Objectives. Immunologic Methods. Objectives. Immunology vs. Serology. Cross Reactivity. Sensitivity and Specificity. Definitions

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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 (ASCP) CM SBB CM Discuss the following testing: Sensitivity Specificity Cross-reactivity Screening test Confirmatory test Objectives as related to immunology Predictive value (positive and negative) Equivalence zone Postzone Prozone 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. Immunology vs. Serology The study of host reactions to foreign substances. The study of serum; in particular, the study of antibodies in serum and other body fluids. Sensitivity and Specificity Reference Test Interpretation # of Patients Negative Negative True Negative 550 Positive Positive True Positive 370 Negative Positive False Positive 5 Positive Negative False Negative 2 Positive reaction with substance that is structurally similar to analyte. Cross Reactivity Sensitivity = number of true positive tests / total number of patients with disease {true pos false neg} [370 / (3702)] x100 = 99.46% Specificity = number of true negative tests / total number of healthy individuals {true neg false pos} [550 / (5505)] x100 = 99.10% Immunologic Methods Antigen/ Antibody Reactions 1

2 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. # of patients Normal Cut-off value Disease Screen vs. Confirm Screening Test: A test used to detect disease. Confirmatory Test: Run to validate the results of the initial screening test. Test Result Value Equilibrium Antigen/ Antibody Reactions Antigen / antibody reactions are readily reversible. Free Ag Free Ab Ag-Ab complex 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. For which antigen will the antibody have greater affinity? The antigen that stimulated antibody production. Cross Reactivity Immunologic Methods Antigen/ Antibody Reactions 2

3 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 Which Ag/Ab combo will have the greatest avidity? Antigen / Antibody Reactions Zone of Equivalence May be visualized when lattice structures form. Sensitization Agglutination or precipitation Prozone antibody excess Postzoneantigen excess Concentration Ratio of Ag/Ab Antigen/antibody reactions are influenced by: Prozone antibody excess Zone of Equivalence Postzoneantigen excess Immunologic Methods Antigen/ Antibody Reactions 3

4 Shielding Zeta Potential Ionic Strength Zeta Potential Other items that influence Ag/Ab binding: ph Reaction time Temperature Number of antigens 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 Location of Antigens Free antigens and cell surface antigens are more available to react than antigens buried within the membrane or within the cell. Interference in ag/ab binding due to the position of other antigens Heat Inactivation Complement 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 56 o C for 30 minutes to inactivate the patient s complement. A known quantity of complement may be added back to the test system. Immunologic Methods Antigen/ Antibody Reactions 4

5 We can use this knowledge of antigen/antibody behavior when designing test methods Basic immunologic methods will be discussed next! Immunologic Methods Antigen/ Antibody Reactions 5

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