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Immunology vs. Serology• Immunology
– Study of immune system
• Serology– Detecting/measuring elements of
humoral immune system (I.e., antibodies)
– Help diagnose infectious disease & immunologic disorders
– Determine immune status
When to Use Serology
• Unable to culture infectious agent
• Confirmation of etiologic agent ID
• Diagnosis of immunologically-related disorders (ex. autoimmune disorders)
• Determine immune status(ex. immunity to Rubella, Hepatitis B)
Antigens
• Molecular weight
• Foreignness
• Complexity
• Structural stability
• Degradability
Antibodies
• IgM
• IgG
• IgA
• IgD
• IgE
Primary Antibody ResponseIn vivo
• First antibody formed? IgM
• IgM & IgG
Secondary Antibody ResponseIn vivo
• IgG predominates (IgM also formed)• Antibody concentration compared to 1°?
Greater in secondary response
Antibody – Antigen Interactions In vitro
• Two steps in the detection of in vitro antigen/antibody reactions
1. Sensitization(initial attraction)
2. Lattice formation(committed, long-lasting relationship)
Sensitization
• Due to affinity– Initial attraction
• Reversible
Affinity = Dissociation
Lattice Formation• Ab >1 binding site
• Ab binds to multiple Ag sites
• Avidity (sum of all attractive forces)
• Stronger bond than sensitization– “lock a door”
Test Condition Influenceson Lattice Formation
• Ratio of available Ab and Ag
Prozone Postzone
Test Condition Influenceson Lattice Formation
• Prozone– No visible
reaction
• If patient antibody is what is being tested for, then false _______ resultnegative
Test Condition Influenceson Lattice Formation
• Postzone– No visible
reaction
• If patient antigen is what is being tested for, then false ________ resultnegative
• pH – usually 7.0• Temperature of Incubation
– Warm reactive, 37°C =
– Cold reactive, 4-22°C =
Test Condition Influenceson Lattice Formation
• Length of reaction– Immunoglobulin class– Strength of Ab-Ag reaction
• Which antibody is the best agglutinator?
Test Condition Influenceson Lattice Formation
750x better agglutinator than IgG
• Ionic strength
Test Condition Influenceson Lattice Formation
Like charges repel each other
• Buffer solutions supply Na+ and Cl-
Test Condition Influenceson Lattice Formation
Sodium ions neutralize negative charges
Sensitization & lattice formation can occur
• Steric Hindrance– Physical or mechanical blocks– Different antibodies directed at separate
antigenic determinants on one antigen…may get in the way of each other
– IgG Ab block sites for IgM Ab– Lack of space around Ag due to
complement, etc.
Test Condition Influenceson Lattice Formation
Specificity vs. Sensitivity
• A test with a high degree of _______ will have a low number of false positives.
• A test with a high degree of _______ will have a low number of false negatives.
• The difference between specificity & sensitivity is…
Sensitivity & Specificity
• Sensitive = detect small amount of antigen or antibody– If it is there, I’ll find it
• Specific = will only be positive or react with particular antigen or antibody– If the test is positive, you can be sure it is
a true positive
• A test with a high degree of __________ will have a low number of false positives.
specificity
Sensitivity & Specificity
• A test with a high degree of _________ will have a low number of false negatives.
sensitivity
• Antibodies made in response to an exposure to the bacteria Brucella sp. (causes a person to develop fevers and become lethargic) also react with the bacteria Francisella tularensis.
• The Brucella antibody ___________ with the Francisella organism.
cross-reacts
• The Brucella antibody a specific antibody.
is not
Sensitivity & Specificity
Titer• Relative concentration of specific
antibody or antigen
• Reciprocal of the highest dilution in which a positive reaction occurs
• When testing acute and convalescent sera, a 4-fold rise in titer is considered evidence of a recent infection
Evidence of Recent Infection• See example on page 26 of your notes
– Acute titer = 1:20 (last dilution tested w/ + rxn)– Convalescent titer = 1:80
• This exhibits a 4-fold rise in titer or a 2-tube difference (using serial 2-fold dilutions)
• The acute and convalescent titers should be tested at same time– Not practical– Often a high single titer determines recent infection
Heat Inactivate Serum
• Removes complement or other substances in serum that are known to interfere with certain serologic tests
• Procedure– 56°C, for 30 minutes
• >4 hrs since last inactivation– Re-inactivate 56°C, for 10 minutes
Summary
• Immunology versus Serology
• Antigens & Antibodies
• Antibody Response
• Antigen-Antibody Interactions
• Other key terms
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