Immunological laboratory
investigation
SEROLOGICAL REACTIONS
Course no. 1
BASIC DIVISION OF IMMUNOLOGICAL
LABORATORY INVESTIGATION
• Serological investigation
– material for investigation
• SERUM or PLASMA
• Cellular investigation
– material for investigation
• PERIPHERAL VENOUS BLOOD
• Other material for immunological investigation
– Cerebrospinal fluid, lymph nodes, organ biopsy material,
bone marrow, bronchoalveolar lavage fluid
SERUM AND PLASMA DEFINITION
SERUM
clear, yellowish fluid that remains after blood has been
allowed to clot and the blood cells (including red blood cells,
white blood cells, and platelets) have been removed
PLASMA
liquid component of blood, making up about 55% of total
blood volume and contains clotting factors (fibrinogen and
other proteins) that are essential for blood clotting
serum is essentially plasma without clotting factors
ANTIGEN – ANTIBODY REACTIONS
IN VITRO
• EPITOP (determinant)
o the specific portion of the macromolecule (antigen), to
which an antibody binds
• PARATOP
o the specific portion of antibody binding site (area of Nterminal
part of variable part of light and heavy chains)
• AFINITY
o the strength of the binding between a single binding site
of the molecule (antibody) and a ligand (antigen)
• AVIDITY
o The overall strength of interaction between two
molecules such as an antibody and antigen
PRIMARY AND SECONDARY PHASE OF
SEROLOGICAL REACTIONS
Primary phase of serological reaction
• specific phase of the reaction, if specific antibody
binds to specific antigen
• It is not visible!
Secondary phase of serological reaction
• visualization of the fact of previously occurred
primary reaction
… resulting complexes are …
• visible
(AGGLUTINATION, PRECIPITATION)
• change of fluid character to colloidal
solution
(TURBIDIMETRY, NEPHELOMETRY)
_________________________________________
• the course of the reaction enable only primary phase of
reaction or only incomplete secondary phase and it is
necessary to visualize the reaction by following
imunohistochemical detection (IMMUNOASSAYS)
PRIMARY AND SECONDARY PHASE OF
SEROLOGICAL REACTIONS
ANTIGLOBULIN ANTIBODIES
polyclonal antisera
• obtained from animals (rabbits, goats, horses) by repeated
immunization by antigen
• markedly polyreactive, because antibody binds to many
epitopes of the antigen but also with other antigens
This is advantageous in „classical“ serological reactions
(agglutination, precipitation)
Examples of secondary antisera:
• RaHuIgG (rabbit anti-human IgG) reacts with human IgG of
various specificities (anti-Rh, anti-microbial antigens)
Clonal selection theory
SENSITIVITY OF THE METHODS
FOR DETECTION OF ANTIBODIES
precipitation
30 g/ml
agglutination
1 g/ml
radioimunoassay and ELISA
1 pg/ml
INTERPRETATION OF LABORATORY
TESTS
• SPECIFICITY
o measures the proportion of negatives that are correctly
identified as such (e.g. the percentage of healthy people
who are correctly identified as not having the condition)
o TRUE NEGATIVE RATE
• SENSITIVITY
o measures the proportion of positives that are correctly
identified as such (e.g. the percentage of sick people
who are correctly identified as having the condition)
o TRUE POSITIVE RATE
POLYCLONAL AND MONOCLONAL
ANTIBODIES
• POLYCLONAL ANTIBODIES
o collection of immunoglobulin molecules that react
against a specific antigen, each identifying a different
epitope
o secreted by different B cell lineage within the body
o OBTAINED BY IMMUNIZATION OF ANIMALS
• MONOCLONAL ANTIBODIES
o Product of a single B lymphocyte with monovalent
affinity, in that they bind to the same epitope
o secreted by a single cell lineage
o OBTAINED BY IN VITRO METHODS
MONOCLONAL ANTIBODIES
• PREPARATION
• prepared by immortalization of B-cells from immunized
mouse
• hybridoma is composed of an antigen-specific B cell and
mouse myeloma cell
• produced antibodies are strictly monospecific and therefore
cannot be used in several „classical“ serological reactions
(agglutination, precipitation)
highly specific agent used for ELISAs, RIAs,
determination of cells surface antigens
Because they react only with a single epitope,
number of „bridges“ is to low to overcome repulsive
forces in classical reactions like agglutination or
precipitation.
LABORATORY USE OF MONOCLONAL
ANTIBODIES
• Immunosuppressive treatment
– anti CD3, CD54, CD20
• Antinflammatory treatment
– Cytokine neutralization (anti-TNFa, anti-IL1, IL6, IL-17)
– Adhesion molecules blocade (anti-LFA-1, …)
• Anti-tumor treatment
– anti-CD20, anti EGF
• Anti allergic treatment
– anti-IgE, anti-IL15
• Anti aggregation treatment
– anti- gpIIb-IIIa – blocks activation of thrombocytes)
CLINICAL USE OF MONOCLONAL
ANTIBODIES
COMPLETE AND INCOMPLETE
ANTIBODIES
• COMPLETE ANTIBODIES
o visible agglutination or precipitation reaction after reaction with
antigen
• INCOMPLETE ANTIBODIES
o despite the fact that the reaction between epitope and antibody
occurred, the agglutinate or precipitate cannot be detected
CAUSES
o because of antigen
o low antigenicity (low numbers of epitope, bad accessibility
of epitopes for antibody binding)
o low number of bridges between antigens, to intense
repulsive forces between antigens
o because of antibody
o monovalent antibodies (IgM x IgG)
SURVEY OF METHOD FOR DETECTION
OF ANTIGEN OR ANTIBODY
• visualization by secondary phase
o AGGLUTINATION (direct, indirect)
o PRECIPITATION (simple, in combination with electrophoresis,
immunofixation)
• visualization by following detection
o IMUNOFLUORESCENCE
o IMUNOANALYSIS (RIA, EIA, and modifications)
o IMUNOBLOT, IMUNODOT
a g g l u t i n a t i o n
principle of reaction
antigen: INSOLUBLE PARTICULATE ANTIGEN
• easy visualization of
occurred reaction
o due to antigen size
o due to reaction in liquid
the action of an antibody when it cross-links multiple antigens
producing clumps of antigen
… AGGLUTINATE
a g g l u t i n a t i o n
factors influencing quality of agglutination
• enough antibodies
o low concentration of antibodies → no agglutination
• antibodies directed to various epitopes
o difference in agglutination between monoclonal and
polyclonal antibodies
• distance between particles
o the force of attraction or repulsion between two electrically
charged particles, in addition to being directly proportional
to the product of the electric charges, is inversely
proportional to the square of the distance between them;
this is known as Coulomb´s law
a g g l u t i n a t i o n
diretc and indirect
direct agglutination
antigen is present on the particle surface
blood groups, direct Coomb´s test, bacterial agglutination
tests for sero-typing and sero-grouping (e.g. Vibrio
cholerae, Salmonella spp)
indirect agglutination
antigen is bind to appropriate macromolecular particle
(red blood cells, polystyrene latex, …)
Latex fixation test, indirect Coomb´s test, detecting cholera
toxins, etc.
a g g l u t i n a t i o n
blood group detection
ANTIGENS OF ERYTHROCYTE SURFACE
polysaccharides
blood group system AB0 (antigen A, antigen B)
blood group system Lewis, P a Ii
glycoproteins
blood group system Rh (antigen D)
blood group system MNSs, Lutheran, Kell, Duffy,
Diego
a g g l u t i n a t i o n
blood group system Rh
COOMB´S TEST
detection of incomplete antibodies against Rh antigen
DIRECT Coomb´s test
detection of in vivo bound antibodies against erythrocytes
INDIRECT Coomb´s test
detection of circulating antibodies against erythrocytes
Coomb´s antiserum
ANTIBODIES AGAINST HUMAN SERUM GLOBULINS
(polyspecific antiserum containing antibodies directed against IgG, complement, light
and heavy chains of immunoglobulins)
THE PRINCIPLE OF THE TEST
if human serum or whole blood is added to anti-human globulin
serum (as used in the Coombs test) the latter will be deprived of
its power to agglutinate red cells sensitized with incomplete Rh
antibody
The procedures used with the reagent are based on the principle of heteroagglutinins
directed against components of human serum. Normal human red blood cells, in the
presence of antibody directed toward an antigen they possess, may become sensitize
but fail to agglutinate due to the particular nature of the antigen and antibody
involved.
Anti-human serum will react with red cells sensitized with gamma globulin (red blood
cell antibody) or components of human complement and cause agglutination of the
red blood cells.
a g g l u t i n a t i o n
Coomb´s test
p r e c i p i t a t i o n
principle of the reaction
antigen: SOLUBLE ANTIGEN OF LOW
MOLECULAR WEIGHT
Reaction between polyclonal antiserum and soluble (molecular)
antigen. A complex lattice of interlocking aggregates is formed. If
performed in a solution the precipitate falls out of the solution.
PRECIPITATION IN …
• in liquids (nephelometry, turbidimetry)
• v gels (immunodiffusion)
Immunodiffusion-I
Gel
Ags diffuse into gel
setting up a
concentration
gradient
Abs diffuse into gel
setting up a
concentration
gradient
Immunodiffusion - II
Gel
Large aggregates
form at the place of
equimolar
concentrations of Ag
& Ab
p r e c i p i t a t a t i o n
… in liquides
NEPHELOMETRYmeas
urement is made by
measuring the light passed
through a sample at an angle
TURBIDIMETRY
loss of intensity of transmitted
light due to the scattering effect
of particles suspended in it
light
PASSED LIGHT
LOSS OF LIGHT INTENSITY
E L I S A
enzyme-linked immunosorbent assay
principle of reaction
detection of antigen or antibody concentration
enzyme is used for visualization of reaction between antigen and
antibody (anti-human Ig conjgated with enzyme)
Use in clinical practice:
• currently the most widely used laboratory method in immunological
and clinical laboratories
• detection of antibodies (antibacterial, antiviral, autoantibody) or
antigens
• the high sensitivity of the assay allows detection of low
concentration analytes
• ELISA is not suitable for detection od analytes with higher concetration
• coating of the ELISA plate with diluted capture antibody or antigen
• incubation and washing of the microtitre plate
• adding of investigated serum with or without antibodies against coated
antigen (creation of immunocomplexes)
• incubation and washing of the microtitre plate
• adding of appropriate dilution of the secondary antibody conjugated with
enzyme (horse radish peroxidase)
• incubation and washing of the microtitre plate
• adding of substrate to well
• incubation and washing of the microtitre plate
• stopping of the enzymatic reaction
• reading of plates on an ELISA microplate reader
E L I S A
enzyme-linked immunosorbent assay
principle of reaction
detection of antigen or antibody concentration
flourochrome is used for detection of antigen or antibodies (conjugate of
animal antibody against antigen or human antibody in IgG, IgA or IgM class with
fluorochrome)
DIRECT IMMUNOFLOURESCENCE
• detection of antigens or antibodies in tissues due to second antibodies
conjugated with flourochrome
• diagnostic approach in SLE, vasculitis, glomerulonephritis, etc.
INDIRECT IMMUNOFLOURESCENCE
• detection of specific antibodies in serum of the patient (antibodies
present in serum bind to antigen in tissue, they are visualized by
animal anti-human antibodies conjugated with flourochrome)
• detection of antibody positivity
I M M U N O F L U O R E S C E N C E
principle of the method
detection of antigen or antibody presence
e l e c t r o p h o r e s i s
principle of the method
• the migration of charged colloidal particles or molecules
through a stationary medium under the influence of applied
electric field usually provided by immersed electrodes
• a method of separating substances, especially proteins, and a
nalyzing molecular structure based on the rate of movement
of each component in a colloidal suspension while under the
influence of an electric field
Application of electrophoresis in clinical practice:
• Analysis and separation of protein mixture, charakterzation of
bacterial or viral surface, diagnosis of monogenic diseases
i m m u n o e l e c t r o p h o r e s i s
principle of the method
1. step
• immunoglobulins migrate through the gel according to the
difference in their individual electric charges
2. step
• antiserum is placed alongside the slide to identify the specific
type of immunoglobulin present
Application of immunoelectrophoresis in clinical practice:
• the results are used to identify different disease entities, and
to aid in monitoring the course of the disease and the therapeutic re
sponse of the patient to such conditions as immunodeficiencies,
autoimmune disease, chronic infections, chronic viral infections, and
intrauterine fetal infections
general name for a number of biochemical methods for separation and
characterization of proteins based on electrophoresis and reaction
with antibodies
i m m u n o f i x a t i o n
principle of the method
1. step
• protein electrophoresis separates proteins based on their size
and electrical charge in 6 lines
2. step
• adding of monospecific antiserum (anti- IgG, IgA, IgM,
kappa, lambda) one to each line
• diffusion of antigen and antibodies in gel – forming of
immunocomplexes – precipitation in gel
Application in clinical practice:
• immunofixation of serum proteins (typing of paraprotein)
• imunofixation of urine proteins – detection and typing of Bence-Jones
protein)
electrophoretic separation of proteins in geles and following
immunoprecipitazion with monospecific antisera
Application of
serum of one
patient into 6
electrophoretic
lines …
… separation of
serum proteins
according to the
size and charge
…
immunofixation
… application of
monospecific
antisera against
IgG, IgA, IgM and
light and heavy  a
 chains …
anti-IgG
anti-IgA
anti-IgM
anti-
anti-
Electrophoreticseparation
… electrophoretic
separation of
serum of 1
patient in 6 lines
IgM IgAIgG 
monoclonal
band
monoclonal
immunoglobulin
detection and
typing
W e s t e r n b l o t
i m m u n o b l o t
principle of the method
• load and separate protein samples on SDS-PAGE
• electrophoretically transfer fractionated proteins onto PVDF
membrane
• block the membrane with neutral protein (BSA or milk casein)
• incubate the membrane with primary antibody specific to target
protein
• incubate the membrane with HRP-labeled secondary antibody
specific to primary antibody
• incubate the blot with HRP substrate and expose to film
Application in clinical practice:
• tests for confirmation of HIV positivity, diagnostic of Borrelia
infections, confirmation of hepatitis B positivity
electrophoretical separation of proteins and their blot to membrane with
following detection with specific antibodies
MOMOCLONAL GAMMOPATHIES
presence of abnormal proteins called monoclonal
proteins or M proteins in the blood
these proteins are produced by a single clone (a group of
identical cells) of plasma cells
(B lymphocytes producing antibodies)
paraprotein = M protein = monoclonal protein
MOMOCLONAL GAMMOPATHY
BIOCHEMICAL POINT OF VIEW
presence of monoclonal immunoglobulin in the blood or urine
(electrophoresis of serum or urine proteins)
CLINICAL POINT OF VIEW
presence of monoclonal immunoglobulin in the blood or urine
(electrophoresis of serum or urine proteins)
MOMOCLONAL GAMMOPATHIES
monoclonal gammopathies can be associated with various
underlying diseases and conditions, and they are typically
identified through blood tests and further diagnostic
evaluations
examples of the key monoclonal gammopathies
monoclonal gammopathy of undetermined significance
(MGUS)
multiple myeloma
Waldenström macroglobulinemia
AL amyloidosis
other less common monoclonal gammopathies
MOMOCLONAL GAMMOPATHIES
MONOCLONAL GAMMOPATHY OF UNDETERMINED
SIGNIFICANCE (MGUS)
is an asymptomatic, premalignant clonal plasma cell proliferative
disorder characterized by the presence of a serum M protein in
persons who lack evidence of
multiple myeloma, macroglobulinemia, amyloidosis, or other related
diseases
it is defined by the presence of a serum M protein < 3 g/dL, bone
marrow plasma < 10%, plus absence of anemia, hypercalcemia,
lytic bone lesions, or renal failure that can be attributed to the
plasma cell proliferative disorder
MOMOCLONAL GAMMOPATHIES
MONOCLONAL GAMMOPATHY OF UNDETERMINED
SIGNIFICANCE (MGUS)
it is the most common plasma cell dyscrasia and is prevalent in
approximately 3% of the general population 50 years of age
and older
the prevalence increases with age
the incidence has been reported as 1.7% in those 50 to 59 years of
age and as over 5% in those older than the age of 70
age-specific incidence is higher in males than females
the main clinical significance of MGUS is its lifelong risk of
transformation to myeloma or related malignancy at a fixed but
unrelenting rate of 1% per year
MOMOCLONAL GAMMOPATHIES
MULTIPLE MYELOMA (MM)
accounts for about 10% of all hematologic malignancies
the diagnosis of MM requires the presence of one or more myeloma
defining events (MDE) in addition to evidence of either 10% or more
clonal plasma cells on bone marrow examination or a biopsy-proven
plasmacytoma
MDE consists of established CRAB features (hypercalcemia, renal
failure, anemia, or lytic bone lesions) and 3 specific biomarkers:
clonal bone marrow plasma cells ≥60%, serum free light chain (FLC)
ratio ≥100 (provided involved FLC level is ≥100 mg/L), and more
than one focal lesion on magnetic resonance imaging (MRI)
Each of the new biomarkers is associated with an approximately 80% risk of
progression to symptomatic end-organ damage in two or more independent
studies
MOMOCLONAL GAMMOPATHIES
MULTIPLE MYELOMA (MM)
clinical manifestation
the clinical presentation of multiple myeloma can vary from person to
person, and the disease can be asymptomatic (without noticeable
symptoms) in its early stages, however as the disease progresses,
various symptoms and complications may develop
bone pain and pathological fractures: One of the hallmark
symptoms of multiple myeloma is bone pain, which is often described as a
deep, aching pain in the bones. This pain can occur in any bone but is most
commonly felt in the back, ribs, hips, and skull. The bone pain is due to the
weakening of bones caused by the growth of myeloma cells. Weakened bones
in multiple myeloma can lead to fractures that occur with minimal or no
trauma, known as pathological fractures.
MOMOCLONAL GAMMOPATHIES
MULTIPLE MYELOMA (MM)
clinical manifestation
fatigue: Many people with multiple myeloma experience significant fatigue
and weakness. This can be related to anemia, a condition in which there is a
shortage of red blood cells, which carry oxygen throughout the body.
recurrent infections: Multiple myeloma weakens the immune system,
making individuals more susceptible to infections. This can lead to frequent or
severe infections, such as pneumonia, urinary tract infections, and skin
infections.
unexplained weight loss: Some people with multiple myeloma
experience unexplained weight loss, which can be a result of the cancer's
impact on metabolism and appetite.
MOMOCLONAL GAMMOPATHIES
MULTIPLE MYELOMA (MM)
clinical manifestation
renal problems: Myeloma proteins can damage the kidneys, leading to
symptoms such as increased thirst, frequent urination, and swelling in the
legs and ankles. This can progress to kidney dysfunction and failure if left
untreated.
hypercalcemia: High levels of calcium in the blood (hypercalcemia) can
occur in multiple myeloma and lead to symptoms like excessive thirst,
frequent urination, constipation, nausea, vomiting, and confusion.
neurological symptoms: In some cases, myeloma-related proteins can
affect nerves, leading to symptoms like numbness, tingling, weakness, and
problems with coordination.
MOMOCLONAL GAMMOPATHIES
MULTIPLE MYELOMA (MM)
clinical manifestation
bleeding and bruising: Multiple myeloma can disrupt the normal
functioning of blood clotting factors, leading to an increased risk of bleeding
and easy bruising.
anemia: Anemia, which is a deficiency of red blood cells, can result in
symptoms such as fatigue, weakness, pale skin, and shortness of breath.
elevated blood protein levels: Blood tests may reveal elevated levels
of certain proteins, such as monoclonal (M) proteins or light chains, which are
produced by myeloma cells and can be detected in the blood and urine.
MOMOCLONAL GAMMOPATHIES
MULTIPLE MYELOMA (MM) TREATMENT
patients eligible for transplantation
typically, patients are treated with approximately 3–4 cycles of
induction therapy with bortezomib, lenalidomide, dexamethasone
(VRd) prior to stem cell harvest
stem cell transplantation
patients eligible for transplantation
initial therapy is with VRd is administered for approximately 8–12
cycles, followed by maintenance therapy with lenalidomide
Thank you for your attention
SEROLOGICAL REACTIONS
Course no. 1