10 Immunoassays and Flow Cytometry Flashcards
Western Blot and Gel based immunoassays are unable to tell what?
Concentration of antibodies in serum
What is affinity?
Strength of binding on one site of a molecule on its ligand
What is avidity?
Strength of binding of multiple binding sites of a molecule on its ligand
What Ig has the highest avidity?
IgM
What tests are non-quantitative analysises of antibody levels?
Immunodiffusion and Immunoelectrophoresis gels
Western Blot
How do most titers show antibody levels?
By serial dilution
What tests are used for blood matching?
Coomb’s test
What does the presence of RBCs in hemmaglutination test indicate?
Negative antibody presence
What does the presence of RBCs in complement fixation assay indicate?
Positive antibody presence, these cells do not get lysed
What does a hemagluttination test look for?
Self antibodies
What does a complement fication assay look for?
Antibody-antigen complexes, if it binds the RBCs are not lysed
No RBC lysis is positive in what test?
Complement
What does self-recognizing antibodies to RBCs indicate?
Autoimmune hemolytic anemia
Direct Coombs test outline:
Patient blood taken
Antihuman antibodies added
Binding of antibodies means RBCs have antigens recognized by that antibody
Indirect Coombs test outline:
Patient Serum obtained Donor blood added Antibody binding occurs If antibody binds, cells clump Positive means NO TRANSFUSION
What is a monospot test for?
Epstein-Barr Virus
What test is positive for antibodies if no lysis occurs?
Complement fixation assay
How are monoclonal antibodies produced?
Fusion of B cells with Myelonas to make eternal antibody factory called a hybridoma
How can we purify antibodies?
Affinity chromatography (Ig binding beads, then wash)
How can we make antibodies easy to detect?
Radiolabeling
Enzyme addition
Fluorescent tags
What is a primary antibody?
Test sample, may or may not bind to antigen
What is a secondary antibody?
Pre-made antibody, known specificity, conjugated to indicator molecule
What is serum that is drawn after infection and Ig’s have been made?
Convalescent Serum
may be of use in treating another person with the same infection
What is serum that is drawn during an active infection?
Acute phase serum
What does acute phase serum contain?
High IgM, Low IgG
High Mannose Binding Protein and C reactive protein
What does convalescent serum contain?
Antibody specific Ig’s
What test uses pre-bound known antibodies in a plate of wells?
ELISA
What does foward scatter in flow cytometry show?
Cell size
What does side scatter in flow cytometry show?
Cell granularity
What is a “gated population” in flow cytometry?
A parameter of cell features that can allow you to focus in on a certain group of cell types within a “gate”
What allows analysis of two types of cell reactions in flow cytometry?
Double staining by Fluorescence of two different colors
What can be used to separate cells?
Fluorescent Activated Cell Sorting in a flow cytometery
What color is FITC?
Green
What color is PE?
Red
Do you ever find monoclonal antibodies in patient serum
(resounding) NO, because of hypermutation
