Question

In a table format, discuss the 3 types of fluorescent immunoassays mentioned in the chapter (direct immunofluorescent assays, indirect immunofluorescent assays, and quantitative fluorescent immunoassays) by including the following categories:

Principle of the assay.

Illustrate how the assay works.

Interpretation of patient results.

List specific diagnostic tests in which the methodology is used.

Answer 1

Test Direct immunofluorescent Indirect immunofluorescent Quantitative immunofluorescent assays assays assays Principle Tagged Ab is directly added Specimen with a known Ag is Can be classified as heterogenous to an unknown Ag fixed to a incubated on a solid phase.or homogenous, depending on the slide Requires incubation and a (from mouse) tagged with aLabel is fluorescent. can be applied wash step Ag are seen as bright apple Specimen Ab binds with green or orange-yellowW against a dark backgrounds. sandwich and localizing Antihuman immunoglobulin fluorophore is added antihuman Ab, forming a Fluorescence type of enzymatic immunoassays to either Ag or Ab. Commonly applied in Fluorescence Polarization Immunoassay (FPIA) Working Solid phase antigen fixed to a Unlabeled patient antibody is Reagent antibody is combined microscope slide is incubated reacted with specific antigen with patient antigen and directly with a fluorescent-resulting in Ag-Ab complex fluorescent labeled antigen. If little labeled antibody. The slide is fixed to a microscope slide. Itor no antigen is present, antibody washed to remove unbound is washed and a labeled antibody. The slide is observed for fluorescence will bind to the labeled antigern antihuman immunoglobulin is Binding causes these molecules to added. After a second wash rotate more slowly in solution, to remove any uncombinedincreasing the amount of polarized anti-immunoglobulin, fluorescence of the sample is determined light. Interpretation If specific antigen is present The amount of fluorescenceWhen patient antigen is present, in the patient sample fluorescence is observed is directly proportional to amount of patient antibodyunbound. These molecule will present. more labeled antigen will be rotate more quickly in solution giving off light in many directions, and polarization will be decreased Application Suitable for detection of Used in Ab identification andSolid-phase heterogenous specific Ag in tissue or body detection of Treponema, fluids. fluorescent assay is employed for antinuclear, chlamydial, and toxoplasma Ab, and Ab to herpes simplex virus (HSV), Epstein-Barr virus (EBV) Cytomegalovirus (CMV) the identification of Ab to nuclear Ag, Toxoplasma Ag, Rubella Ag, and numerous other virus Ag Can also be used to detect important biological compounds like cortisol, progesterone thyroxine