Term
| Explain what equivalence means in these test? What two zones surround it? |
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Definition
-It is basically the point where there is neither much unbound antigen, nor unbound Ig binding sites, causing the maximum agglutination/precipitance
-It is also the "window period" we shoot for in most tests
-Antigen-excess is before this; all Ig is bound, but free Ag
-Antibody-excess is after; all Ag is bound, but free Ig
-The normal disease progression would go through all 3 of these steps, with the Ig being cleared afterward |
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Term
| When do we get agglutination as apposed to precipitance? Which Ig does these best? |
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Definition
-We get precipitance with soluble, and agglutination with insoluble antigen
-IgM because of so many sites (IgA is in second) |
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Term
| What two particles are commonly used for agglutination based tests? What are each commonly used for and how does it work (for one of them)? |
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Definition
-RBCs and latex beads
-Latex beads are often used to diagnose cerebrospinal infections; we complex antibodies to latex beads and see if we get agglutination when placed in CSF
-RBC agglutinations are important in ABO blood-typing, diagnosing Epstein-Barr virus, and Coombs test |
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Term
| How do the two types of Coombs test work and what is it useful for? Which is more specific? |
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Definition
Direct Coombs test; detect antibody bound to RBCs
-Treat blood with Coombs reagent (anti-human γ-globulin) and look for agglutination
-Used to identify maternal anti-Rh antibodies already bound to infant RBCs or bound antibodies in patients with autoimmune hemolytic anemia
Indirect Coombs test; detect free antibody (in mother)
-Test Rh-negative mothers for anti-Rh antibodies of the IgG isotype (cross placenta) by mixing her serum with Rh+ RBCs and then using Coombs reagent
-This one is more steps, but also more specific |
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Term
| What is DFA, what do we use if for, and how does it work? Name something we use it for? |
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Definition
-DFA = direct fluorescent antibody test
-Used to detect antigen in tissue samples
-Treat tissue with fluorescently labeled antibodies against a particular antigen and then use a microscope
-We use it to detect herpes |
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Term
| What is IFA, what do we use if for, and how does it work? Name something we use it for? |
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Definition
-IFA = indirect fluorescent antibody test
-Used to detect antibodies in tissue samples
-We treat the tissue with fluorescently labeled anti-Igs and then view under microscope
-Use it for some autoimmune diseases, and Epstein-Barr virus |
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Term
| What are RIA and ELISA? What is their major advantage? What is the difference between them? How do they work? What is a common use? |
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Definition
-Radioimmunoassay and enzyme-linked immunoabsorbent assay (aka EIA sometimes)
-They are extremely sensitive and can detect very small amounts of material (hormones, drugs, etc.)
-The main difference is in the labeling; either use enzyme or radiolabeled anti-Ig (with enzyme producing color)
-Also ELISA is newer and used rather than RIA these days
-ELISA is used to screen for HIV, and many other things
The process is as follows (for indirect method);
-Microtiter plates are loaded with p24 capsid antigen
-Patient serum is passed over the plate
-Labeled anti-human γ-globulin is added
-Finally, enzyme substrate is added (in case of ELISA)
-Usually there are a bunch of serial dilutions with positive and negative controls
-Not the best as far as specificity however, because sometimes you may have a bit of antibody that by chance has some binding to the test antigen |
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Term
| What is the confirmation test for HIV? |
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Definition
| -Western blot (major use is this purpose) |
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Term
| What is western blot and how does it work? |
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Definition
-Its main use is as confirmation of HIV (because ELISA has a higher false positive rate; i.e. low specificity)
-It works exactly the same as RIA and ELISA, except now we use several types of more specific antigen separated out onto the plate by gel electrophoresis and look for a score of 2/3 for a +test |
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Term
| How does flow cytometry work, what machine does it use, what do we use it for, and what does the data look like? |
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Definition
-It is used to rapidly analyze cell types in a complex mixture based on their binding to fluorescent dyes using a fluorescence-activated cell sorter (FACS) (actually sorts them incase you want to use them separately)
-Basically, it deflects the cells depending on fluorescent color and intensity and generates a graph with CD markers on each axis and dots representing cells;
-Quadrant 1 is cells with both marker, 2 is y-marker only, 3 is cells with no marker, and 4 is x-marker-only cells |
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Term
| What is, in general, the difference between direct and indirect test? |
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Definition
-Direct is looking for the antigen directly or Igs bound to antigen (either way, there is antigen)
-Indirect tests look for free Ig, then we have to bind it to lab antigen; more steps, but also a higher sensitivity |
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Term
| Which cells should be indicated more intensely in flow cytometry; CD8 or CD4 cells? What is this fact helpful in diagnosing? |
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Definition
-There should be about double the TH cells as CTL
-HIV will switch that ratio to be more CTL because they are killing off the TH cells |
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Term
| What expresses CD40L? When is it missing? |
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Definition
-Activated T cells
-In hyper IgM syndrome |
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Term
| Following cards are from Lima's L8-9; |
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Definition
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Term
| Linear determinant? What recognizes it? |
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Definition
-Formed by sequential AAs (in a line)
-Accessible when denatured, but may or may not be accessible when in native conformation
-B and T cells can recognize this one |
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Term
| Conformational determinant? Recognized by? |
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Definition
-It is an epitope formed by AAs brought into close proximity by protein folding
-Only works when protein is in correct conformation
-B cells cells can recognize them, but T cells cannot |
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Term
| Overall, what are T cell limitations on recognizing antigen? |
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Definition
| -Has to be linear, MHC bound, and proteins only |
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Term
| Describe Kd as far as antibody binding is concerned? |
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Definition
-Kd (dissociation constant) = free/bound
-So for tight binding we want very small Kd |
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Term
| What is cross reactivity? |
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Definition
-This is just referring to the fact that antibodies only react with one epitope, but that the same epitope may exist on completely different antigens/pathogens
-Stated otherwise, an antibody often can react to multiple antigenic particles |
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Term
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Definition
-Substances which increase the immunogenicity (immune reaction generating ability) of an antigen non-specifically
-In other words, adjuvant are crap we add to vaccines to boost the bodies use of the included antigens... think of it as vaccine lube lol |
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