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