Section 3: Labeled Immunoassays Flashcards
Labeled immunoassay
-a marker or label is attached to an antibody or antigen, thus increasing the sensitivity
unlabeled immunoassays
-are cheap and easy to perform but limited by their sensitivity (visualization of precip or agglunt.)
Radioimmunoassay (RIA)
-was the first labeled immunoassay developed by Rosalyn Yalow in 1959 to detect insulin
- Competitive RIA (patient Ag competes with radiolabeled Ag)
-Ag binds to a fixed amount of Ab
-Concentrations measured at lower levels than possible by eye
-1000 fold increased sensitivity
What are some of the various labels and markers that can be used?
-radiolabel (radioactive isotope) (RIA)
-Enzymes (enzyme immunoassay) (EIA/ELISA)
-Fluorescent compounds (FIA)
- Chemiluminescent label
-Colloid particles
What are the two types of assays?
- Heterogeneous Assay
- Homogeneous Assay
Heterogeneous Assay
-requires separation of bound label from a free label (solid phase for facilitating the reaction, followed by a wash)
-use solid phase attachment of Ab or Ag to a microtiter plate, slide, or bead that allows for rapid separation of bound label from free
Homogeneous Assay
-does not require separation or washing because the binding of an Ag to Ab directly affects the activity of the label
colloid immunoassays
-detected by eye after separation (immunochromatography)
ex: point of care testing, home pregnancy tests
Types of heterogeneous immunoassays
- direct
- indirect
- competitive
- sandwich or capture
- western blot
Direct Immunoassay
-labeled antibody binding to antigen or labeled antigen binding to antibody
(Direct)
-directly labeled targets (ex: primary antibody)
Indirect immunoassay
-unlabeled antigen and unlabeled antibody are used in conjunction with a labeled detection (or secondary) antibody
Anti-Human immunoglobulin
-key reagent in indirect immunoassay
- can detect all or specific isotypes of Ab (IgM, IgG, IgE)
-various labels can also be used in indirect assays
capture immunoassay (sandwich)
-captures antigen between 2 antibodies, usually, one antibody (capture Ab) is bound to a solid-phase medium and the other antibody is labeled (labeled Ab)
EX: immunochromatographic sandwich assay: home pregnancy test
-looking for antigens
double capture
-an assay that can test for patient antibodies (West Nile virus)
-was developed to detect IgM preferentially over IgG for the diagnosis of a current infection
-looking for antibodies
Why do patients with rheumatoid arthritis produce false positives?
Rheumatoid factor is a human antibody (predominantly IgM) but can be other ones, that respond to the fc portion of human IgG. So if you use that assay to detect IgM and you have human antibodies here, if RF Is there it is going to bind to stuff indiscriminately and nonspecific to the assay
Why will capture assays not work?
- Ag is too small
- Ag has only one epitope
- Ag has multiple epitopes that cause steric hindrance of the reaction
steric hindrance
when a reaction is inhibited because of the size and or shape of molecules involved in the reaction
Competitive Immunoassay
-because less substrate is created when patient Ag or Ab competes with kit reagent, an inverse relationship exists between substrate and amount of patient analysis
-we have a bead or substrate in the assay that acts as the solid phase or substrate. We coat that bead with our antigen and add our antibody complex with our label. If nothing we get a strong inhibited signal. Bead and antibody do not change but patient samples compete with those binding sites and sequester them off. Wash-off will have a decreased signal
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Multiplex (microbead assay)
-microbeads coated with Ag-specific to reflex testing of a positive ANA are used to examine patient samples for multiple antibodies simultaneously
-each bead has its own internal fluorescent signature
-used on cytokines, can detect antibodies or auto nuclear antibodies (ex; part of lupus testing)
-read a combination of colors, for a true read you need not just one color
Homogenous assays
-do not require the separation of bound analytes from free ones (easier than heterogeneous)
-also bound to Ag in a homogeneous assay causes a direct change in the visualization compound
-used for small molecules (drugs and therapeutics)
Fluorescent polarization
-fluorochrome is placed on the small molecular analyte of interest
-polarized light used as an excitation wavelength
-fluorochrome attached to small molecules will freely rotate and emit light in a variety of directions (depolarize)
-If Ab binds to molecule and fluorochrome, the rotation is slowed and the plan-polarized light remains polarized
-competitive assay: drug in the patient’s sera competes with labeled drug molecules for binding to kit Ab. The more drug in the patient sera the more free-labeled drug and thus less polarized light is detected
-Rapid Rotation = light is depolarized (the more patient sample we add the less polarized the light is)
- Slow Rotation = light is polarized
interfering substances
-some inhibiting substances affect many test, like using plasma from EDTA tubes
Rheumatoid factor
-Ab to the Fc region of IgG
-false positive for any capture assay
Human heterophilic antibodies
-are antibodies to animal Ig present in people who work or are exposed to those animals regularly
-false positive for capture assays (detection Ab of animal origin)
Human Anti-Mouse Antibody (HAMA)
-Specific type of human heterophilic Ab that is produced in response to therapeutic or diagnostic mouse monoclonal antibody
Enzyme: detection method
-spectrophotometer
-fluorimeter
- luminometer
Fluorescent: detection method
fluorimeter
radioimmunoassay: detection method
-gamma or scintillation counting
chemiluminescence: detection method
luminometer
The double capture assay was developed to detect IgM
preferentially over IgG for the diagnosis of a current
infection…. why?
Persistent infections or reinfections will produce IgG antibodies (class switch would have occurred). To diagnose current or acute infections that are new the best way to do would be the detection of IgM.
inhibition assay readout
-the antigen concentration is inversely related to the readout
