Immunologic Tests Flashcards
immunologic tests
- confirm diagnosis
- often necessary before treatment or referral
immunologic tests indications
- indicators of infection and/or inflammation
- pathogen detection
- auto-antibody detection
- blood and tissue typing
- immune deficiency testing
indications for immunologic tests in optometric care
- ocular infections (conjunctivitis, viritis)
- autoimmune disease (uveitis, keratoconjunctivitis sicca, amuarosis fugax (sudden loss of vision))
- ocular allergies
available in-office immunologic tests in optometric care
- adenoviral conjunctivitis
- Sjorgren’s syndrome
- non-specific dry eye
- environmental allergies
immunologic tests categories
- serology
- diagnostic immunology (immunodiagnostics)
serology
- study of blood serum and other bodily fluids
- clinically: diagnostic identification of antibodies, enzymes, or minerals
diagnostic immunology (immunodiagnostics)
- antigen or antibody detection through the use of Ag:Ab interactions
- Ag or Ab identified by radiolabel, enzyme, or fluorescent label
monoclonal antibodies (mAb)
-Ag-specific Abs lab-grown via hybridoma
uses of monoclonal antibodies
- immunodiagnostics
- cancer treatment
- autoimmune disease therapy
naked mAbs
independent Abs
conjugated mAbs
Abs joined with another molecule
serologic tests
- precipitation tests
- agglutination tests
- labeled antibody tests
- complement fixation tests
- viral neutralization tests
complement fixation tests
- mix sample serum with test solution
- then add to solution containing complement
- if Abs are present in sample serum, those Abs will bind the Ags, preventing Ag from activating complement
- presence of Abs in serum prevent complement activation
viral neutralization tests
- put patient sample and virus in an egg
- presence of Abs in serum prevent virus growth
- viral growth will occur if the patient sample does not have the Abs
precipitation tests:
tests for _____
- Abs
- viruses
- bacteria
precipitation tests:
characteristics
- soluble target molecule and known Ag or Ab
- precipitate (immune complexes) often visible to naked eye
- requires large amounts of Ag or Ab for positive test
- low sensitivity
precipitation tests:
types of tests
- gel immunodiffusion
- immunoelectrophoresis
agglutination tests:
tests for ____
- Abs
- viruses
- bacteria
agglutination tests:
characteristics
- target may be bound to a carrier protein (increases visibility)
- cross-linking causes clumping
- easy to see and interpret
- fast
- can be very sensitive
hemagglutination
- clumping of red blood cells 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
- e.g., determining IgG titer after vaccination
agglutination tests for Treponema pallidum (syphilis pathogen)
- fast and cheap but not very specific
- Venereal Disease Research Laboratory (VDRL) test
- Rapid Plasma Reagin (RPR)
Venereal Disease Research Laboratory (VDRL) test
- serum Ab detection via flocculation
- can be negative in latent syphilis or after treatment
Rapid Plasma Reagin (RPR)
- same Ag used as VDRL, but bound to carbon particle
- flocculation is visible without a microscope
labelled assays
- radioimmunoassay (RIA)
- enzyme-linked immunosorbent assay (ELISA)
- immunofluorescence
- western blot
- flow cytometry
radioimmunoassay (RIA)
- competitive immunoassay
- detects hormone levels (T3, T4)
- also tests for: SLE Abs, HBsAg, drugs in plasma
enzyme-linked immunosorbent assay (ELISA)
- test for Ag or Ab - with visible color change
- fast and sensitive
- in vitro disease diagnostics: Abs in sample (HIV, SLE, Grave’s, myasthenia gravis); drug in sample (cocaine, opiates, marijuana); hormone in sample (pregnancy, thyroid function, steroids)
sandwich ELISA
1) monoclonal Ab attached to solid surface
2) sample solution added
3) Ab-enzyme conjugate added
4) substrate added
5) target Ag causes color change
6) can titrate to find concentration in sample
rheumatoid factor
- auto-Abs that binds Fc region of IgG
- detected via ELISA
RF+ autoimmune diseases
- 70% of patients with RA are RF+
- SLE
- Sjogren’s syndrome
- only 5% of Juvenile Rheumatoid Arthritis (JRA)
lateral flow immunoassay
- competitive or sandwich assay
- qualitative, not quantitative
- simple device used for at-home or in-office testing (home pregnancy tests, drug tests, HIV tests)
- sample migrates across a series of capillary beds
- sample pad –> conjugate pad –> reaction stripes –> wick
lateral flow assays for ocular disease
- RPS Adeno Detector (viral conjunctivitis, adenoviral antigen)
- RPS Inflammatory (dry eye, matrix metalloproteinase-9)
immunofluorescence and Antinuclear Antibodies (ANA)
- auto-Abs that bind host DNA, RNA, etc.
- indirect fluorescent antibody (IFA): mix serum sample with cells fixed to slide, add fluorescently labeled Abs
ANA+ autoimmune diseases
- 80% of patients with eye involvement of JRA
- 95% with SLE
- 30% of rheumatoid arthritis
- 40-70% with Sjogren’s syndrome
- 60-90% scleroderma
Western blot
- confirmatory test
- labeled antibodies bind to protein sample or labeled Ag bind Ab sample (HIV testing)
- very sensitive and very specific
- used to confirm ELISA (bovine spongiform encephalopathy, Lyme disease, hepatitis B, HIV)
automated analysis by flow cytometry
- automatic analysis
- fluorescently tagged mAbs
- cell surface protein expression
- intracellular protein expression
- characterizing cell populations
treponemal test for treponema pallidum
- Fluorescent Treponemal Antibody Absorption Test (FTA-ABS)
- expensive, confirmatory test (would use this if someone had a positive RPR)
- looking for organism-specific Abs (IgG and IgM)
- add sample serum to slide with fixed pathogen
- add fluorescently tagged anti-human mAb
- look for anti-treponeme Abs on surface of treponemes
blood tests for inflammation
- C-reactive protein (CRP): acute inflammation
- Erythrocyte sedimentation rate (ESR): acute and chronic inflammation
C-reactive protein (CRP)
- non-specific (tells you someone has inflammation but not why)
- produced by the liver during an acute inflammatory response
- associated with complement activation
- CRP levels are associated with elevated disease risk (heart attack, stroke, severe macular degeneration, colon cancer, type II diabetes)
- detected via various immunodiagnostic assays (ELISA)
Erythrocyte sedimentation rate (ESR)
- non-specific
- measure of RBC sedimentation
- rate at which RBCs settle out of blood in one hour
- treated to prevent coagulation
- acute phase reactants (fibrinogen) accelerate RBC sedimentation
- results affected by age and sex
- good for monitoring disease
