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Immunologic Methods

CLS 420 Clinical Immunology & Molecular Diagnostics. Immunologic Methods. Part One Antigen-Antibody Reactions. Objectives. Discuss the following as related to immunology testing: Equivalence zone Postzone Prozone Sensitivity Specificity Cross-reactivity Screening test

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Immunologic Methods

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  1. CLS 420 Clinical Immunology & Molecular Diagnostics Immunologic Methods Part One Antigen-Antibody Reactions

  2. Objectives • Discuss the following as related to immunology testing: • Equivalence zone • Postzone • Prozone • Sensitivity • Specificity • Cross-reactivity • Screening test • Confirmatory test

  3. Objectives • Explain how the following physical factors can affect antigen-antibody reactions: • Ionic strength • pH • Reaction time • Temperature • Concentration ratio of antigen and antibody

  4. The study of host reactions to foreign substances. The study of serum; in particular, the study of antibodies in serum and other body fluids. Immunology vs. Serology

  5. Sensitivity & Specificity • Sensitivity: How well a test is able to detect minute quantities of an analyte. • Specificity: The ability of a test to detect one unique analyte.

  6. Cross Reactivity • Reactions with substances that are structurally similar to an analyte.

  7. Predictive Value • The probability that a laboratory test result will correctly diagnose or rule out a particular disease. • Predictive Value - Positive is the probability that a positive test result is associated with disease. • # of true positives / (# of true positives + # of false positives) • Predictive Value - Negative is the probability that a normal test result is not associated with disease. • # of true negatives / (# of true negatives + # of false negatives) Cut-off value “Normal”Disease # of patients Test Result Value

  8. Screen vs. Confirm • Screening Test: A test used to detect disease, usually in someone who is asymptomatic. They are highly sensitive but are not as specific as the confirmatory tests. • Confirmatory Test: Highly specific tests that are run to validate the results of the initial screening test.

  9. Antigen/ Antibody Reactions

  10. Antigen / Antibody Binding • Antigen and antibody bind with “Lock and Key” fit. • Affinity – the total attractive force that draws antibody to antigen. • Avidity – how “tightly” antigen and antibody bind. AG AB

  11. Equilibrium • Antigen / antibody reactions are readily reversible. • Free Ag + Free Ab Ag-Ab complex

  12. Antigen / Antibody Reactions • May be visualized when lattice structures form. • Sensitization • Agglutination or precipitation Y Y Y Y Y Y

  13. Zone of Equivalence Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Postzone-antigen excess Prozone – antibody excess

  14. Antigen/antibody reactions are influenced by:

  15. Ionic Strength • Shielding – charges that surround the Fab portion of an antibody, blocking antigen/antibody binding • Zeta potential – the difference in electrical charge between the surface of a cell and the outer layer of the ionic cloud that surrounds the cell in an electrolyte solution. • Keeps cells too far apart to allow lattice formation

  16. Zeta Potential Y Y Y Y - - - - - - - - - - - - - - ++++++++++++++ Y Y J - - - - - - - - - - - - - ++++++++++++++ - - - - - - - - - - - - - - -

  17. Ag/Ab binding is influenced by: • pH • Reaction time • Temperature • Number of antigens

  18. Dosage • Antibodies that react stronger with cells that have homozygous expression of an antigen than they do with cells that have heterozygous expression are said to show dosage • Cells with homozygous antigen expression have twice as much antigen available to participate in the reaction than do cells with heterozygous antigen expression

  19. If there is an anti- antibody, which of these cells will yield the stronger reaction with it? Homozygous Heterozygous

  20. Location of Antigens • Free antigens and cell surface antigens are more available to react than those buried within the membrane or within the cell. • Interference in ag/ab binding due to the position of other antigens Y Y Y Y

  21. Concentration Ratio of Ag/Ab Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Postzone-antigen excess Prozone – antibody excess Zone of Equivalence

  22. The End Specific, basic immunologic methods are discussed in the PowerPoint, “Immunologic Methods, Part 2”.

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