WK 5- IMMUNE RESPONSES IN HEALTH AND DISEASE Flashcards
What is a titre
Tires is the relative amount of a specific antibody present in a sample-> it is the reciprocal value of the highest sample dilution given a positive reaction in a serological test. The further the sample is able to be diluted (and remain coloured) indicates a high titre and a high antibody conc.
What is antiserum
Serum collected from an animal that has been exposed to a particular antigen and therefore contains antibodies for that antigen-> antiserum is able to be used to test for antibodies within the body (as the antiserum will bind to the antibodies it has been primed against)
What are the two approaches for diagnosing disease by immunoassays
- Testing for specific antigens
OR - Testing for antigen-specific antibodies
What is a monoclonal antibody
Every antibody in the re-agent is identical, all derived from a single B cell clone and is used to detect an antibody
What is an antigen
Antigen is a molecule on the surface of a cell that identifies it as being foreign or self
What is an antigen-specific antibody
Antigen specific antibodies are Ab that are specific for one particular antigen-> if you were wanting to test for presence of HIV, would use HIV-specific antibodies as they would attack/bind to HIV antigens
What does polyclonal mean
Polyclonal serum is produced when you immunise an animal with Ag that contains numerous epitopes, so the host animal will generate antibodies against several of these epitopes (creating a large variety of Abs)
What is humoral immunity
Immunity that is mediated by cells found in extracellular fluids such as secreted antibodies, complement proteins, and certain antimicrobial peptides.
What is a serological assay
Assays performed on serum, plasma or any other body fluid (eg. synovial)-> allows you to measure acute phase proteins, complement levels, cytokine levels, Ig, Ab to microbial antigens, autoantibodies and allergy IgE
What is cell enumeration and phenotyping
Allows you to determine the types of cells that are present in the sample-> T vs B cells, CD4 vs CD8
What is an In-Vivo assay
Done inside of the patient eg. Allergy skin test (place allergens on the skin and wait for a reaction)
What is the ELISA test used for
used to test for antigen or antibody
How does the ELISA test work
Solution containing antigen is placed on the bottom of a well-> primary antibody (ie. pt serum/plasma) is added->if there are antibodies in the serum/plasma, they will bind to the antigen in the well-> antispecies antibody (antibody from an animal) is added to the well-> the antispecies antibody will bind to the antibody (that has bound to the antigen)-> the binding will cause a colour change that will indicate a positive test
In an ELISA test, what does a dark colour indicate and what does no colour indicate
Dark colour indicates a high presence of antibodies
No colour indicates no antibodies are present for that particular antigen
When testing Ab levels to determine Ag exposure, what does;
a) IgG and IgM ab negative
b) IgG and IgM ab positive
c) IgG ab neg, IgM ab pos
d) IgG ab post, IgM ab neg
a) Either there is no exposure or it is too early to detect in acute phase
b) Current or recurrent infection
c) Very early acute phase or false positive IgM
d) Past exposure to infection
What does a high IgG mean
High when there was a past exposure to an antigen
When is IgM high
IgM is high when there is an acute infection
What is a paired serum
Paired serum is when a serum test is taken two weeks apart- conducted in order to analyse titre levels- if the titre level has continued to rise it indicates the infection is still present
What is a precipitation test - give an example
-Soluble antigens cause antibodies to bind and precipitate
Eg. testing for CRP - Binding of an antibody to CRP causes precipitation, indicates a positive test and CRP presence
What is an agglutination/haemaglutination test
Involves the interaction of antibodies and antigens-> one arm of Y of antibody will bind to antigen, other arm of Y may cross link and bind to other antigen→ causing clumping and agglutination of antibodies-> this process is used in haemaglutination
What does a negative haemaglutination test look like
no antibody in sample will mean there is nothing to clump them together and the RBC fall to the bottom in a ‘button’ formation
What is a direct coombs test/how does it work
Direct; Addition of anti-human Ig antibodies to washed foetal RBC→ if the mothers antiRh antibodies are present on the fetal RBC then there will be agglutination
What is an indirect coombs test/how does it work
Maternal serum with is incubated with Rh+ RBC→ add anti-human Ig antibodies → if anti-Rh antibodies are present in maternal serum there will be agglutination
What is the difference between an indirect and a direct coombs test
Direct assesses presences of anti-RH antibodies on surface of FETAL RBC
Indirect assesses whether there are antibodies in SERUM
What is an ELISPOT assay
Is like an ELISA, except involves measuring cytokines not antibodies
How does an ELISPOT assay work
population of T cells are stimulated with antigen, and then settle onto plastic plate coated with antibodies to the particular cytokine being studied→ if the activated T cell is secreting that particular cytokine, it is captured by the antibodies on the plastic plate→ T cells are then removed and anti-cytokine antibody is added and will attach to the cytokine:antibody complex and reveal the circle of each activated t cell
What does a positive ELISPOT assay look like
A dark spot allows you to determine frequency of t cell secreting that particular cytokine–> darker the spot, more cytokines released
What is a ficoll gradient
-ficcol combined with centrifuging will cause the PBMC (peripheral blood mononuclear cells→containing
lymphocytes and monocytes) to separate from the RBC and polymorphonuclear cells (granulocytes)
-allows you to collect the cells you are wanting to analyse
How does western blotting work
- dissociate virus and separate different proteins→ 2. blot proteins to a piece of filter paper→ 3.add persons serum and if they have antibodies against proteins they will bind→ 4. introduce anti-species anti-body and observe for colour change
How does western blotting work
- dissociate virus and separate different proteins→ 2. blot proteins to a piece of filter paper→ 3.add persons serum and if they have antibodies against proteins they will bind→ 4. introduce anti-species anti-body and observe for colour change
What is a point of care test
Can be used bedside/in clinic-> provides rapid results (ie. preg test)
How is a point of care test done (Immunochromatography)
- add sample (blood/urine/saliva) to filter paper→ 2. antibodies in that sample can interact with antigens imbedded in that paper →3. anti-species antibody is added to observe colour change
What is flow cytometry
Is performed on cell suspension→ most commonly used medium is blood
-Antibodies are marked with flourescent markers–> antibodies will bind to antigens on cells and allows the cells to be identified/counted
How does flow cytometry work
- take blood, incubate with antibodies, wash off excess antibodies→ 2. cells pass in single file past the laser→ the colour of the emission tells you what cell it is (ig. Every cell that has a green label will be detected by a specific receptor)→ 3. allows you to work out how many cells and antigens are present
What is a forward and side scatter
Allows you to determine the size and structure of cells by how much light is scattered
How does forward/side scatter work
-if the cell has lots of granules inside, the scattered light will be detected by lots of different receptors (side scatter)→ allows you to determine which cell is which (based on size and intracellular contents- granules)
Which cell would have the largest side scatter and why
Neutrophils- have the largest amount of granules inside, and are the largest WBC cell
What is a limitation to forward/side scatter
On scatter, CD4/CD8 look the same so antibodies are used to determine which is which → eg staining with anti-HLA identifies the HLA molecules
