Seminars - Some Basic First Few Weeks Stuff (Dustin) Flashcards
Polyclonal vs Monoclonal Antibody Definitions
- Polyclonal: product of many different B cell clones. Antibodies recognize different epitopes of same antigen
- Monoclonal: only 1 type of B cell products it, and antibodies are specific for only one epitope
What is avidity in terms of antibodies?
Combined strength of many different bonds between antibodies and antigens.
IgM has high avidity because it’s a pentamer with 10 binding sites, whereas IgG only has 2 binding sites and thus lower avidity
What are some good uses for immunodiffusion?
Detection of solule antigens or antibodies from body fluids
e.b. bacterial, viral antigens, transferrin, Ig classes
What are some things that can be detected by serum electrophoresis?
- Multiple myeloma: most important. Shows narrow M spike in the gamma range (monoclonal gammopathy)
- Chronic Liver Failure
- Chronic inflammation
- Polyclonal gammopathies: many things with inflammation like hepatitis, AIDS, RA, endocarditis etc show broad increases in the gamma range
Immuno electrophoresis can be used to help diagnose:
- Multiple myeloma
- Rheumatoid Arthritis
- Chronic Inflammation
Direct and indirect agglutation are used to test for:
Direct: ABO blood group, Widal test, typhoid fever
Indirect for both ABO and Rh (Rh does not produce direct response) - used in Coombs test
What is the direct and indirect Coombs test and what do they test for?
- Direct Coombs: Fetal red blood cells are coated to see if they’re Rh+ coated with maternal anti-Rh IgG. Just one step: add anti-human antibodies to see if they agglutinate
- Indirect Coombs: take maternal serum and test if it has anti-Rh antibodies by first adding Rh+ RBCs, then adding the anti-human antibodies to see if they agglutinate
What lab test is more commonly used for ABO blood groups besides the two-sided ABO test?
Micro-column Agglutination Test
Tube has antibodies against IgG, IgM, and C3d. Add patient blood and centrifuge.
Direct and/or indirect agglutination occurs.
What are some diagnostic uses of passive agglutination?
- Rheumatoid factor latex test: IgM antibodies that attack Fc receptor of IgG
- hCG latex test (pregnancy)
- CRP latex test: test for inflammation
How do we generate monoclonal antibodies in the lab?
Inject a mouse with antigen, wait for it to produce antibodies to that antigen, isolate the mouse’s B cells
Now fuse those B cells with an immortal myeloma cell
Get hybridoma cell that can produce those antibodies indefinitely
When doing immunohistochemistry, are the primary antibodies monoclonal or polyclonal?
What about the secondary antibodies?
Primary are monoclonal: only recognizes very specific antigen
Secondary are polyclonal: recognize many epitopes of the first Ab, are better able to give a strong signal
What is the difference in method between indirect ELISA and sandwhich ELISA?
Indirect ELISA: starts with antigen bound to the test container. Test to see if you have antibodies to that antigen in your sample. Add secondary antibodies that label with an enzyme -> color product.
Sandwhich ELISA: starts with antibodies bound to the test container. Captures any antigens you want to test. Then add secondary antibodies to form “sandwhich” and also have enzyme -> color product
How does ELISPOT work?
What is an example of something it could be used to diagnose?
ELISPOT is similar to Sandwich ELISA, but tests the products secreted by living cells. Has Ab against specific products of cells. Shows up as dark spots when colored enzyme added.
Example: test for latent TB by having living T-cells exposed to TB and seeing if they activate and begin secreting IFN gamma (ELISPOT has anti-IFN Abs).
What is the difference between RIA and IRMA?
RIA: Radioimmuno Assay: Similar to Competitive ELISA, but with radioactive labeling instead of enzyme. Radiolabelled antigen competes for an unlabelled Ag binding site. More radiosignal means weaker competition (negative result)
IRMA: Immunoradiometric Assay: Similar to Sandwich ELISA. Stronger signal = more antigen
What is the advantage of confocal microscopy vs traditional light microscopy?
Confocal microscopy allows 3D imaging.
The light is focused on specific points and a laser-scanning microscope can read those with multiple images that combine to show the object in 3 dimensions
What type of specimens are immunofluorescence ideal for?
Sections / microscope slides with cells
IF uses immunohistochemistry (normally indirect but potentially also direct) to label antigens, light them up or color them to be visible with microscopy. For example, can identify B cell lymphoma with anti-CD20 Ab
Scatter plot: try to identify which cells are where
What type of graph/test is this?
What is the purpose of this particular graph?
If the CD3+ section shifts upwards (higher peak), what does that mean? What if the CD3+ peak is the same, but it all shifts to the left?
FCM histogram
- Shows difference between T cells and others that are CD3-, with the ratio of antigen presenting cells.
- Higher peak = more T cells expressing CD3+
- Shifts to the left = less antigen is expressed per T cell
CD3 is X axis, CD4 is Y axis
So which box represents Th cells and which represents Tc cells?
Th is top right box
Tc is bottom right box, also maybe some γδT
Others are various cells that are negative for both, or a few CD4+ non-T cells (some monocytes, macrophages, etc have CD4)
What is this graph a representation of, and what technique is done to make this graph?
What important information does it give you?
Cell Cycle Analysis** via **Propidium Iodide (PI) Staining
Stains DNA, shows # of DNA molecules. The normal 46 are in G0G1 stage. Some are replicating and have 2x. Very few are between, in S phase. Others have less than 46 and are in apoptosis.
The below graph shows cancer with S phase and G2/M elevation. Particularly diagnostic for bone marrow cancer, acute lymphoblastic leukemias.
