires no washing to remove unbound complexes. Antigen-antibody measured directly
-latex agglutination
Heterogeneous - requires washing
- removes background signal from unbound
- less false positive
- more sensitive
EMIT - commonly used for…
Drug testing
-mnemonic - EMIT acquit
Some components of pre-analytical, analytical, and post-analytical to consider when doing an immunoassay
Pre-analytical example = draw time and detection of Hepatitis Antibody to core, surface or envelope antigens
Analytical = have to do right test, right reagent, right kit to detect marker, right enzymes, right substrates, controls, QC
Post-analytical = ELISA result, confirmatory testing
-presumptive reporting when patient needs immediate results
Which immunoassay is most subjective and requires interpretation by 2 CLSs?
Fluorescent techniques
ANA
FTA-ABS
FPIA
Nephelometry - what is it used to detect? Based on what principle?
Ig quantitation, antigen-antibody complexes
Light scatter/reflected back
-more scatter = more concentrated sample (more complex)
Fluorescent microscopy - the excitation light that hits the specimen is called… The light that is emitted from the specimen is called…
Incident light - hits specimen, excites electron, releases emission light
Fluorescence
Incident vs fluorescence light - which wavelength is smaller or higher? Which wavelength has higher energy?
Incident = smaller = higher energy
-495 nm
Fluorescence = higher
-520 nm
Labeled fluorescent antibodies are called
Fluorophores or fluorochromes
The ability of specimens to absorb and re-radiate light is called… How do fluorescent microscopes deal with this?
Photoluminescence
Separate weaker fluorescent signal from brighter excitation lights
What is epifluorescence
Combination of excitation and emission wavelengths travel through specimen, emitting fluorescence
-objective lens both release excitation light and capture emission light
