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Immunologic Methods

Immunologic Methods. Part One Definitions Part Two Antigen-Antibody Reactions. CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP) CM ktrudell@NebraskaMed.com. Objectives. Discuss the following as related to immunology testing: Sensitivity Specificity

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Immunologic Methods

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  1. Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)CM ktrudell@NebraskaMed.com

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

  3. Objectives • Explain how the following physical factors can affect antigen-antibody reactions: • Concentration ratio of antigen and antibody • Ionic strength • pH • Reaction time • Temperature • Describe heat inactivation of patient serum, including method and purpose.

  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 and Specificity • Sensitivity = number of true positive tests / total number of patients with disease {true pos + false neg} • [370 / (370+2)] x100 = 99.46% • Specificity = number of true negative tests / total number of healthy individuals {true neg + false pos} • [550 / (550+5)] x100 = 99.10%

  6. Cross Reactivity • Positive reaction with substance that is structurally similar to analyte.

  7. Predictive Value • Predictive Value – Positive: the probability that a positive test result is associated with disease • Predictive Value – Negative: the probability that a normal test result is not associated with disease. Cut-off value “Normal”Disease # of patients Test Result Value

  8. Screen vs. Confirm • Screening Test: A test used to detect disease. • Confirmatory Test: Run to validate the results of the initial screening test.

  9. Antigen/ Antibody Reactions

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

  11. Affinity • Antigen and antibody bind with “Lock and Key” fit. • Affinity – the attractive force between an Fab piece of an antibody and a single epitope on an antigen.

  12. Cross Reactivity • For which antigen will the antibody have greater affinity? • The antigen that stimulated antibody production.

  13. Avidity • How “tightly” the antigen and antibody bind. • The sum of forces binding multivalent antibody to multivalent antigen. • Number Fab pieces • Number of identical epitopes

  14. Y Y Y Y Y Y Y Y Y Y Y Y Which Ag/Ab combo will have the greatest avidity?

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

  16. 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

  17. Antigen/antibody reactions are influenced by:

  18. 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

  19. Ionic Strength • Shielding • Zeta Potential Y - - + - + - + - - + - - - - + + - + - - + - + - + - - - + - + - - - + - + + - - - - + - + -

  20. Zeta Potential Y Y Y Y Y Y - - - - - - - - - - - - - - ++++++++++++++ J - - - - - - - - -

  21. Other items that influence Ag/Ab binding: • pH • Reaction time • Temperature • Number of antigens

  22. Given that and are alleles, if there is an anti- antibody, which of these cells will yield the stronger reaction with the antibody? Homozygous Heterozygous

  23. 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

  24. Heat Inactivation • Complement that is naturally present in a patient’s serum may interfere with tests that use complement as a reagent. • When a test needs a controlled amount of complement, the patient’s serum is heated at 56oC for 30 minutes to inactivate the patient’s complement. • A known quantity of complement may then be added back to the test system.

  25. We can use this knowledge of antigen/antibody behavior when designing test methods… Basic immunologic methods will be discussed next!

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