BTN-207

Immunotechnology

Immune - ology

Immunity : meaning “exempt”

All mechanisms used by the body as protection against

environmental agents that are foreign to the body

Immune system

System of many biological structures and processes

within an organism that protects against disease
 The Immune System

Immunology deals with understanding how the body distinguishes

between what is “self” and what is “nonself”
 Immunology

Immunity: Innate and Aquired (Active/Passive)

Immunity: Cellular and Humoral

Immunization: Initial contact with a foreign agent

Vaccination: Process of inducing Acquired Immunity

AGAINST?
Introduction Major Infectious Disease Pandemics

Plague
14th Century Ebola
Malaria 2014-
1890s HIV/AIDS
Small pox Typhoid
1983- SARS
1600-1700s 1900s
2003
Infectious Diseases Timeline
Tuberculosis Anthrax H1N1
1600s- Cholera Polio 2001 2009
1800s 1890s

Spanish Influenza
1918

Leprosy Yellow Fever Chicken pox

BC 1793 Measles
Mumps
Diptheria
Hepatitis
Meningitis
Dengue
Chikungunya
 Vaccination: Beginning of Immunology Research

Edward Jenner: 1798

Small pox

The term vaccination is derived from Latin words vacca (Cow) and vaccinia (cowpox)
Movie: Small pox and Edward Jenner
Vaccination: Beginning of Immunology Research

French German
Louis Pasteur Robert Koch
1822-1895 1843-1910
History Pasteur’s Experiments

1879 Chicken cholera

History Pasteur’s Experiments

1881 Cattle anthrax 1885 Rabies

Joseph Meister 9 yr old boy

Movie: Louis Pasteur

Rabies
Cattle anthrax
Chicken cholera
Institut Pasteur

1887 Paris
History Immunology

Robert Koch

(Father of Bacteriology/Microbiology)

Bacillus anthracis caused anthrax

Mycobacterium tuberculosis caused tuberculosis
Vibrio cholera caused cholera

Institute of Infectious Diseases (1891)

Berlin
(renamed Robert Koch Institute)
History Development of Human Vaccines

Small pox 1798 18th century

Rabies 1885 19th century

Typhoid 1896
Cholera 1896
Plague 1897

Diptheria 1923 20th century (first half)

Tetanus 1926
Pertusis 1926
Tuberculosis 1927
Yellow Fever 1935
Influenza 1936
Typhus 1938

Polio 20th century (second half)

Measles
Mumps
Rubella
Varicella
History Immunology

Emil Von Behring

Joined as Assistant to Robert Koch at Institute of Infectious Disease, Berlin

Developed Serum against Diptheria (Corynebacterium diphtheriae)

Also developed Serum against Tetanus (Clostridium tetani)

Shibasaburo Kitasato
 History Serum Therapy (Passive Immunization)

Behring and Kitasato 1890

They immunized rats, guinea pigs and rabbits with attenuated forms of the infectious agents causing
diptheria and tetanus

Sera produced by these animals was injected into animals previously infected with fully virulent bacteria

The ill animals could be cured through administration of the serum.

They called the substance antitoxin and their treatment serum therapy

With this serum therapy, Behring and Kitasato were the first to use passive immunization methods in the
fight against infectious diseases

They realized that they needed to

immunize large animals, such as
horses and sheep, to produce
enough antitoxin to protect humans

Von Behring would win the first Nobel Prize in

medicine in 1901 for his work on diphtheria
 Antibodies or Igs

Paul Ehlrich coined the term “Antibodies” in 1891

Originally referred to as ‘antitoxins’ by Behring and Kitasato (1890)

In 1899, Laszlo Detre named the hypothetical substances halfway between bacterial
constituents and antibodies "substances immunogenes ou antigenes" (antigenic or
immunogenic substances)

Antigen: Immune responses arise as a result of exposure to foreign stimuli

The compound that evokes the response is referred to as antigen

An antigen is any
molecule capable of
eliciting either an
immune response, or
an immune reaction
 Antibodies or Igs

Serum Therapy Antitoxins or Antibodies present in serum could protect

What is serum?
 Components of the Immune System: Blood

Erythrocytes (RBCs)
Leukocytes (WBCs)
Thrombocytes (Platelets)
Plasma

TLC/DLC

Leukocytes Granulocytes and Agranulocytes

ls: 0.5 – 1%
hils: 1 – 4%
hils: 40 – 60%

cytes (B, T & NK cells): 20 – 40%Monocytes: 2 – 8% (DCs and Macrophages)

Components of the Immune System: Blood
Introduction Serum Therapy: Isolation of Antibodies

Serum Proteins:

1.Albumin
2.Globulin The globulins are a family of globular proteins that have higher molecular
weights than albumins

All globulins fall into one of the following four categories:

Alpha 1 globulins
Alpha 2 globulins
Beta globulins
Gamma globulins

Arne Tiselius & Elvin Kabat (1939)

They immunized rabbits with protein ovalbumin (albumin of egg whites)

Collected rabbit blood and isolated serum

The serum obtained was divided in 2 aliquots

Electrophoretic separation of one serum aliquot

Introduction Serum Therapy: Isolation of Antibodies

When serum is separated in electric field, proteins will migrate towards positive

Revealed 5 major fractions:

Serum albumin
Alpha 1
Alpha 2
Beta
Gamma globulins

The slowest in terms of migration is gamma-globulin

Prior to electrophoresis, second aliquot of serum was mixed with antigen (ovalbumin) and
antigen antibody immune precipitation complex was removed

Remaining serum (minus antibody) was electrophoresed again

There was a significant drop in gamma globulin peak

Thus gamma-globulin peak was identified as containing serum antibodies

Timeline Immunoglobulins or Antibodies

1945: Linus Pauling. Lock and key concept of antigen-antibody interaction

1948 Eisen demonstrated antibodies are divalent

1950s Svedburg developed ultracentrifugation. Molecular weight of an antibody was found to be 150 kDa

1950. Porter selectively cleaved antibody molecules by enzyme Papain

It split the Ig molecule in 3 fragments of approx equal size
Timeline Immunoglobulins or Antibodies

2 of fragments retained antibodies ability to bind antigen, but they could not precipitate antigen from solution
unlike the intact Antibody molecule

These 2 fragments referred to as Fab (fragment antigen binding)

Each considered univalent, one binding site, and both identical to each other

The 3rd fragment could be crystallized out of the solution, and was called Fc (crysallizable fragment)
It could not bind antigen

1959. Edelman showed that when antibody molecule is first reduced and proteolyzed,
A different set of 4 products were formed

When gamma globulin was reduced by mercaptoethanol treatment, this molecule fell apart in 4 chains

Two identical heavy chains of 50 kDa each

Two identical light chains of 25 kDa each
Timeline Immunoglobulins or Antibodies

1963. Porter injected Fab and Fc from rabbits into goat to raise anti-sera
Antisera to Fab could react with L or H chain, but antisera to Fc only reacted with H chain

Based on this result, Porter suggested the Y shaped prototype structure of antibody

Ag binding sites

The two heavy chains are linked to each other by disulfide bonds and each heavy
chain is linked to a light chain by a disulfide bond
Timeline Immunoglobulins or Antibodies

Papain Digestion
Porter

Pepsin Digestion of antibody molecule results in 2 fragments

Divalent F(ab)2 consisting of 2 fab regions joined by disulphide bond and Fc fragment
 Antibody or Ig Structure
Two identical heavy chains of 50 kDa each
Two identical light chains of 25 kDa each

In any given immunoglobulin molecule, the two heavy chains

and the two light chains are identical, giving an antibody
molecule two identical antigen-binding sites, and thus the ability
to bind simultaneously to two identical structures

Each H or L chain will have a series of

domains approx 110 amino acids long

Light chain: 2 domains

Heavy chain: 4 domains

Variable VH/VL
Constant
Antibody or Ig Structure

The variability in sequence is limited to

approximately the first 110 amino acids,
corresponding to the first domain, whereas
the remaining domains are constant between
immunoglobulin chains of the same isotype.

Hinge region

Flexible stretch of polypeptide

Joining 3 globular portions of Ig
Cysteine & Proline

The disulfide bonds that link the two heavy

chains lie in the hinge region between the
CH1 and CH2 domains
Timeline Immunoglobulins or Antibodies

Papain Digestion

Before Hinge

Pepsin Digestion of antibody molecule results in 2 fragments

Divalent F(ab)2 consisting of 2 fab regions joined by disulphide bond and Fc fragment

After Hinge
 Antibody or Ig Structure

Antibody Light chains

Types of Light Chains (biochemical and serological diff)

Almost all species have 2 major classes of light chains

Kappa κ
Lambda λ

VK, CK, VL, CL

Any one individual of a species will have both types of light chains, kappa and lambda,
but the ratio of kappa to lambda varies with species

In any one Ig molecule or antibody, both light chains will be of any one type
 Antibody or Ig Structure

Types or class of Heavy chains define Ab effector function

(serological, size differences)

IgM μ
IgG γ
IgA α
Different in constant Regions
Effector function IgD δ
IgE ε
Figure 4-18
Antibody or Ig Structure
 Antibody or Ig Structure
Subclasses of Heavy chains
Type of heavy chain determines class and subclass of antibody

IgM 970 kDa 5-10 days

IgG (IgG1, IgG2, IgG3, IgG4) 150 kDa 21 days
IgA (IgA1, IgA2) 385 kda 5 to 6 days
IgD 180 kDa 2 days
IgE 190 kDa 2 days
Ig Class
Ig Class
Introduction Antibodies or Igs

The gamma globulin band as seen in

conventional serum protein electrophoresis
consists of 5 immunoglobulins

In normal serum,

70-80% is immunoglobulin G (IgG),

20-15% is immunoglobulin A (IgA),
10-5% is immunoglobulin M (IgM),
0.2% is immunoglobulin D (IgD),
and a trace is immunoglobulin E (IgE).
Ig Structure: Localized regions
 Ig Structure: Localized regions
Hypervariable regions are present within variable region of both H & L chains

Hypervariable regions are also called complementarity determining regions

CDR1, CDR2 and CDR3

Intervening peptide segments are called framework regions (FRs)

FR1, FR2, FR3, and FR4
Structure Immunoglobulins/Antibodies

Peptide sequences with exceptional variability,

around amino acids 30, 50, 95
Structure Immunoglobulins/Antibodies

Each protein domain (VL or CL) is constructed from two β sheets, which are elements of protein
structure made up of strands of the polypeptide chain (β strands) packed together

Each domain of 2 beta sheets packed tightly against each other in a compressed beta barrel
 Ig Structure
Framework regions form Beta sheets that provide structural framework of Ig domain

Hypervariable sequences correspond to 3 loops at outer edge of Beta barrel,

Juxtaposed in the folded domain

Antigen Binding Site

Hypervariable loops of Heavy and Light chains are

paired together to form antigen binding site

Total 6 CDR (three from H and three from L chain)

Structure Immunoglobulins/Antibodies
Ig Terms
Ag-Ab Interaction
Introduction Immunoglobulins or Antibodies

Immunoglobulins, also known as antibodies, are glycoprotein molecules produced by B cells

An antibody, or immunoglobulin (Ig), is a large, Y-shape protein produced by B/plasma cells

Antigen recognition molecules of B cells are called Immunoglobulins

Membrane bound Ig on B cell surface is called B-cell receptor (BCR)

Immunoglobulin of same antigen specificity secreted by differentiated B cells is Antibody

Secretion of Antibodies which bind pathogens or their toxins is main effector function of B cells
 Isohemaglutinins
An isoantibody normally present in the serum of an individual that causes the
agglutination of the red blood cells of another individual of the same species

Isohemagglutinins are antibodies against blood group antigens, usually of the IgM isotype

People with type O blood will have isohemagglutinin titers against both type A and B blood
groups but those with type AB blood have neither
Lecture 3
Introduction Infectious Diseases and Immunity

Host Immunity

Ability of an organism to protect or resist from a pathogenic infection

Cellular
Active Immunity

Passive Immunity

Humoral
Introduction Innate & Adaptive Immunity
Introduction Humoral & Cellular Immunity
Introduction Humoral & Cellular Immunity
Components of the Immune System

Primary Lymphoid Organs

Bone marrow
Thymus

Secondary Lymphoid Organs

Spleen
Peyer’s patch
Lymph nodes
Tonsils
Components of the Immune System
B cells
T cells
An idiotope is the unique set of antigenic determinants (epitopes) of the variable portion
of an antibody.

In some cases it can be the actual antigen-binding site, and in some cases it may
comprise variable region sequences outside of the antigen-binding site on the antibody
itself.

Thus each antibody would have multiple idiotopes; and the set of these individual
idiotopes is termed the idiotype of the antibody.
The variable part of an antibody including the unique antigen binding site is known as the idiotype.

The idiotype is one or more antigenic determinants specific to the variable region
of a particular antibody
If a separate antibody is produced that has specific binding capabilities to an idiotope of
the previously described antibody, it is said to be an "anti-idiotypic antibody"

When one antibody binds to an idiotope of another antibody it is referred to

as an anti-idiotypic antibody.