Immunologic Tests Flashcards
Why do immunologic tests?
Confirm diagnosis, often necessary before treatment or referral.
Why would you use an immunologic test in optometric care?
Ocular infections
Autoimmune Disease
Ocular allergies
2 Immunologic test categories
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)
mAb
Ag-specific Ab grown in lab via a hybridoma.
Hydridoma
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
Naked mAbs: independent Abs.
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.
Two types of mAbs
Naked mAbs: independent Abs.
Cojugated mAbs: Abs joined with another molecule
5 types of serologic tests
Precipitation tests Agglutination tests Labeled Ab tests Complement fixation tests Viral neutralization tests
Complement fixation test (serologic)
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.
Viral neutralization tests (serologic)
Presence of Ab in serum prevent virus growth.
Ab + Ag = no virus growth
Ag without Ag = virus growth.
Precipitation tests for what ?
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.
Types of precipitation tests
Gel immunodiffusion
Immunoelectrophoresis
Agglutination tests for what?
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
Which test is more sensitive? Precipitation or agglutination
Agglutination
Hemaaglutination
Clumping of RBC after mixing of anti-RBC antibodies.
Blood typing for transfusion.
Titration and agglutination
Concentration of Abs or Ag in serum
Titer
Reciprocal of greatest dilution with reaction.
Agglutination tests for treponema pallidum (syphilis)
Fast and cheap, but not very specific.
2 types: VDRL (venereal disease research lab)
or Rapid plasma reagin (RPR)
How does VDRL and RPR work to detect the syphilis pathogen
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.
5 types of labelled assays
Radioimmunoassay (RIA) Enzyme linked immunosorbent assay (ELISA) Immunofluorescence Western Blot Flow Cytometry
Radioimmunoassay
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.
ELISA
Test for Ag or Ab with visible color change.
Fast and sensitive.
3 types:
Direct
Indirect
Capture assay sandwich
Sandwich ELISA
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
Rheumatoid Factor
Auto Ab (IgM) that binds Fc region of IgG. Creates immune complex that settles out in the body.
Detected using ELISA
Lateral Flow immunoassay (sideways ELISA) Type of sandwich Elisa
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)
Immunofluorescence and antinuclear antibodies (ANA)
Auto abs that bind host DNA/RNA.
Western Blot
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.
Which test is used to confirm ELISA?
Western blot. Confirmatory test that is very sensitive and very specific.
What is automated analysis by Flow Cytometry used for?
Fluorescently tagged mAbs used to characterize cell populations, cell surface protein expression, or intracellular protein expressions.
Blood tests for inflammation (2)
C-reactive protein (acute inflammation)
Erythrocyte sedimentation rate (ESR) for acute and chronic inflammation
Both are non-specific
C-reactive protein blood test
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
Erythrocyte sedimentation rate (ESR) blood test
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.
