• MEDICAL MICROBIOLOGY

Course for medical students

• Summer semester 2024
Professor G. Zhelezova, M.D., Ph.D.

Department of Biology, Medical Genetics and Microbiology

Medical Faculty
SU “St. Kliment Ohridski”

Email: [email protected]
 Lecture 12
Humoral Immune Response

• 12.1. Antibodies – structure, classes and

biological functions. Dynamics of the
humoral immune response.
• 12.2. Monoclonal antibodies –
production, types, application
 Specific Humoral, Antibody-Mediated Immune
Response (AMI)

• Induced by antigens
• Safety function (second line of defense)
• Highly specific, MHC restricted, with
immunological memory
• Main cells of humoral immunity - B
lymphocytes
• Effector molecules - antibodies
 Definition of Antibodies

• Glycoprotein molecules, belonging to the

group of serum proteins known as globulins
• Antibodies, called immunoglobulins (Igs) are
produced by plasma cells in response of Ag
stimulation
• Constitute 20-25% of the total serum
proteins
• Bind specifically to an antigen making
complexes
 Distribution of Proteins in the Serum
in an Electrical Field
+ -

Albumin
Amount of protein

Globulins

α1 α2 β γ

Mobility
 B and T Cell Receptors
• B cells can recognize much broader range of
chemical structures than the antigen receptors
of T cells
• They can be proteins, lipids, carbohydrates,
nucleic acids, as well as simple, small chemical
groups
• Antibodies can recognize diverse microbes and
toxins
• In striking contrast, most T cells see only
peptides
Antigen Recognition and Proliferation
 Types of B Cells
• Conventional B cells, cells of adaptive immunity
• Immature B cells – surface IgM
• Mature B cell – surface IgM and IgD
• After the contact with Ag:
– 1. clonal proliferation -> plasma cells - secrete Ab
– 2. memory cells
• Unconventional B cells (very small number in
adults)
• CD5+
• Secrete natural antibodies, class IgM, low
specificity, part of innate immunity
 Basic Structure of Immunoglobulin Molecules

• All are built on the same general principle

• Small differences in the structure determine

significant functional differences

• There are five classes or isotypes of

immunoglobulins: IgG, IgA, IgE, IgM and IgD

• The change in Ig isotype production is called

heavy-chain class (or isotype) switching
 Basic Structure of Immunoglobulin Molecule
• Two heavy chains (H-chains) and two light chains
(L-chains)

• Each of chain consists of variable regions and

constant regions

• Constant regions have the same amino acids for

all immunoglobulins

• Variable region is the section of the molecule

that interacts with the antigenic epitopes
 Variable and Hypervariable Region

• The variable regions of Abs are different in

their arrangement of amino acids

• They correspond to different antigens

• Hypervariable region – that part of variable

region, corresponding to epitope
 Basic Structure of the Immunoglobulins
Heavy and light chains
Domains - structural Disulfide bond
units, composed of
approximately 110
amino acids
Carbohydrate
Constant (C) and
variable (V) domains
CH1

Hypervariable regions VH
(HVR) CH2 CH3
CL
Hinge region (with VL Hinge region
some flexibility)
Carbohydrate
 Immunoglobulin Fragments
• In the presence of enzyme papain Ig is spited into
2 parts:
• Fc-fragment:
– One part
– The Fc regions of all antibodies are the same for each
species; they are constant rather than variable

• Fragment antigen binding (Fab)

– Two identical Fab fractions which can bind with
antigens
– They are variable
 Ag-Ab Binding

• “Lock and key“ concept

• Epitope (antigenic determinant ) and paratope
(variable part of the Ab) make complex

• Highly specific binding

• Basis for highly specific diagnostic immune

reactions
 IgG
• Monomer (bivalent)
• Subclasses from 1 to 4
• 75-80% in serum (8-16 mg/mL)
 Ig G Functional Zones

Ag
binding

Complement
binding site
Binding to Fc
receptors
Placental
Transfer – FcRn
on trophoblast
cells
 IgG Functions

• Oppsonization of viruses and bacteria -

enhancement of phagocytosis
• Complement activation - interaction with some
proteins of the complement system
• ADCC (bind with NK-cells)
• Neutralization of toxins and viruses
• Immobilization of bacterial movement
• Transport across the placenta → passive
immunity of newborn
• Feedback inhibition of B cells immune response
 IgG (cont.)

• Ig G is a predominant antibody in
secondary immune response (or late
immune response)
• Important defense against bacteria
and viruses
• Serum half-life - 23 days
• Used for serotherapy
 IgA
• 10-15 % in serum (0.6-4 mg/ml)
• Two subtypes – A1 and A2
• Two forms:
• 1. Serum IgA (monomeric form)
– IgA1 predominates in serum (~80%)
• 2. Secretary IgA (SIgA) (dimeric form)
– IgA2 percentages are higher in secretions than in serum
(~35% in secretions)
• sIgA presents in secretions (tears, saliva, barest milk)
and in mucous of all mucous membranes
• Provide local defense against pathogens of
respiratory, intestinal and urogenital tract
• Serum half-life - 6 days
IgM Pentameric Form

J chain

Cµ4
additional
domain
 IgM
• 8-10% in the serum (0.5-2 mg/ml)
• Pentameric form (10 identical binding sites: 10
valences)
• The most efficient Ig in agglutination,
opsonization and in complement fixation and
activation
• First Ig synthesized by the fetus
• The predominant antibody in primary immune
response (It appears first after Ag contact
(indicates about acute infection)
• Native membrane B cell receptor (with IgD)
• Serum half-life - 6 days
 IgE

• Monomeric form (monovalent)

• Less than 0.05 %
• Ability to bind with mast cells → mast cells
activation -> release of vasoactive substances
-> immediate type of hypersensitivity
• Defense against helminthic parasites
• Ig of allergic and parasitic diseases
• Serum half-life - 2 days
IgE

Cε4
 IgD

• Monomeric form
• Secreted form - none
• Plasma concentration – trace
• Marker of B cell maturation (with IgM
membrane immunoglobulin receptor)
IgD

Tail Piece
Determination of Immunoglobulins in the
Practice
• Determination of total immunoglobulins
from different classes
• Part of diagnosis of some internal
diseases
• Determination of specific
immunoglobulins of different classes
• For diagnosis of specific infectious
diseases (Ex. Hepatitis B, HIV, etc.)
 Immunoglobulin Class Switching
(Heavy-Chain Isotype Switching)
• Immunoglobulin Class Switching - the process by
which the isotype changes (but not the specificity)

• Isotype switching depends on the cytokines,

expressed by helper T cells

• Different cytokines induce switching to different

antibody isotypes

• In this way the immune system can respond in the

most effective way to microbes
Primary and Secondary Humoral Immune Response
Primary response Secondary resopse

Lag after immunization Usually 5 -10 days Usually 1-3 days

Peak response Smaller Larger

Antibody isotype Usually IgM>IgG Realative increase in IgG and

under certain situations, in
IgE or IgA (isotype switching)

Antibody affinity Lower average affinity Higher average affinity

Dynamics of Antibody Responses Induced by T-
Independent Antigen

IgM Ab
Ab Titer

1o Ag 2o Ag

Days After Immunization

 Dynamics of Antibody Responses Induced by T-
Dependent Antigen

Total Ab IgM Ab IgG Ab

Ab Titer

1o Ag 2o Ag

Days After Immunization

 Immunoglobulin Class Switching

• IL-2 and IFNγ stimulate production of IgG

and IgM

• IL-4, IL-5, IL-6, IL-10 stimulate production of

IgM, IgG, IgE, IgA

• IgA production is stimulated by IL-5 и TGF-β

General Function and Usage of Immunoglobulins

• Defense through the immune response

• Diagnosis of different infections

• Passive immuno Treatment (sero therapy)

Immunologic Methods for Diagnosis of Infectious
Diseases
• Based on highly specific binding between Ag
and Ab
• “Lock and key“ concept
• Result is the formation of immune complexes
• With help of known Ags it is possible to
detect unknown Abs in the serum or vice
versa
 Primary Association Between Antibody and
Antigen

• The binding forces between Ag-Ab are

relatively week (no covalent bonds)

• Mainly van der Waals, electrostatic or

hydrophobic forces

• Thus, the interaction requires very close fit

between an epitope and the paratope
 Affinity of Ag-Ab Binding
• The strength of the reaction (association)
between an antigenic determinant and their
respective epitope is called affinity
Strong Weak

Ab Ab

Ag Ag
 Cross-reactivity
Antibodies produced against one antigen may bind other,
structurally similar antigens
Such binding to same or similar epitopes is called a cross-
reaction; has been observed on many levels in the
nature
Cross-reaction

Anti-A Anti-A
Anti-A Ab Ab
Ab

Ag B Ag C
Ag A
Same epitope Similar epitope
 Molecular Mimicry
• Molecular mimicry - an antigenic similarity
between unrelated macromolecules

• It is believed that it play a role in the

pathogenesis of some autoimmune diseases

• A false positive results in immunological

diagnostic tests are possible
 Immune Methods for Diagnosis –Serological
diagnosis
• Serological diagnosis of the infection diseases
is based on the detection of unknown Abs in
the serum of patient with help of known
Ags

• Serological diagnosis of the infection diseases

which is based on the detection of unknown
microbial Ag in the serum of the patient with
help of known Abs is also possible
How to Express the Results from Serodiagnostics

• It depends on the test and on the disease

• 1. Positive or negative result (qualitative result)
• 2. Diagnostic titer (semi-quantitative result)
• Increasing of the titer in 4 or more times in the
pared sera of the same patient is indication of
the specific disease
• A titer is a measurement of the amount of a
substance in a solution on the basis of dilution
• A titer measurement is expressed as a ratio,
such as 1:40 (1 part substance plus 39 parts
diluent)
How to Express the Results from Serodiagnostics
(cont.)
• 3. Determination of amount of antibody as a
number - Ex. 50 ng/ml (quantitative)
• ELISA results are reported very often as a
number
• “Cut-off" point between a positive and a
negative result
 Diagnostic Value of Antibodies
• Antibodies are only witnesses for infectious
process
• They have high specificity and sensitivity
• Non-specific reactions are possible (cross-
reacting antibodies)
• Abs are result of infection or vaccination
• Determination of Ig-class is important for
differentiation of acute from chronic or late
infection or past immunization
 Immunological Methods for Diagnosis
(Serological Methods)
• Serological methods - agglutination,
precipitation, complement-fixation test (CFT),
ELISA, Immunoblot, Immunofluorescence,
etc.
• Diagnosticum – known killed microbes or Ags
(O-diagnosticum, H-diagnosticum, erytrocytic
diagnosticum, etc.)
• Diagnostic antiserum is a serum containing
specific Abs of known specificity
 Studied Clinical Samples

• Serum – the most commonly studied clinical

sample
• CSF
• Urine
• Joint fluid
• Tears
• Saliva
 Agglutination
• When an antigen is mixed with a
corresponding antibody, interactions may
lead to formation of large, stable, visible
structures

• This is because the antigen may be bound by

bivalent antibodies, each arm of which can
recognize adjacent Ags

• The result is net formation or agglutination

Agglutination
 Polyclonal Immune Responses
• The normal immune response is polyclonal
(immunoglobulins are heterogeneous)
– Against different epitops of antigens (antigens are multivalent)
– Abs are from different classes (isotypes)

• Monoclonal immune response in the case of pathology as

Myeloma disease

• High level of homogeneous (monoclonal)

immunoglobulins, product of only one pathologic clone B
lymphocytes
 Monoclonal Antibodies

• Identical antibodies produced by a clone of B

cells

• In pathology – myeloma monoclonal

antibodies

• In laboratory – hybridomas that produce

monoclonal antibodies
 History of Monoclonal Technology
• G. Köhler and C. Milstein create technology for the
production of monoclonal antibodies in 1975 (the
hybridoma technology)
• Won the Nobel Prize in Physiology or Medicine in
1984, "for work on the immune system and the
production of monoclonal antibodies“
• One clone of B cells makes an antibody of only one
specificity, called monoclonal antibodies (MoAb)
• One of the most important technical advances in
immunology, with far-reaching implications for
clinical medicine and research
 The Principle of Hybridoma Technology
• Preparation of a new cell (hybridoma) by fusing a B
lymphocyte from an immunized animal with a
myeloma cell
• B cells, which have a short lifespan in vitro, are
obtained from an animal immunized with an antigen
• Myeloma cells (tumors of plasma cells), which can be
propagated indefinitely in tissue culture
• Hibridoma cell gives rise to clones of cells producing
antibodies with the same specificity
• By this means, monoclonal antibodies against
virtually any antigen can be produced
Hybridoma
Different Types of Monoclonal Antibodies

• Mouse MoAb

• MoAb chimeras: a combination of a human

constant region and murine variable region

• Humanized MoAb: contain human constant

region and only hypervariable region of
mouse

• Recombinant MoAbs
 Monoclonal Antibodies Used for Diagnosis

• Diagnosis of infectious agents

• Determining amounts of hormones,

cytokines, tumor markers and others

• Determination of surface molecules on

different cells:
– CD markers on the cells of immune system,
Markers of leukemia and its subtypes, etc.
Monoclonal Antibodies Used for Therapy

• Autoimmune diseases
• Tumors
• Infections
• Transplantations
• Example: OKT3 murine monoclonal antibody
(MoAb) against CD3 which acts to modulate the
T cell receptor (TCR) complex, inactivating both
naive T cells and activated cytotoxic T cells
• Although a potent immunosuppressive agent, it
has several significant side effects