Diagnositc Immunology Flashcards
Serology orders
Interactions between antigen and antibodies
Useful for
- screening a population for the presence of antibodies
- identification of infectious agents/antigens
- diagnosis B-cell cancers or autoimmunity/hypersensitivities
Immunodianostic assays
Detect presence and amount of an antibody and/or antibody
Titer = relative concentration for he antigen/antibody in contention
- MUST KNOW ONE OF ANTIGEN/ANTIBODY before running a titer (cant diagnose unknown)
Five main types of diagnostic assays
Nephelometry: detection of immune complexes in liquid w/out precipitates.
Precipitation reactions: detect insoluble reactions between antigens and antibodies which form precipitates
Agglutination reactions: detect aggregates from the interactions between antibodies and antigens with a specific particle attached to one or the other.
Labeled immunoassay: measures signals from a “label” placed in the solution that is specific for a select antigen/antibody
Agglutination reactions
Most common with IgM antibodies due to the pentimer structure
- best structure for agglutination
Generated from cross linking between double antibodies and insoluble antigens they code for
involve a reaction between a soluble antibody and an insoluble antigen
direct Coombs test
Blood sample is taken from patient suspected for hemolytic anemia induced by autoimmunity
Coombs reagent is then mixed with washed blood sample without plasma (usually codes for selected antigen/antibody suspected)
If agglutination occurs, suggests presence of the blood type antigen or autoantibodies present
often used for autoimmune reaction diagnosis such as RA and hemolytic diseases
Indirect Coombs test
MOST common type
- screens for blood typing and Rh- pregnancy reactions
Patient serum is drawn and donor blood is mixed into a solution
Patient antibodies will bind to donors blood (signals incompatible match)
Coombs reagent is mixed in with the solution and agglutination will occur if the patient antibodies in the serum bound to the donor blood
Immunoassay labeled
Labeling antibodies or antigens allows for visualization of an antigen/antibody
- produces a light if the target is present
Types of labels:
- fluorophores
- Enzymes/substrates
Flow cytometry
Cells are bound with fluorescent-labeled antibodies
- antibodies are specific for cell-specific surface markers (CD 3/4/8/20)
often used in HIV diagnosis
Slide agglutination and agglutination inhibition
Cheap and quick way to determine blood type, previous bacterial infections and latex allergies
Agglutination inhibition =. Mix patients blood in with beads that possess specific antigens
- no agglutination = positive
- agglutination = negative
Hemagglutination
Routinely preformed on RBC typing and Rh factor
Done in wells
- positive sign = watery and widespread
- negative sign = solid and clumped in the middle
Types of labels in immunoassays
Fluorophores
- flow cytometry
- western blot
- immunoflurorescence microscopy
Enzymes
- ELISA
- western blot
Flow cytometry
Cells are stained with fluorescent-labeled antibodies to specific CD markers and bind to their specific cells
Cells can then be introduced to cytokines or not and activity can be noted
Direct vs indirect immunoflurorescence
Direct = primary antibody directly binds to cells/tissue detects presence of antigen
Indirect = secondary antibodies bind to primary antibodies detecting specific tissue antigens.
Enzyme-Linked-Immunosorbent Assays (ELISA)
Colorless enzyme solutions are mixed with separated antibody/protein samples
Positive sign for the protein that binds to the enzyme = colored via substrates formed (usually blue or red)
Used for HIV testing, drug screening, infections, hormone levels.
3 types of ELISA assays
3 types of ELISA assays
Direct assay: antigens and other sample components are placed in the well/ area first and then the primary antibody binds to what it reacts with
- primary antibody possesses the enzyme
Indirect assay: primary antibodies coat the well/area first and then the antigens are placed in the well, followed by a secondary antibody specific for an antigen being looked for
- secondary antibody possesses the enzyme
In both situations, after washing free floating antibodies, the enzyme substrate is then administered
Sandwich assay: similar to an indirect assay except uses specific Biotin molecules on the primary antibody. Then avidin molecules are bound to the enzyme with multiple avidin binding to biotin.
- allows for greater signal amplification and specificity (strongest one)
Indirect vs direct detection in ELISA
Indirect detection, multiple secondary antibodies can bind to a primary anti body(only done in indirect or sand which assays)
- amplifies signal and allows for greater sensitivity
Radioimmunoassay
Radio-labeled secondary antibodies that bind to primary antibodies in the patient serum if present
Used to detect and quantify antibody titers
RAST
Radio allegosorbent test which tests for specific IgE in patient titer
Used when patient cannot have dermal allergy testing and tests for allergies
Examples = psoriasis/eczema
Western blots
Identify and determine the real-time quantity of a certain protein/antibody in a patient sample
Used to confirm ELISA assays and for both HIV and Lyme diseases
Nephelometry
Determines concentration of antibodies in a patient titer sample by measuring the Ag;Ab immune complexes in the liquid phase
Uses light to do this. And is usually used to confirm measurements of IgM, IgG and IgA
Precipitation reactions
Measures if patient titer has antibodies for a specific antigen
Need to have an optimal concentration of both antigen and antibody (equivalence zone) in order to get appropriate amounts of precipitation
- if patient has the antibody then precipitates will form a precipitin ring around the equivalence zone
Radial immunodiffusion
Used for haemolytic anemias and complement disorders
Tests for serum haptoglobin levels
- If hemolytic anemia is present, haptoglobin levels will be very lower = small and condensed
Double immunodiffusion
A branch off of radial immunodiffusion
Detects antigen/antibody presence in patient serum
If present produces a precipitin line from both wells will occur
- used pretty much only for fungal infections, specifically coccidioidomycosis
