immunologic methods part one definitions part two antigen-antibody reactions cls 420 clinical...
TRANSCRIPT
Immunologic MethodsPart One
DefinitionsPart Two
Antigen-Antibody Reactions
CLS 420Clinical Immunology and Molecular DiagnosticsKathy 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
Reference Test # of Patients
Negative Negative 550
Positive Positive 370
Negative Positive 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.
# 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
YY YY
Y
Y
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YYY
YWhich Ag/Ab combo will have the greatest avidity?
Antigen / Antibody Reactions
• May be visualized when lattice structures form.– Sensitization– Agglutination
or precipitation
YY Y
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Y
Zone of Equivalence
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Y Y
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Y
Prozone – antibody excess
Postzone-antigen excess
Antigen/antibody reactions are influenced by:
Concentration Ratio of Ag/Ab
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YY
Y Y
Y
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Prozone – antibody excess
Postzone-antigen excess
Zone of Equivalence
Ionic Strength
• Shielding
• Zeta Potential
Y
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+
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- -
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Y YYYYYJ
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 those buried within the membrane or within the cell.
• Interference in ag/ab binding due to the position of other antigens
YY
YY
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!