Lecture 22 11/9/23 Flashcards
What is an immunological assay?
test that makes use of antigen-antibody interactions
What are the clinical applications of immunological tests?
-confirming diagnosis of infectious disease
-assess patient’s immune status
-determine exposure to a pathogen
-assess immunity in response to a vaccine
What is the role of a qualitative test?
determine if a particular substance is present in the clinical sample
What is the role of a quantitative test?
assess how much of the tested antigen/antibody is present
What is a “stat” test?
-test with a quick turnaround time
-ordered when result is needed quickly
What is a “routine” test?
-test with a less immediate turnaround for patients with less of a need for immediate results
-results available within hours to days
Which section of the antibody is labelled/tagged in immunological assays?
the Fc segment
What is an enzyme label?
-enzyme will cleave a substrate
-cleavage produces a detectable product (light, color, etc)
What is a fluorescent label?
-fluorophore that absorbs light at a specific wavelength
-emits light at a different wavelength
What is a colored bead label?
-large synthetic particle
-color can be read visually when enough aggregate together
What are the characteristics of ELISA assays?
-performed in 96-well plate
-use enzyme labels (horseradish peroxidase)
-read using spectrophotometer
-color change is positive
-can be qualitative or quantitative
What are the steps of a direct ELISA?
-patient specimen fixed
-antigen-specific antibody with enzyme label applied
-unbound antibodies rinsed off after incubation
-substrate added; if cleaved by enzyme label, color change occurs
What is measured with a direct ELISA?
antigen within a patient specimen
What is measured with an indirect ELISA?
antibody within a patient specimen
What are the steps of an indirect ELISA?
-purified known antigen attached to solid phase
-patient serum added
-unbound antibodies washed off after incubation
-enzyme-labeled secondary antibody added
-unbound antibodies washed off after incubation
-substrate added; color change indicates cleavage by enzyme/positive
What are the characteristics of immunofluorescence assays?
-use fluorescent label
-read with fluorescence microscope
-performed on glass slides with 8-12 samples
-fluorescence is positive indicator
-can be qualitative or quantitative
What are the characteristics of a direct fluorescence antibody assay?
-detects viral antigens in a specimen
-patient specimen fixed to slide
-fluorescent labelled antibodies added to specimen
What are the characteristics of an indirect fluorescence antibody assay?
-detects antibodies that a patient has formed against a virus
-known viral antigens fixed to slide
-patient serum added
-fluorescently labeled antibodies added that attach to patient antibodies
What is the role of serum protein electrophoresis?
separate and quantify different protein fractions in blood serum
What is the gamma globulin fraction mostly made up of?
immunoglobulins (antibodies)
What is hypergammaglobulinemia?
abnormal increase in antibody synthesis
What are the two types of hypergammaglobulinemia?
-monoclonal
-polyclonal
What are the characteristics of monoclonal gammopathies?
-increase in a single type of antibody
-associated with immunoglobulin-secreting cancers/myelomas
-monoclonal peak has narrow base with peak that is as sharp/sharper than albumin peak
What are the characteristics of polyclonal gammopathies?
-increase in all major antibody types
-result from long term immune stimulation
-globulin peak has a broad and diffuse base
What is flow cytometry?
technique used to detect and measure physical and chemical characteristics of a population of cells
What is forward scattered light (FSC) proportional to?
cell-surface area or size
What is side-scattered light (SSC) proportional to?
cell granularity/internal complexity
Which conditions warrant the use of a flowcytometer?
-differentiation between B and T cell lymphoid neoplasms
-differentiation between reactive and neoplastic expansions of lymphocytes
-identification of specific subsets of lymphocytes
-prognosis in lymphoma
