Immuno Diagnosis

IMMUNODIAGNOSIS
Johnson Welishe
Outline
 Serological tests
 ELISA assays
 Flow Cytometry
 Rapid Diagnostic Tests
Objectives
 Explain the basis of immunodiagnostic laboratory
procedures
 Briefly discuss clinically important immunodiagnostic tests

Critical Definitions
 Phlebotomy-Is the act of drawing or removing blood from
the circulatory system in order to obtain a sample for
analysis and diagnosis
 Turn around time (TAT)- Refers to the time between

ordering a test or submitting a specimen to a lab and
reporting results
 Sensitivity of a clinical test- Refers to the ability of a

test to correctly identify those patients with the disease
 Specificity of a clinical test- Refers to the ability of a

test to correctly identify those patients without the disease
Serological tests
 Serology is the scientific study of serum and other bodily fluids e.g.
serum and saliva
 Serum is obtained thru centrifugation which separates blood into different

compartments i.e. Serum, Buffy coat (WBC’s), packed cells (RBC’s)
 These are blood tests that look for antigen in your blood and focus on
proteins made by your immune system (antibodies)
Types of Serological tests
Agglutination tests
 Agglutination is the visible expression of the aggregation of Antigen & Antibody

reactions
 Agglutination tests are easy to perform and have a wide range of applications in
the clinical diagnosis of non-infectious immune disorders and infectious disease
Principle
 When the particle Ag’s interact with the appropriate Ab, they clump
together and eventually form masses that become large enough to be seen
Antigen antibody reaction when antigen is in
particulate form e.g. bacterial cell, antigen coupled
with latex beads
It is simple, inexpensive & sensitive
Slide Agglutination tests

 Many bacterial isolate identification
 Blood grouping and cross matching
Tube test
 Serological diagnosis of typhoid, Brucella, typhus,
atypical pneumonia
Types of agglutination tests
Latex agglutination
 Is a clinical method to detect presence of Antigens or Antibodies in a variety of

bodily fluids such as Saliva, Urine, CSF or blood
 Here sample is mixed with latex beads coated with specific Antigen or Antibody
 If suspected substance is present, latex beads will clump together (agglutinate)

Examples of Latex agglutination tests
Rapid Plasma Reagin test (RPR)
 Applied in screening for Syphilis infection
Principle
 A Cardiolopin lecithin-cholesterol antigen coated with carbon
 Particle is mixed with patient’s serum. Then flocculation reaction is

observed macroscopically in the presence of Cardiolipin Ag.
Hemagglutination test
 Type of Agglutination involving Red Blood Cells
 Applications of Hemagglutination include blood typing
Blood typing
 Can be determined by using antibodies that bind to the A
or B blood group antigens in a blood sample
 If antibodies that bind blood grp A are added and
agglutination occurs, the blood is either type A or AB
 To determine btn type A or type AB, antibodies that bind
the B group are added and if agglutination does not occur,
the blood is type A
 If agglutination does not occur with either antibodies that
bind to Type A or B antigens, then neither antigen is
present on blood cells, meaning its group O
Transfuse No Transfusion
ELISA
 ELISA (enzyme-linked immunosorbent assay) is a plate-based assay
technique designed for detecting and quantifying substances such as
peptides, proteins, antibodies and hormones.
 Provides useful measurement of Antigen or Antibody concentration
 Can be used to detect presence of antigens that are recognized by an

antibody or test for antibody that recognize antigen
Steps
 Coat microtiter plate wells with antigen
 Block all unbound sites to prevent false positive results (non specific
binding)
 Add primary antibody e.g. rabbit monoclonal antibody
 Add secondary antibody conjugated to an enzyme e.g. anti-mouse IgG
 Reaction of substrate with enzyme to produce a coloured product thus

indicating a positive reaction
 Read optical densities (OD’s) of microtiter wells from ELISA reader

Advantages of ELISA
The advantages of ELISA include
 specificity, sensitivity, rapidity, inexpensiveness, and safety.
 Enzyme: horseradish peroxidase, HRP
 Substrates:
diaminobenzidine (DAB)
3,3’,5,5’-tetramethylbenzidine (TMB)
Flow Cytometry
Flow cytometry is a technology that simultaneously measures and then

analyses multiple physical characteristics of single particles, usually cells, as
they flow in a fluid stream through a beam of light.
The properties measured include a particle’s:
-Relative size
-Relative granularity or internal complexity
-Relative fluorescence intensity

These characteristics are determined using an optical-to-electronic coupling system that
records how the cell or particle scatters incident laser light and emits fluorescence.
A flow cytometer is made up of three main systems:
-The fluidics system transports particles in a stream to the laser beam for
interrogation.
-The optics system consists of lasers to illuminate the particles in the sample stream &
optical filters to direct the resulting light signals to the appropriate detectors.
-The electronics system converts the detected light signals into electronic signals
that can be processed by the computer
Light Scatter
Occurs when a particle deflects incident laser light.
The extent to which this occurs depends on the physical properties of a

particle i.e. size & internal complexity.
Factors that affect light scattering are:
-The cell's membrane
-Nucleus
-Any granular material inside the cell
-Cell shape and surface topography

Forward Scatter
 Forward scattered light (FSC) is proportional to cell-surface area or size.
 FSC is a measurement of mostly diffracted light and is detected just off the
axis of the incident laser beam in the forward direction by a photodiode
Side scatter
 Side-scattered light (SSC) is proportional to cell granularity or internal

complexity.
 SSC is a measurement of mostly refracted and reflected light that occurs

at any interface within the cell where there is a change in refractive index
 Correlated measurements of FSC and SSC can allow for differentiation of
cell types in a heterogeneous cell population.
 Major leucocyte subpopulations can be differentiated using FSC and SSC
Gating
 A gate or a region is a boundary drawn around a subpopulation to isolate
events for analysis or sorting.
 Data for events within this gate can then be displayed in subsequent
plots
Dot plots
Rapid Diagnostic Tests
 Also called Immunochromatographic assays, or simply strip tests, They

are a logical extension of the technology used in latex agglutination tests.
The benefits include:
 User-friendly platform (no need for technical expertise or

instrumentation)
 Long-term stability over a wide range of climates.
 Rapid point of care (POC) testing
 Allows for mass screening

Application
 Sample is added to the proximal end of the strip, the sample pad
 The sample migrates thru this region to the conjugate pad, where a
particulate conjugate has been immobilized.
 This particle has been conjugated to one of the specific biological components
of the assay, either antigen or antibody depending on the assay format .
 The sample re-mobilizes the dried conjugate, and the analyte in the sample
interacts with the conjugate as both migrate into the next section of the strip,
which is the reaction matrix.
 This reaction matrix is a porous membrane, onto which the other specific
biological component of the assay has been immobilized.
 These are typically proteins, either antibody or antigen, which have been laid
down in bands in specific areas of the membrane where they serve to
capture the analyte and the conjugate as they migrate by the capture lines.
 If antigen is present, some labelled antibody-antigen complex will be trapped
and accumulate on the test line.
 Excess-labelled antibody is trapped and accumulates on the control line. A
visible control line indicates that labelled antibody has traversed the full
length of the strip, past the test line, and that at least some free antibody
remains conjugated to the dye and that some of the capturing properties of
the antibodies remain intact.
References
 Comlex Immunology and Microbiology. Kaplan Medical
 Cambridge Essential Clinical Immunology. Jan. 2009

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IMMUNODIAGNOSIS

 Briefly discuss clinically important immunodiagnostic tests

 Turn around time (TAT)- Refers to the time between

 Sensitivity of a clinical test- Refers to the ability of a

 Specificity of a clinical test- Refers to the ability of a

 Serum is obtained thru centrifugation which separates blood into different

 Agglutination is the visible expression of the aggregation of Antigen & Antibody

Slide Agglutination tests

 Is a clinical method to detect presence of Antigens or Antibodies in a variety of

 If suspected substance is present, latex beads will clump together (agglutinate)

 Applied in screening for Syphilis infection

 A Cardiolopin lecithin-cholesterol antigen coated with carbon

 Particle is mixed with patient’s serum. Then flocculation reaction is

 Provides useful measurement of Antigen or Antibody concentration

 Can be used to detect presence of antigens that are recognized by an

 Add primary antibody e.g. rabbit monoclonal antibody

 Add secondary antibody conjugated to an enzyme e.g. anti-mouse IgG

 Reaction of substrate with enzyme to produce a coloured product thus

 Read optical densities (OD’s) of microtiter wells from ELISA reader

 Enzyme: horseradish peroxidase, HRP

Flow cytometry is a technology that simultaneously measures and then

The properties measured include a particle’s:

-Relative granularity or internal complexity

-Relative fluorescence intensity

A flow cytometer is made up of three main systems:

Occurs when a particle deflects incident laser light.

The extent to which this occurs depends on the physical properties of a

Factors that affect light scattering are:

-The cell's membrane

-Any granular material inside the cell

-Cell shape and surface topography

 Forward scattered light (FSC) is proportional to cell-surface area or size.

 Side-scattered light (SSC) is proportional to cell granularity or internal

 SSC is a measurement of mostly refracted and reflected light that occurs

 Also called Immunochromatographic assays, or simply strip tests, They

The benefits include:

 User-friendly platform (no need for technical expertise or

 Long-term stability over a wide range of climates.

 Rapid point of care (POC) testing

 Allows for mass screening

 Cambridge Essential Clinical Immunology. Jan. 2009

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