Labeled Immunoassays Flashcards
What is the purpose of labeled immunoassays?
Testing that is used for antigens or antibodies that may be small in size or present in very low concentrations.
What are the two major formats of immunoassays?
Heterogenous and homogenous.
Describe heterogenous immunoassays.
The separation of bound and free components. A wash process is needed (otherwise false positives occur).
Describe homogenous immunoassays.
The lack of separation or wash phase.
Which type of immunoassay (homogenous/heterogenous) is less prone to analytical errors?
Homogenous.
What are some labeling techniques that can be used in immunoassays?
Radioactivity, enzymes, chemiluminescence, and fluorescence.
Describe the process of noncompetitive immunoassays.
Antibody or antigen is passively absorbed to a solid phase; unknown patient Ab/Ag can react with and be captured by antibodies. After washing, a second antibody with a label is added.
In noncompetitive immunoassays, the amount of labeled measured is ___ proportional to the amount of patient antigen.
Directly.
Describe the process of competitive immunoassays.
All the reactants are mixed together at the same time. Labeled antigens then compete with unlabeled patient antigens for antibody binding sites.
The patient sample “competes” with labeled antigen.
In competitive immunoassays, the amount of bound label is ___ proportional to the concentration of labeled antigen.
Inversely.
The radioimmunoassay method (RIA) can measure trace amounts of analytes such as ___ and ___ that are small in size.
hormones; vitamins.
In radioimmunoassay method (RIA) labeled in bound phase is ___ proportional to the amount of patient antigen present.
Indirectly.
Enzyme immunoassays use ___ as labels.
Enzymes.
In enzyme immunoassays, what is the purpose of the substrate?
What enzymes use to produce breakdown products that are chromogenic.
Describe the principle of competitive ELISAs.
Sample antigen competes with enzyme-conjugated antigen for a limited number of binding sites on antibody molecules attached to a solid phase.
In competitive ELISAs, the product signal is ___ proportional to the concentration of the test substance.
Inversely.
In competitive ELISA, the more antigen in the sample, the ___ reference antigen detected, indicating a ___ signal.
less; weaker.
Describe the principle of noncompetitive ELISA.
Excess solid-phase antigen binds patient antibody, and a second labeled antibody is added.
In noncompetitive ELISAs, the results are ___ to the amount of enzymatic activity detected.
Proportional.
Capture (Sandwich) ELISA, is best for antigens that have what?
Multiple determinants.
What is the principle behind the Capture (Sandwich) ELISA method?
Excess solid-phase antibody binds patient antigen, and a second labeled antibody (to the antigen) is added after a wash step. Antigen is ‘sandwiched’ between two antibodies.
With Homogenous ELISA, if reagent antigen binds to purchased antibody, what happens to the active site and enzyme?
The active site is closed and the enzyme is no longer active.
With homogenous ELISA, if a patient binds to the purchased antibody, what happens with the enzyme?
The reagent is still active.
Homogenous ELISA contains ___ proportional results.
directly.
