Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical...
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![Page 1: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/1.jpg)
Immunologic MethodsPart One
DefinitionsPart Two
Antigen-Antibody Reactions
CLS 420Clinical Immunology and Molecular DiagnosticsKathy Trudell MLS SBB(ASCP)CM
![Page 2: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/2.jpg)
Objectives
• Discuss the following as related to immunology testing:– Sensitivity– Specificity– Cross-reactivity– Screening test– Confirmatory test– Equivalence zone– Postzone– Prozone
![Page 3: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/3.jpg)
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.
![Page 4: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/4.jpg)
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.
![Page 5: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/5.jpg)
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%
![Page 6: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/6.jpg)
Cross Reactivity
• Positive reaction with substance that is structurally similar to analyte.
![Page 7: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/7.jpg)
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
![Page 8: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/8.jpg)
Screen vs. Confirm
• Screening Test: A test used to detect disease.
• Confirmatory Test: Run to validate the results of the initial screening test.
![Page 9: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/9.jpg)
Antigen/ Antibody Reactions
![Page 10: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/10.jpg)
Equilibrium
• Antigen / antibody reactions are readily reversible.
• Free Ag + Free Ab Ag-Ab complex
![Page 11: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/11.jpg)
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.
![Page 12: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/12.jpg)
Cross Reactivity
• For which antigen will the antibody have greater affinity?– The antigen that
stimulated antibody production.
![Page 13: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/13.jpg)
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
![Page 14: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/14.jpg)
YY YY
Y
Y
YY
YYY
YWhich Ag/Ab combo will have the greatest avidity?
![Page 15: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/15.jpg)
Antigen / Antibody Reactions
• May be visualized when lattice structures form.– Sensitization– Agglutination
or precipitation
YY Y
Y
Y
Y
![Page 16: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/16.jpg)
Zone of Equivalence
Y
Y
Y
YY
Y Y
Y
Y
Y
Y
Y
Y
YY
YY
YY
Y
Y
Y
Y
YY
Y
Y
Prozone – antibody excess
Postzone-antigen excess
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Antigen/antibody reactions are influenced by:
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Concentration Ratio of Ag/Ab
Y
Y
Y
YY
Y Y
Y
Y
Y
Y
Y
Y
YY
YY
YY
Y
Y
Y
Y
YY
Y
Y
Prozone – antibody excess
Postzone-antigen excess
Zone of Equivalence
![Page 19: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/19.jpg)
Ionic Strength
• Shielding
• Zeta Potential
Y
-
+
++
+
+
+
-
--
-++
--
--
- -
-
--
-+
+++
+
--
-
-
-
-
-
--
++
++
--
---
![Page 20: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/20.jpg)
Y YYYYYJ
Zeta Potential
![Page 21: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/21.jpg)
Other items that influence Ag/Ab binding:
• pH• Reaction time• Temperature• Number of antigens
![Page 22: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/22.jpg)
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
![Page 23: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/23.jpg)
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
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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.
![Page 25: Immunologic Methods Part One Definitions Part Two Antigen-Antibody Reactions CLS 420 Clinical Immunology and Molecular Diagnostics Kathy Trudell MLS SBB(ASCP)](https://reader035.fdocuments.us/reader035/viewer/2022062421/56649da05503460f94a8c6f6/html5/thumbnails/25.jpg)
We can use this knowledge of antigen/antibody behavior when
designing test methods…
Basic immunologic methods will be discussed next!