Labelled Immunoassays Complement Neutralization Molecular Methods

MT309 Lec Immunology and
Serology
Antigen-Antibody Reactions
Prepared by: Nhoelyn E. Burcao RMT, MSMT© Prepared by: Nhoelyn E. Burcao RMT, MSMT©
Objectives:
At the end of the discussion, students are able to:
1. Explain the basic principles of immunoflourescence,
radioimmunoassay, enzyme immunoassay and
chemiluminescence
2. Describe the role of indicator labels in immunoassays.
Levels of Ab-Ag interactions
Primary Reactions Secondary Reactions

• Immunofluorescence • Precipitation
• Radioimmunoassay • Agglutination
• Enzyme immunoassay • Flocculation
• Chemiluminescense • Neutralization
• Complement Fixation
Immunochromatography
T C
Sample Well
Y
YY Test Control
YYYYYY
U GCU U GC U
G
C
U GC
G G
C C
G
C
G C
C
Y YY
U
U
U GC
U
U
G
T C
Sample Well Test Control
YYYYYY
UC GC
G U
G
C
U
U
G
C
U
U
G
G
C
Neutralization
Tests
NEUTRALIZATION REACTIONS
• Principle: An antibody neutralizes toxin. After neutralization

antibody is not available to react in indicator system.
• Results:
• POSITIVE REACTION: NO agglutination or NO hemolysis
• NEGATIVE REACTION: Agglutination or hemolysis ( antibody not bound
in origin)
• Generally, positive control samples used should show no
reaction and negative control samples show a reaction.
• Example: Antistreptolysin O test (ASO)
INHIBITION or NEUTRALIZATION REACTIONS
• Results
• NO agglutination or NO hemolysis = positive reaction
• Agglutination or hemolysis = negative reaction (antibody not bound in
original reaction and is available to react with indicator cells)
• Examples
• Inhibition : hemagglutination inhibition test for rubella
• Neutralization : antistreptolysin O test (ASO)
ASO test
• E.W. Todd
• Serum (strep Ab) + reagent (strep Ag) +indicator cells →no hemolysis
• No hemolysis : (+) strep Ab : previous strep infection
• Serum (NO strep Ab) + reagent (strep Ag) + indicator cells →

hemolysis
• Hemolysis : (-) strep Ab : No previous strep infection
PSGN : Post Streptococcal Glomerulonephritis
• Strep infection : skin or throat -→ sequalae : PSGN
• Kidney malfunction
• Document previous strep infection
• ASO titers
• Significant titer : ≥ 166 Todd units
• Schick test: skin test used to detect immunity to diphteria
• Dick test: skin test to detect immunity to scarlet fever
• Animal protection test: mixture of toxin and serum →
laboratory animal → toxin neutralize if animal will show no
sign of illness
Complement
Fixation
Serum with Ab Serum without Ab
+ Ag
+ Complement
Ag-Ab-Complement Ag-Complement
+ RBC-Ab
(indicator)
No Complement Available Complement Available

No Hemolysis Hemolysis
POSITIVE RESULT NEGATIVE RESULT
Y
YAntibody Y Y Y Y
(Sample)
+
Ag – Ab – Complement Ag – Ab – Complement
→ Complex → Complex
Antigen
(Reagent) + Y Y
+ Y Y
RBC
(indicator)
Complement RBC UNBOUND
(indicator)
POSITIVE Result: No Hemolysis
X
Antibody
(Sample)
Y Y
+
Free Complement
→ → RBC – Complement
Antigen
(Reagent) + Complex
+ Y Y
Complement RBC
(indicator)
Negative Result: Hemolysis
LABELLED
IMMUNOASSAYS
METHODS
Labelled Immunoassays
• Used to detect or demonstrate antigen-antibody complexes
• Designed for antigens and antibodies that may be small in
size or present in very low concentration
• The presence of antigens or antibodies is determined
indirectly by using a labelled reactant to detect whether or
not specific binding has taken place
• Constituents
1. Labelled analytes
1. RIA : gamma emitting isotope; beta emitting isotope
2. EIA : horseradish peroxidase; alkaline phosphatase
3. FIA : fluorescein; rhodamine
2. Monoclonal Antibodies
3. Standards or calibrators
• Label detection
• RIA : scintillation counter
• EIA : spectrophotometer
• FIA : spectrofluorometer; fluorometer; fluorescence
microscope
RADIOIMMUNOASSAY
• 1960 : Berson and Yalow

• Rapid, sensitive
• Detects small amounts of Ag and Ab
• Disadvantage: radiation sickness, increased incidence of
neoplasm
RADIOIMMUNOASSAY
Competitive binding assay
• Patient antigen and labelled antigen are incubated with
known amount of specific antibody (unlabelled and labelled
antigen compete for binding with antibody)
• Wash to remove unbound antigen
• Radioactivity counted on a gamma counter; compare to
standard curve
• The lower the radioactive count, the higher the
concentration of unlabelled antigen
RADIOIMMUNOASSAY
Spectrofluorometer
Application of RIA
• Examples :
1. tests for hepatitis antigens and antibodies
2. Radioimmunosorbent Test (RIST) measures
total IgE concentration
3. Radioallergosorbent Test (RAST) measures IgE
to specific allergens
ENZYME IMMUNOASSAY
• Monoclonal or polyclonal antibody adsorbed on solid surface
(bead or microtiter plate)
• Add patient serum; if antigen is present in serum it binds to
antibody coated bead or plate
• Add excess labelled antibody; forms antigen-antibody-labelled
antibody “sandwich”
• Add substrate, incubate and read absorbance
• Washing required between each step
• Absorbance is DIRECTLY proportional to antigen concentration
ENZYME IMMUNOASSAY
• Application of EIA
1. HIV testing
2. Serum HCG (pregnancy)
3. tests for hepatitis antigens and antibodies
4. antibodies to bacteria
Indirect ELISA
Direct ELISA
Competitive ELISA
Solid Phase Non-Competitive RIA/ELISA
• Ab detection
• Immobilize Ag Ab in
Labeled
• Incubate with sample Patient’s
Anti-Ig
• Add labeled anti-Ig sample
• Amount of labeled Ab
Immobilized Ag
bound is proportional to
amount of Ab in the sample Solid
Phase
• Quantitative
Solid Phase Non-Competitive RIA/ELISA
• Ag detection Labeled
• Immobilize Ab Ag in
Ab
Patient’s
• Incubate with sample sample
• Add labeled antibody Ag
• Amount of labeled Ab bound is Immobilized
proportional to the amount of Solid
Ag in the sample Phase
ENZYME MULTIPLIED IMMUNOASSAY (EMIT)
• Used to measure concentrations of small molecules such as drugs
and hormones
• Principle :
• Add patient serum to an enzyme-drug conjugate; also add anti-
drug antibody
• Add enzyme substrate and incubate
• Positive test : color formation
• Drug in patient’s serum combined with anti-drug antibody
• Sites on the enzyme portion of the conjugate remain
available to bind with substrate --- color produced
ENZYME MULTIPLIED IMMUNOASSAY (EMIT)
• Negative test : NO color formation
• Anti-drug antibody attached to the drug in the enzyme-
drug conjugate and blocks the active sites on the enzyme;
substrate unable to bind to enzyme
• No color produced
Enzyme-multiplied immunoassay technique (EMIT) system diagram
Fluorescence Immunoassay
• 1941 : Albert Coons : showed that antibodies could be
labelled with molecules that have the property of
fluorescence
• Antibody molecules tagged with a fluorescent dye when

they form immune complexes , emits a light detectable with
the use of a fluorescence microscope
IMMUNOFLUORESCENCE
Direct
• Add fluorescein-labelled antibody to patient tissue, wash
and examine under fluorescent microscope
Indirect
• Add patient serum to tissue containing known antigen,
wash, add labelled antiglobulin, wash and examine under
fluorescent microscope
Example
• Testing for antinuclear antibodies (ANA)
• Fluorescent Treponemal Antibody (FTA-Abs)
IMMUNOFLUORESCENCE
• Qualitative to Semi-
qualitative
Fluorochrome
• Direct Labeled Ab
• Add fluorescein-labelled
antibody to patient
tissue, wash and examine
under fluorescent
microscope Ag
• Ab to tissue Ag is labeled Tissue Section
with fluorochrome
IMMUNOFLUORESCENCE
• Indirect
• Add patient serum to tissue
containing known antigen, wash, Fluorochrome
add labelled antiglobulin, wash Unlabeled Labeled Anti-Ig
and examine under fluorescent Ab
microscope
• Ab to tissue Ag is unlabeled Ag
Tissue Section
• Fluorochrome-labeled anti-Ig is
used to detect binding of the first
Ab.
The vimentin + cells in
the epithelial layer are
immune cells (CD45 + ).
Immunofluorescence
staining of high
mammographic density
(HMD) samples with 4 ′
,6-diamidino-2-
phenylindole ( a ),
vimentin ( b ) and pan-
CD45 immune cell
marker ( c ) are shown as
well as the overlay of all
stain ( d ). ( e - g ) High-
power images of the
selected region in (D)
From: https://www.researchgate.net/figure/277722373_fig8_Fig-8-The-vimentin-cells-in-the-epithelial-layer-are-
immune-cells-CD45
Application of IFA
• Testing for antinuclear antibodies (ANA)
• Fluorescent Treponemal Antibody (FTA-Abs)
FLUORESCENCE POLARIZATION IMMUNOASSAY (FPIA)
• Principle:
• Add reagent antibody and fluorescent tagged antigen to
patient serum
• Positive test
• Antigen present in patient serum binds to reagent
leaving most tagged antigen unbound
• Unbound labelled antigens rotate quickly reducing
amount of polarized light produced
FLUORESCENCE POLARIZATION IMMUNOASSAY (FPIA)
• Negative test
• If no antigen is present in patient serum, tagged antigen
binds to reagent antibody
• Tagged antigen-antibody complexes rotate slowly giving
off increased polarized light
CHEMILUMINESCENCE
• Emission of light caused by chemical reaction producing an excited
molecule that decays back to the original ground state measured
using illuminometer
• Chemiluminescent labels can be attached to an antigen or antibody
• Acridinium esters
• Chemiluminescent substance→ oxidation using hydrogen peroxide or
enzyme→ produces intermediates at higher energy
• The intermediates spontaneously return to their original state→
giving energy in the form of light
FLOW CYTOMETRY
• Principle
• Forward light scatter indicates cell size or volume
• 90’C side scattered light indicates granularity
• Common uses :
• DNA analysis
• Reticulocyte counts
• Leukemia/lymphoma classification
FLOW CYTOMETRY
• Method of choice for T and B cell analysis
• Principle
• Incubate specimen with one or two monoclonal antibodies
tagged with fluorochrome
• Single cells pass through incident light of instrument (laser)
which excites fluorochrome and results in emitted light of
different wavelengths
• Intensity of fluorescence measured to detect cells possessing
surface markers for the specific monoclonal antibodies that
were employed
Flow Cytometry
Molecular Methods
WESTERN BLOT
WESTERN
BLOT
WESTERN BLOT
WESTERN BLOT
WESTERN BLOT
WESTERN BLOT
WESTERN
BLOT
WESTERN BLOT
WESTERN BLOT
SOUTHERN BLOT
POLYMERASE CHAIN REACTION
EVALUATION OF TEST METHODOLOGIES
SENSITIVITY
• Analytical sensitivity – ability of a test to detect very small
amounts of a substance
• Clinical sensitivity – ability of a test to give positive result
if patient has the disease
SPECIFICITY
• Analytical specificity – ability of test to detect substance
without interference from cross reacting substances
• Clinical specificity – ability of a test to give negative result
if patient does not have disease

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Labelled Immunoassays Complement Neutralization Molecular Methods

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MT309 Lec Immunology and

Primary Reactions Secondary Reactions

• Principle: An antibody neutralizes toxin. After neutralization

• Serum (NO strep Ab) + reagent (strep Ag) + indicator cells →

No Complement Available Complement Available

• 1960 : Berson and Yalow

• Antibody molecules tagged with a fluorescent dye when

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