Immunologic Tests

Question 1

Why do immunologic tests?

Answer 1

Confirm diagnosis, often necessary before treatment or referral.

Question 2

Why would you use an immunologic test in optometric care?

Answer 2

Ocular infections
Autoimmune Disease
Ocular allergies

Question 3

2 Immunologic test categories

Answer 3

Serology: Cells and coagulations removed. Tears, saliva, semen. Used for diagnostic identification of antibodies, enzymes, or minerals.

Diagnostic Immunology (immunodiagnostics)
Antigen or antibody detection through Ab:Ag interactions.
Ag/Ab identified by
-Radiolabel
-Enzyme (could indicate molecule you are looking for with colors)
-Fluorescent label (after introducing certain wavelength of light)

Question 4

mAb

Answer 4

Ag-specific Ab grown in lab via a hybridoma.

Question 5

Hydridoma

Answer 5

Created when you inject a mouse with antigen you are looking for. Mouse will develop plasma cells + Abs for that Ag. Plasma cells are removed and fused with myeloma cancer cell. Will then proliferate (like cancer cells) and produce Abs (like plasma cell)

used to create mAb

Question 6

Naked mAbs: independent Abs.

Answer 6

Specific for wide range of different antigens. Can then determine presence of naked mAb by using another mAb that binds Naked Ab as its Ag.

Question 7

Two types of mAbs

Answer 7

Naked mAbs: independent Abs.

Cojugated mAbs: Abs joined with another molecule

Question 8

5 types of serologic tests

Answer 8

Precipitation tests
Agglutination tests
Labeled Ab tests
Complement fixation tests
Viral neutralization tests

Question 9

Complement fixation test (serologic)

Answer 9

Presence of ABs in serum prevent complement activation, which prevents RBC lysis.

If Ab are present, the Ab will bind the Ag and prevent complement from happening. No RBC will be lysed.

If Ab are not present, the Ag will activate complement, which will lyse RBC.

Question 10

Viral neutralization tests (serologic)

Answer 10

Presence of Ab in serum prevent virus growth.

Ab + Ag = no virus growth
Ag without Ag = virus growth.

Question 11

Precipitation tests for what ?

Answer 11

Ab, viruses, bacteria

Precipitate (immune complexes) often visible to the naked eye. Requires large amounts of Ag or Ab for positive test.

Low sensitivity. Lots of false positives.

Question 12

Types of precipitation tests

Answer 12

Gel immunodiffusion

Immunoelectrophoresis

Question 13

Agglutination tests for what?

Answer 13

Virus, bacteria, and Abs.
Target Ag may be bound to a carrier protein- makes it easier to visualize.
Cross linking causes clumping, easy to see and interpret. Fast and more sensitive than precipitation

Question 14

Which test is more sensitive? Precipitation or agglutination

Answer 14

Agglutination

Question 15

Hemaaglutination

Answer 15

Clumping of RBC after mixing of anti-RBC antibodies.

Blood typing for transfusion.

Question 16

Titration and agglutination

Answer 16

Concentration of Abs or Ag in serum

Question 17

Titer

Answer 17

Reciprocal of greatest dilution with reaction.

Question 18

Agglutination tests for treponema pallidum (syphilis)

Answer 18

Fast and cheap, but not very specific.
2 types: VDRL (venereal disease research lab)
or Rapid plasma reagin (RPR)

Question 19

How does VDRL and RPR work to detect the syphilis pathogen

Answer 19

VDRL: Serum Ab detection via flocculation (foamy agglutination) Must use light microscope to see flocculation.

RPR: Same Ag used as VDRL, but bound to carbon particle. Flocculation is visible without microscope.

Question 20

5 types of labelled assays

Answer 20

Radioimmunoassay (RIA) 
Enzyme linked immunosorbent assay (ELISA) 
Immunofluorescence 
Western Blot 
Flow Cytometry

Question 21

Radioimmunoassay

Answer 21

Radioactive- must use extra precautions.
Competitive immunoassay that detects hormone levels of T3 and T4.

Also tests for SLE, Hepatitis B Ag, or drugs in plasma.

Question 22

ELISA

Answer 22

Test for Ag or Ab with visible color change.
Fast and sensitive.

3 types:
Direct
Indirect
Capture assay sandwich

Question 23

Sandwich ELISA

Answer 23

mAb attached to solid surface
Sample solution added (Ag)
Ab enzyme conjugate added
Substrate added (colorless)

Solid surface + mAb + Ag + Ab with enzyme + substrate = color

Question 24

Rheumatoid Factor

Answer 24

Auto Ab (IgM) that binds Fc region of IgG. Creates immune complex that settles out in the body.

Detected using ELISA

Question 25

Lateral Flow immunoassay (sideways ELISA) Type of sandwich Elisa

Answer 25

Competitive/Sandwhich type of ELISA
Qualitative (great for yes or no responses)
Home pregnancy test, HIV, drug tests.
Tests for viral conjunctivitis and dry eye (Matrix metalloproteinases)

Question 26

Immunofluorescence and antinuclear antibodies (ANA)

Answer 26

Auto abs that bind host DNA/RNA.

Question 27

Western Blot

Answer 27

Confirmatory test.
Labeled antibodies bind protein sample
Or
Labeled Ag bind Ab sample. HIV testing.

Very sensitive and very specific.
Used to confirm Elisa.
Uses electrophoresis.

Question 28

Which test is used to confirm ELISA?

Answer 28

Western blot. Confirmatory test that is very sensitive and very specific.

Question 29

What is automated analysis by Flow Cytometry used for?

Answer 29

Fluorescently tagged mAbs used to characterize cell populations, cell surface protein expression, or intracellular protein expressions.

Question 30

Blood tests for inflammation (2)

Answer 30

C-reactive protein (acute inflammation)
Erythrocyte sedimentation rate (ESR) for acute and chronic inflammation

Both are non-specific

Question 31

C-reactive protein blood test

Answer 31

Used to determine acute inflammation that is on going. Non-specific. Associated with complement activation.
CRP is produced by the liver.
CRP levels are associated with elevated disease risk such as heart attack, stroke, cancer.

Detected via ELISA

Question 32

Erythrocyte sedimentation rate (ESR) blood test

Answer 32

Used to determine acute and chronic inflammation.
Non specific.
Measure of RBC sedimentation- rate at which RBCs settle out of blood. Treated to prevent coagulation.

Acute phase reactants (fibrinogen) accelerates RBC sedimentation.