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

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 ReferenceTest# of Patients Negative 550 Positive 370 NegativePositive5 Negative2 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. # of patients Test Result Value Cut-off value “Normal” Disease

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

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

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

Zone of Equivalence Y Y Y Y Y YY Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Prozone – antibody excess Postzone- antigen excess

Antigen/antibody reactions are influenced by:

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

Ionic Strength Shielding Zeta Potential Y

Y Y Y Y Y Y J

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 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

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 56 o C 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!