Module 13: Immunoassays Flashcards
method of immunoassay
antigen-antibody reactions to identify and quantitate analyte in clinical ssamples
Labels often attached to regain components to produce a measurable signal
how sensitive is immunoassay
high levels of specificity and sensitivity
analytes commonly measured by immunoassay
hormones therapeutic drugs vitamins serological markers (hepatitis antibodies) metabolites (folate, ferritin) cancer markers (CA 125, PSA) cardiac markers (CK-MB, troponin)
label
a reagent component that produces a measurable signal
capture antibody
reagent antibody designed to capture the analyte of interest
conjugate
a reagent antibody with an attached label
tracer
a reagent antigen with an attached label
radioactive isotopes
labels involve attaching radioactive isotopes to either reagent antigens or antibodies, often isotopes of iodine or cobalt
Fluorescent
labels are fluorescent substances (flurophores) that will be excited by certain short, high energy wavelengths of light and then emit longer, lower energy wavelengths
Enzymes and substrates
enzyme itself does not produce a signal
Substrate added to reaction, which the enzymes catalyzes into a specific product
Chemiluminesscent
labels emit light as a result of chemical reaction
chemilunimescent component attached to reagent component and often a trigger is added to start the chemical reaction
Best sensitivity among immunoassay methods
competitive
called Limited reagent
only a low, fixed quantity of reagent capture antibody is added and only one antibody is used
Labelled tracer competes with analyte in sample for limited binding sites
Indirect relationship between analyte and signal produced
Non competitive
2 reagent antibodies One is the capture antibody Second is conjugate antibody Often a direct relationship "sandwich assay"
Heterogeneous
assays that use labels that produce the same signal whether bound or not require a separation step
End with 2 solution
Homogeneous
assays that employ labels that will produce different signals when bound versus when free do not require a separation step
End with 1 solution
Simultaneous methods
all the reagent and sample are added together at once (one step)
Sequential methods
sample is added first to interact with reagent capture antibody, allowed to incubate, and then the competing reagent labelled antigen is added (two step)
Radioimmunoassays (RIA)
due to safety concerns of use of radioactive materials, is not common
Immunoradiometric assay (IRMA)
radioactive labelled antibody was used instead of labelled antigen
Enzyme immunoassays (EIA)
use enzyme labelling system to produce measurable signal
Ex. ELISA
Cloned Enzyme Donor Immunoassay (CEDIA)
once an enzyme-labelled antigen is bound to an antibody, the enzyme fragment cannot combine with the other half of the enzyme to form an active intact enzyme
Advantage of not requiring separation step but $$$$
Fluorescent Polarization immunoassay (FPIA)
uses fluorescent labelled antigen
when exposed to polarized excitation light, the bound fluorescent tracer will emit light that is still polarized int he same plane as the excitation light
High dose effect
if there are very high levels of analyte in sample, all capture binding sites may become saturated and excess analyte will remain in solution. Excess may be washed away during separation step and not measured
Results in falsely decreased result
Termed “High dose hook effect” due to apparent of obtained graph of analyte concentration vs signal
Dilute sample and retest to avoid this
Heterophile Antibodies
antibodies produced against the antibodies of other species
If patient has developed these to the specific species used as a source of reagent antibody, there can be interference
Common heterophile antibody
human anti-mouse antibody (HAMA)
Techniques used to help identify or account for heterophiles antibody interferences
repeat testing using different method
repeat test using different sample type (serum vs urine)
running serial dilutions of sample to determine linear response
Use of blocking agents designed specifically to address known interferences
