Immunological Diagnostics

Question 1

Antigens

Answer 1

• indicates active infection

- not always circulating in the blood

Question 2

Antibodies

Answer 2

• active infection
• exposure and subsequent immunity
• vaccination

Question 3

DNA

Answer 3

• active infection

- presence does not always equal causation

Question 4

What are the 4 things we can test for?

Answer 4

• antigens
• antibodies
• DNA
• cell surface markers

Question 5

Sensitivity

Answer 5

High sensitivity picks up any remote chance that an animal is infected/affected
- ex: pancreatic snap tests

Question 6

Specificity

Answer 6

Something can have high sensitivity, but low specificity

- chance for cross reactivity

Question 7

Primary binding tests

Answer 7

Directly detects antigen binding to antibody (immune complexes)
- one of reactants (either ag or ab) must be chemically labeled for detection

Question 8

What are the chemical labels used for primary binding tests?

Answer 8

• radioisotopes
• fluorescent dyes
• enzymes

Question 9

What do primary binding tests detect?

Answer 9

Either antigen or antibody

Question 10

Immunofluorescence assays

Answer 10

Direct or indirect fluorescent antibody tests

Question 11

Immunoenzymes assays

Answer 11

Enzyme-linked immunosorbent assays

• ELISA
• western blot
• immunohistochemistry

Question 12

Radioimmnoassays

Answer 12

Uses radioisotopes

Question 13

Chemiluminescent immunoassays

Answer 13

Better than RIA

- commonly used in labs

Question 14

Epitope

Answer 14

Something on the antigen that the antibody recognizes and binds to

Question 15

Antigen-antibody recognition

Answer 15

Specific recognition between antigen and a monoclonal antibody specific for a certain target
- target can be the patient antibody or the patient antigen

Question 16

Patient antigen reacts with test antibody

Answer 16

• patient antigen in blood

- patient antigen in tissue

Question 17

Patient antibody reacts with test antigen

Answer 17

Patient serum applied to test antigen

Question 18

IFA

Answer 18

Immunofluorescense assays

• direct fluorescent antibody: detects specific Ag in patient sample
• indirect fluorescent antibody: detects patient antibody specific to Ag

Question 19

Direct fluorescent antibody

Answer 19

Patient tissue or fluid with suspected antigen is fixed to a slide and incubated with commercial antibody
- ex: rabies detection, FeLV test

Question 20

Indirect fluorescent antibody

Answer 20

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

Question 21

ELISA

Answer 21

Enzyme-linked because an enzyme is conjugated to an antibody

• enzyme changes color when mixed with a substrate
• can detect patient antibodies or antigens

Question 22

Immunosorbent

Answer 22

Relating to or denoting techniques making use of the absorption of antibodies by insoluble preparations of antigens

Question 23

ELISA method

Answer 23

• 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

Question 24

Color change is proportional to amount of ______

Answer 24

Antibody in patient’s serum

Question 25

Disposable immunoassays

Answer 25

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

Question 26

Immunochromotography

Answer 26

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

Question 27

Western Blot

Answer 27

Identifies a specific protein in a complex mixture | - use clinically as a confirmation of other test results (rule out false positives)

Question 28

Western Blot method

Answer 28

- 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

Question 29

IHC

Answer 29

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

Question 30

What is the difference between direct and indirect IHC methods?

Answer 30

Direct: primary Ab labelled Indirect: secondary Ab (antiglobulin) labelled

Question 31

RIA

Answer 31

Highly sensitive - expensive - detection of trace drugs

Question 32

Antibody titration

Answer 32

Testing for amount of circulating antibodies present in the serum to a given pathogen - performed for infectious diseases (lepto, tick-borne diseases)

Question 33

Using titers

Answer 33

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

Question 34

Serum neutralization

Answer 34

Incubate different concentrations of serum with virus

Question 35

Microscopic agglutination tests

Answer 35

Serum dilutions incubated with the organism | - titer is the highest dilution that causes agglutination of 50% of the organisms

Question 36

Polymerase chain reaction

Answer 36

Amplifies DNA specific to the organism you are looking for - positive PCR: target DNA found in patient sample - risks: contamination, sensitivity and specificity issues

Question 37

PCR DNA extraction

Answer 37

- blood: mycoplasma, hepatozooan - urine: leptospirosis - bone marrow - fluids: feline infectious peritonitis

Question 38

RT-PCR

Answer 38

Reverse transcriptase - used when starting sample is RNA - must reverse transcribe RNA to DNA before amplification

Question 39

Quantitative

Answer 39

Amplify and quantitate the amount of target DNA | - can get absolute copy number or perform relative quantification

Question 40

PCR only works when ___

Answer 40

Actual organism is present in patient blood or sample

Question 41

Flow cytometry

Answer 41

Identifies cell surface antigen (NOT immune complex!) | - most commonly used for immunophenotyping (lymphoma, leukemia)

Question 42

Immunophenotyping

Answer 42

Useful for assessing leukocyte populations - lymphoma: all B or T cells - leukemia: look for markers of mature leukocytes - use for transplantations