Labeled Immunoassay Flashcards
Labeled immunoassays are designed
for antigens and antibodies that may be
_________ in size or present in _______.
small in size
very low concentrations
The presence of such antigens or
antibodies is determined indirectly by using
a ______ to detect whether or not
specific binding has taken place.
labeled reactant
substance to be measured
ANALYTE
Examples of analytes
Examples include bacterial and
viral antigens, hormones, drugs, tumor
markers, specific immunoglobulins,
and many other substances.
all the reactants are mixed together
simultaneously
Competitive Immunoassay
In Competitive Immunoassay the amount of bound label is ______ proportional to the
concentration of the labeled antigen
inversely proportional
Typically, an antibody, often called capture antibody, is first passively absorbed to a solid phase
Noncompetitive Immunoassay
In Noncompetitive Immunoassay, a ___ is added to the reaction after washing
second antibody with a label
In Noncompetitive Immunoassay, the amount of label measured is ____ proportional to the amount of patient antigen
directly proportional
requires a step to physically separate free from bound analyte
Heterogeneous Immunoassay
no separation step is necessary
Homogeneous Immunoassay
Examples of solid-phase vehicle for separation
Polystyrene test tubes
Glass or polystyrene beads
Microtiter plates
Magnetic latex beads
Nitrocellulose membranes
The last step common to all immunoassays
detection of the labeled analyte
pioneered the first type of immunoassay
Yalow and Berson
It was used to determine the level of insulin anti-insulin complexes in diabetic patients
radioimmunoassay (RIA)
Used as a label in radioimmunoassay (RIA)
radioactive substance
Radioactive elements have nuclei that ______, emitting ___ and ____
decay spontaneously
matter and energy
the most popular radioactive label and has a half-life of 60 days
125 I
125I is easily incorporated into protein molecules, and it emits ______, which is detected by a ________
gamma radiation
gamma counter
RIA was originally based on the principle of
competitive binding
Used to measure total IgE
Radioimmunosorbent Assay (RIST)
Used to measure IgE to specific allergens
Radioallergosorbent Assay (RAST)
Radioimmunoassay
DISADVANTAGES
• Problems with disposal
• Short shelf-life of labels
• There is the need for expensive equipment
are naturally occurring molecules that catalyze certain biochemical reactions
Enzymes
Enzymes react with suitable substrates to produce breakdown products that may be
chromogenic, fluorogenic, or luminescent
Typically used enzymes
Alkaline phosphatase
Horseradish peroxidase
Glucose-6-phosphate dehydrogenase
β-D-galactosidase
Most often used; highest turnover, high sensitivity, easy to detect enzymes
Alkaline phosphatase
Horseradish peroxidase
The first enzyme immunoassays (EIAs) were ______ based on the principles of RIA
competitive assays
This is typically used for measuring small antigens that are relatively pure
Heterogenous Enzyme Immunoassay
Competitive EIA
Heterogenous Enzyme Immunoassay :Noncompetitive EIA are often referred to as _____, because the enzyme-labeled reagent does not participate in the initial antigen–antibody binding reaction
indirect enzyme-linked immunosorbent assays (ELISA)
Used to detect antibodies to HIV, EBV, HBV, and HCV
Heterogenous Enzyme Immunoassay
Noncompetitive EIA
Also called sandwich immunoassays
Heterogenous Enzyme Immunoassay
Capture Assays
Designed primarily for point-of-care or home testing
Membrane-based Cassette Assays
The membrane in Membrane-based Cassette Assays is usually ___ - able to easily immobilize proteins and nucleic acids
nitrocellulose
Another type of rapid assay combines all the steps (separate addition of patient sample, wash reagent, labeled antigen or antibody, and the substrate) into one
immunochromatography
It has been used to identify microorganisms (S. pyogenes and S. agalactiae), test for pregnancy, for troponin in a heart attack, and for hepatitis B surface antigen
Immunochromatography
Immunochromatography Test results are most often
Qualitative
Is any antigen–antibody system in which no separation step (of bound and free analyte) is necessary
Homogenous Enzyme Immunoassay
Used primarily in the determination of low-molecular-weight analytes such as hormones, therapeutic drugs, and drugs of abuse in both serum and urine
Homogenous Enzyme Immunoassay
Homogeneous assays are based on the principle of ______ as specific antigen–antibody combination occurs
change in enzyme activity
demonstrated antibodies could be labeled molecules that fluoresce
Albert Coons
