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 Cross-reactivity Screening test Confirmatory test 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 # of Patients Negative Positive Negative Negative Positive Positive 550 370 5 Positive Negative 2 •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% Cross Reactivity • Positive reaction with substance that is structurally similar to analyte. 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 # of patients “Normal” Disease Test Result Value Screen vs. Confirm • • Screening Test: A test used to detect disease. Confirmatory Test: Run to validate the results of the initial screening test. Antigen/ Antibody Reactions Equilibrium • 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. Cross Reactivity • For which antigen will the antibody have greater affinity? – The antigen that stimulated antibody production. 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 • May be visualized when lattice structures form. Y – Sensitization – Agglutination or precipitation Zone of Equivalence Y Y Y Y Y Y Prozone – antibody excess Y Postzoneantigen excess Antigen/antibody reactions are influenced by: Concentration Ratio of Ag/Ab Y Y Y Y Y Y Prozone – Zone of antibody excess Equivalence Y Postzoneantigen excess Ionic Strength • Shielding - +- + + -+- - - + + -+-+ - +-+- + - - + - + • Zeta Potential Y Zeta Potential Y 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 Y Y • 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 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. We can use this knowledge of antigen/antibody behavior when designing test methods… Basic immunologic methods will be discussed next!