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 Response In vivo First antibody formed?IgM IgM & IgG

Secondary Antibody Response In 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 Influences on Lattice Formation Ratio of available Ab and Ag ProzonePostzone

Test Condition Influences on Lattice Formation Prozone –No visible reaction If patient antibody is what is being tested for, then false _______ result negative

Test Condition Influences on Lattice Formation Postzone –No visible reaction If patient antigen is what is being tested for, then false ________ result negative

pH – usually 7.0 Temperature of Incubation –Warm reactive, 37°C = –Cold reactive, 4-22°C = Test Condition Influences on Lattice Formation Length of reaction –Immunoglobulin class –Strength of Ab-Ag reaction

Which antibody is the best agglutinator? Test Condition Influences on Lattice Formation 750x better agglutinator than IgG

Ionic strength Test Condition Influences on Lattice Formation Like charges repel each other

Buffer solutions supply Na + and Cl - Test Condition Influences on 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 Influences on 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