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!