Module 5 Flashcards
tools used in order to understand and help fight infections
-ELISA
-Flow Cytometry
-Monoclonal Antidies
ELISA
Enzyme-linked immunosorbent assay (ELISA) is a fundamental tool of clinical immunology based on the principle of antigen-antibody interaction
Flow Cytometry
method of detecting and quantifying different cell types in a mixed cell suspension.
analyzes cell suspensions and translates this information into datasets
Monoclonal Antibodies
developed by Georges Kohler and Cesar Milstein in 1975
-antibodies that are produced by a single clone of a B cell that are specific for a single epitope
ELISA can be modified to detect:
pepties, proteins, antibodies, hormones and other molecules
why is ELISA highly specific?
based on principle of antibody-antigen interaction an the antibody binding site is very specific to one antigen
1st step of indirect ELISA
bottom of wells are coated with an antigen that is specifically recognized by the antibody you wish to measure (primary antibody)
What happens between steps in indirect ELISA
wells are washed to remove any excess antigen not attached to the bottom of the well
step 2 of indirect ELISA
sample containing antibody to be measured is added to well. Primary antibodies, if present, will bind to the antigens attached to the bottom of the well
Step 3 of indirect ELISA
an enzyme-conjuated secondary antibody is added to the well. THis secondary antibody will bind to the Fc portion of the primary antibodies already presnt in the well. The secondary antibodie used specifically recognizes antibodies from a particular animal
step 4 indirect ELISA
the substarte of the enzyme attached to teh secondary antibody is added to the well. The reaction of the substrate (a chromogen) and the enzyme produces a coloured product which can be measured by absorbance
chromogen
a substance that can be readily converted into a dye or other coloured compound
What does ELISA measure
- a coloured reaction product by absorbance with the help of a machine
Indirect ELISA
detects or quantifies antibody. Ex, to determine the presence of serum antibodies against HIV
Enzyme-conjugated secondary antibody
a secondary antibody specifically binds to the primary antibody. (has enzyme attached to it)
how can flow cytometry be used to diagnose AIDS/HIV
used to identify and count these specific T-cells in a blood sample
How does Flow cytometry work
-single file stream of cells passed through laser light
-scattering of laser light unique to each cell type
-measuring FSC allows for discrimination of cells by size
-FSC intesnsity proportional to diameter of cell
-SSC provides infro about internal complexity of cell
-also used to det. proportion of cells expressing a particular antigen via stain
FSC and SSC measured in conjunction
allow for detection of specific heterogenous population
What does flow Cytometry measure
-physical properties of a cell
-detecg specific antigens on or inside a cell
what is readily determined via flow cytometry
-total number of cells in suspension
-number of cells of a particular type in the suspension
-overall composition of suspention
what is flow cytometry used to determine
complete blood counts
when would one use flow cytommetry
used to count and analyze size, shape and properties of individual cells within a heterogenous population of cells
Aneuploidy
the presence of an abnormal number of chromosomes
Main diagnostic test of flow cytometry used to diagnose cancer
detecting DNA sneuploidy, analyzing cell cycles, and th e immunophenotypical characterization