Immunological

Test Methods

Fang ZHENG MD. PhD.

郑芳
Prof. Clinical Immunology
Tianjin Medical University
Nature of Ag-Ab Reactions
Immunologic test methods
 Nature of Ag-Ab Reactions
Concept:
Specific binding reaction
between antigen and antibody
Molecular Basis:
Complementary binding of the
antigenic epitope and antibody
hypervariable regions
Nature of Ag-Ab Reactions
 Characteristics of Ag-Ab reaction

• Specificity
• Non-covalent Bonds

• Multiple Bonds
• Reversible
Nature of Ag-Ab Reactions
• Specificity
 Specificity refers to the ability of an
individual antibody combining site to react with
only one antigenic determinant, or the ability of a
population of antibody molecules to react with
only one antigen.
 In general, there is a high degree of specificity
in Ag-Ab reactions.
Nature of Ag-Ab Reactions

• Non-covalent Bonds
– Hydrogen bonds
– Electrostatic bonds
– Van der Waal forces
– Hydrophobic bonds
Nature of Ag-Ab Reactions

 Most immunologic tests use

combination of the techniques to evaluate
humoral and cell-mediated immune
responses or their individual components.
All tests are based on Ag-Ab reactions
Immunologic test methods

1. Precipitation Reactions
2. Immunoelectro-diffusion
3. Agglutination Reaction
4. Radioimmunoassay
5. Immunofluorescence
6. Enzyme-Linked Immunosorbent Assay
7. Complement fixation
1.Precipitation
 Concept:
Refers to the precipitation phenomena
of soluble antigen and the
corresponding antibody specific
binding occurs under appropriate
conditions

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 1.Precipitation
 When a soluble antibody reacts with a
soluble antigen, cross-linking occurs between
the antibody and the antigen-- Lattice
formation.

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 1.Precipitation
 Lattice formation : presence of multiple epitopes
on the surface of Ab and Ag– cross-linkage

( Lattices or ( no precipitate if an Ag
large aggregates ) contains only a single copy of
each epitope ) 11
1.Precipitation

A. Liquid Phase Precipitation

B. Gel Phase Precipitation

 Single diffusion test

 Double gel diffusion test

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1.Precipitation

A. Liquid Phase Precipitation

 Involve soluble
antigens with
antibodies.
 soluble Ags and Abs to
precipitate if found in
equivalent amounts.

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Introduction
絮 状 沉 淀 示 意 图 Ag diluted multi-
proportion/tube

Ag + serum
1:4 1:8 1:16 1:32 1:64 1:128
Identical
amount of Ab

Ab
Shaking
Ag 37℃

Precipitation

Jog

optimal proportion
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1.Precipitation
Antigen and
antibody visible
reaction than
need to follow
certain
relationship

Precipitation
reactions in fluids
yield a precipitin
curve.

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1. Precipitation
B. Gel Phase Precipitation
 It relies on the tendency of Ag and Ab to
diffuse in agar matrix, and to form a precipitin
line where they meet.

Single diffusion (radial immunodiffusion)

Double gel diffusion

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 1. Precipitation
• Antibody is
1)Single diffusion incorporated into the
agar gel as it is
 Method poured, and different
– Ab in gel, Ag in hole, 24-48h dilutions of the
antigen are placed in
holes punched into
the agar.
• As the antigen
diffuses into the gel it
reacts with the
antibody, and when
the equivalence point
is reached a ring of
precipitation is
formed
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 1. Precipitation
1)Single diffusion
Significance
 Diameter of ring is proportional Ab in
to the concentration of Ag gel
Ag Ag Ag Ag
 By running different
concentrations of a standard
antigen, one can generate a
standard cure from which one

Diameter2
can quantitate the amount of an
antigen in an unknown sample.
Thus, this is a quantitative test.
 i.e. to measure the Ab (Ig) levels
Ag Concentration
in the serum.
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 1.Precipitation
2) Double gel diffusion

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 1. Precipitation
2) Double gel diffusion

Ag, Ab double diffuse freely

Precipitation line or arc formation
18--24hrs --results

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 1. Precipitation
2) Double gel diffusion
 One of the most often used method for detecting
unknown Ags or Abs, simple and useful
 Formation of a single precipitin line provides a
rough quantitative estimate of Ag or Ab.
 It is not sensitive.

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2.Immunoelectrophoresis
 Incorporated electrophoresis with
double diffusion to identify and
measure serum immuno-globulins
and other proteins.

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2.Immunoelectrophoresis

+ -  A mixture of Ags are placed in

Ag
an agar gel. The Ags are
electrophoresed so that they are
separated according to their
charge.
Ag  After electrophoresis, a trough
Ab
is cut in the gel and Abs are
added.
 As the Abs diffuse into the agar,
Ag precipitin lines are produced in
the equivalence zone when an
Ab
Ag/Ab reaction occurs

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2.Immunoelectrophoresis

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2.Immunoelectrophoresis
Clinical application
 This test is used for the qualitative analysis
of complex mixtures of Ags,
eg, the analysis of components in a patient'
serum.
eg, the presence/absence of specific proteins or Ig
classes.
This test can also be used to evaluate purity
of isolated serum proteins.
 Diagnosis of immunodeficiency or immuno-
proliferative disorders 25
3. Agglutination
 Agglutination occurs when large, insoluble Ag
particles, or Ags attached to the particles are clumped
together by Abs.
 Visible clumping by interaction between Abs and
particulate Ags (RBC, latex particles).
 Agglutinin is used to describe Abs that agglutinate
particulate antigens.
 All Abs can theoretically agglutinate particulate Ags,
depending on the cross-linking of polyvalent Ags,
similar to precipitation.
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3. Agglutination

 types :
a) Direct
b) Indirect or passive

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3. Agglutination

 Direct : antibody is against to a cell or

an insoluble particulate native antigen
 Indirect or passive : soluble antigen
attached to blood cells, bacteria, or latex
particles, which are inert carrier particles.

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3. Agglutination
 Involve particulate
antigens and
antibodies.
 Antigens
On a cell (direct).
Attached to latex
spheres (indirect or
passive).

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3. Agglutination
Application
 To detect specific Abs, such as Abs in rheumatoid
arthritis
To assess bacterial infections: i.e. salmonella
infections
To classify the type of RBCs for blood transfusion
To determine the use of illegal drugs

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4.Radioimmunoassay (RIA)

 Radioimmunoassays (RIA) are assays which

are based on the measurement of
radioactivity associated with immune
complexes. In any particular test, the label
may be on either the antigen or the antibody.
 First discovered by Dr. Berson & Yalow in
1960, (1977 Noble prize to Yalow)

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4.Radioimmunoassay (RIA)
 The principle involves competitive binding of
radio-labeled Ag and unlabeled Ag to the
limited supply of a high affinity Ab.
 Commonly used marker radionuclide: 125I, 131I,
3H, 32P

 To determine the amount of Ag in a serum

sample.
 It is One of the most sensitive techniques:
measuring at <0.001 ㎍/ml

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 4.Radioimmunoassay (RIA)
 Principle

Labeled antigen ( Ag * ) and

Unlabeled antigen (Ag) and limit
amount of antibody (Ab)
competitive binding
Ag * and Ag have equivalent
Ability to bind with Ab

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4.Radioimmunoassay (RIA)

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4.Radioimmunoassay (RIA)
 Radiolabeled Ag is
added to sample, and it
binds to the Ab;
Various amounts of
unlabeled Ag are then
added to the mixture;
 competition between
radiolabeled &
unlabeled Ags for
binding sites on Ab
A standard curve is
made
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 4.Radioimmunoassay (RIA)

A RIA to detect hepatitis B virus in blood samples

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4.Radioimmunoassay (RIA)

A standard curve to determine the concentrations

of HBsAg in unknown serum. 37
5. Immunofluorescence

 A histochemical technique
for the identification of Ags
and Abs on fixed tissue and
cells

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5. Immunofluorescence
 A histochemical technique
Abs are conjugated with a fluorescent
dye (fluorescein, phycoerythrin (PE),
FITC)
If Abs bind to specific Ag’s, they can be
illuminated with UV light, and emit
bright colors

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5. Immunofluorescence

 Direct Immunofluorescence

 Indirect Immunofluorescence

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5. Immuno fluorescence
 Direct
Ab (against tissue Ag) is labeled with
fluorochrome
Fluorochrome
Labeled Ab

Ag
Tissue Section

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5. Immuno fluorescence
Indirect
Ab against tissue Ag is
Fluorochrome
unlabeled Labeled Anti-Ig
Unlabeled
Fluorochrome-labeled Ab

anti-Ig is (2nd Ab) used

Ag
to detect binding of the Tissue Section

first Ab.

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5. Immuno fluorescence

B cells indirectly stained with rhodamine-conjugated

2nd Ab under a fluorescence microscope.
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5. Immunofluorescence

Specificity (Ag-Ab reaction),

Sensitivity (precise detection and
demonstration of the tissue
antigens )
Direct-viewing (fluorescence dye)

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6. Enzyme-Linked Immunosorbent Assay

 Enzyme Linked Immunosorbent assays

(ELISA) :
based on the measurement of an enzymatic
reaction associated with immune complexes.
 In clinical laboratory ELISA is the most
commonly used method to measure serum
proteins, hormones, drugs metabolites,
quantitatively

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6. Enzyme-Linked Immunosorbent Assay

 In any particular assay the enzyme may

be linked to either the antigen or the
antibody.
 ELISA tests depend on enzyme
conjugated to a secondary Ab catalyzing a
specific substrate to produce a color
reaction.

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6. Enzyme-Linked Immunosorbent Assay

 Catalysis of the substrate produces a color

change, quantified by a spectrophotometer.
 The amount of substrate catalyzed is directly
proportional to the amount of Ag/Ab in the
serum sample.
 a standard curve based on known [C]’s of
Ag/Ab can be prepared and an unknown [C}
can be detected

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6. Enzyme-Linked Immunosorbent Assay

 Direct ELISA
– Ab to tissue Ag is labeled with Enzyme

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6. Enzyme-Linked Immunosorbent Assay

 Direct ELISA

 Sandwich ELISA
Use to test Ag

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6. Enzyme-Linked Immunosorbent Assay

Indirect ELISA
Use to test Ab

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6. Enzyme-Linked Immunosorbent Assay

 Sensitive
 Reproducible
 qualitative or quantitative
 Safe
 Simple

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7. Complement Fixation Test

 Complement activation
 Used to determine the presence and
extent of Ag-Ab reaction.

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7. Complement Fixation Test

Indicator system hemolysis

 If hemolysis
doesn’t occur, C
- must have been
depleted in the
original reaction;
Complement i.e. the unknown
+ antibody was
present in the
sample.
 The unknown
antibody is
assayed.
RBC
hemolysin 53
Sensitivity of various immunoassays

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 Thank you
for your attention !