Immunological Diagnostics Flashcards
Antigens
- indicates active infection
- not always circulating in the blood
Antibodies
- active infection
- exposure and subsequent immunity
- vaccination
DNA
- active infection
- presence does not always equal causation
What are the 4 things we can test for?
- antigens
- antibodies
- DNA
- cell surface markers
Sensitivity
High sensitivity picks up any remote chance that an animal is infected/affected
- ex: pancreatic snap tests
Specificity
Something can have high sensitivity, but low specificity
- chance for cross reactivity
Primary binding tests
Directly detects antigen binding to antibody (immune complexes)
- one of reactants (either ag or ab) must be chemically labeled for detection
What are the chemical labels used for primary binding tests?
- radioisotopes
- fluorescent dyes
- enzymes
What do primary binding tests detect?
Either antigen or antibody
Immunofluorescence assays
Direct or indirect fluorescent antibody tests
Immunoenzymes assays
Enzyme-linked immunosorbent assays
- ELISA
- western blot
- immunohistochemistry
Radioimmnoassays
Uses radioisotopes
Chemiluminescent immunoassays
Better than RIA
- commonly used in labs
Epitope
Something on the antigen that the antibody recognizes and binds to
Antigen-antibody recognition
Specific recognition between antigen and a monoclonal antibody specific for a certain target
- target can be the patient antibody or the patient antigen
Patient antigen reacts with test antibody
- patient antigen in blood
- patient antigen in tissue
Patient antibody reacts with test antigen
Patient serum applied to test antigen
IFA
Immunofluorescense assays
- direct fluorescent antibody: detects specific Ag in patient sample
- indirect fluorescent antibody: detects patient antibody specific to Ag
Direct fluorescent antibody
Patient tissue or fluid with suspected antigen is fixed to a slide and incubated with commercial antibody
- ex: rabies detection, FeLV test
Indirect fluorescent antibody
Commercial antigen is pre-placed on slide and incubated with patient serum
- bound Ab is detected with FITC-labeled antiglobulin (antiglobulins bind antibodies)
- fluorescence indicates patient has Ab to specific antigen
ELISA
Enzyme-linked because an enzyme is conjugated to an antibody
- enzyme changes color when mixed with a substrate
- can detect patient antibodies or antigens
Immunosorbent
Relating to or denoting techniques making use of the absorption of antibodies by insoluble preparations of antigens
ELISA method
- add patient serum to plate of commercial Ag
- bound Ab is identified by adding solution with antiglobulin that is linked to enzyme
- when a solution with enzyme substrate is added, a color change is produced
Color change is proportional to amount of ______
Antibody in patient’s serum
Disposable immunoassays
Nylon membrane coated with Ab
- patient sample with suspected Ag is applied
- if Ag binding occurs, a positive result is detected by a dot or + sign
Immunochromotography
Lateral flow test
- antigen solution (patient sample) flows thru a porous strip and meets labeled commercial Ab to form an immune complex
- positive indicated by a blue or pink color
Western Blot
Identifies a specific protein in a complex mixture
- use clinically as a confirmation of other test results (rule out false positives)
Western Blot method
- electrophoresis of a protein mixture on gels –> resolves proteins to a single band
- blotting of bands to a nitrocellulose membrane via electric current
- enzyme or immunoassay used to visualize transferred protein
IHC
Enzymes are used as the label and are conjugated to Ab or antiglobulin to locate specific antigen in tissue
- diagnosis of disease and determination of tissue type in tumors (ex: actin Ab used to determine if skeletal muscle in origin)
- bound Ab detected by brown color
What is the difference between direct and indirect IHC methods?
Direct: primary Ab labelled
Indirect: secondary Ab (antiglobulin) labelled
RIA
Highly sensitive
- expensive
- detection of trace drugs
Antibody titration
Testing for amount of circulating antibodies present in the serum to a given pathogen
- performed for infectious diseases (lepto, tick-borne diseases)
Using titers
Antibody levels checked at initial presentation
- disease usually likely if initial titers are extremely high
- recheck 2-3 weeks later (can be falsely decreased from antibiotics)
- 4-fold rise in titers confirms infection
Serum neutralization
Incubate different concentrations of serum with virus
Microscopic agglutination tests
Serum dilutions incubated with the organism
- titer is the highest dilution that causes agglutination of 50% of the organisms
Polymerase chain reaction
Amplifies DNA specific to the organism you are looking for
- positive PCR: target DNA found in patient sample
- risks: contamination, sensitivity and specificity issues
PCR DNA extraction
- blood: mycoplasma, hepatozooan
- urine: leptospirosis
- bone marrow
- fluids: feline infectious peritonitis
RT-PCR
Reverse transcriptase
- used when starting sample is RNA
- must reverse transcribe RNA to DNA before amplification
Quantitative
Amplify and quantitate the amount of target DNA
- can get absolute copy number or perform relative quantification
PCR only works when ___
Actual organism is present in patient blood or sample
Flow cytometry
Identifies cell surface antigen (NOT immune complex!)
- most commonly used for immunophenotyping (lymphoma, leukemia)
Immunophenotyping
Useful for assessing leukocyte populations
- lymphoma: all B or T cells
- leukemia: look for markers of mature leukocytes
- use for transplantations
