A presentation on Immunochemistry

1. IMMUNOCHEMISTRY
PaPART B: Immunoglobulins and The Complement System
(CLI 320)
BY
Taiwo T. Adeniyi
Dept Of Medical Biochemistry

2. What is Immunology?
• Immunology is the branch of biomedical science that deals with the response
of an organism to antigenic challenge and its recognition of what is self and
what is non-self.
• It deals with the defense mechanisms including all physical, chemical and
biological properties of the organism that help to control its susceptibility to
foreign organisms, materials etc.
• It is the physiological functioning of the immune system in states of health
and disease, as well as malfunctioning of the immune system in
immunological disorders.

3. Immune System
• The immune system fights germs on the skin, in body tissues and
body fluids such as blood etc
• It is made up of Innate (general) immune system and adaptive
(acquired or specialized) immune system
• The two systems work closely together and take on different tasks.

4. INNATE AND ADAPTIVE IMMUNITY
• Innate immune system
• Forms the first line of defense
against intruders
• Quick response, non-specific and
static resistance
• Provides protection offered by
• 1. the skin and mucous membrane
• 2. the immune system cells and
proteins
• Adaptive immune system
• Body’s second line of defense, once the
innate system has been overwhelmed.
• It is specific to the infective agent
• Has ability to store information as
memory to show enhanced response to
subsequent challenge
• Made up of T cells, B cells and
antibodies
• The adaptive immune system has two
main mechanisms (a) Cellular immunity
and (b) Humoral immunity

5. 3 Salient Teatures of Immunological Reactions
• A) Recognition of self from non-self
• B) Specificity of the reactions: when injecyed with 100 different proteins,
the body will produce 100 different antibodies. This is called specificity.
Very small differences at molecular level are recognized.
• C) Memory of the responses: if the same antigen is introduced for a second
time, body will react immediately . This memory is the basis of vaccination
• ANTIGENS: Any substance which invokes an immunological response is an
antigen or immunogen. E.g. certain components of the cell membrane . The
difference between antigens A and B of RBC is an extra N-acetyl group on A

6. Immune Response
• The bone marrow generate lymphocytes
• The lymphocytes that pass through and processed by the thymus gland
are called T-lymphocytes.
• T-lymphocytes can directly kill the target cells and are the effector cells
for the cell mediated immunity or cellular immunity
• Certain other cells originated from bone marrow and processed by the
Bursa of fabricus in avians are called B-cells or B-lymphocytes.
• (The Bursa equivalent organs in human beings are gut associated and
lung associated lymphoid organs)
• Plasma cells belonging to the B-lymphocytes secret immunoglobulins.
• The B cells govern Humoral (antibody mediated) immunity
• In peripheral blood, 80% lymphocytes are T-cells and 15% are B-cells

7. Cellular Immunity
• The followings are the major activities of T-lymphocytes
• 1. Immunity against Infections: immunity against bacteria, many
viruses and almost all parasites
• 2. Rejection of allograft: Body tries to reject transplanted organs
mainly by T-cell mediated mechanisms
• 3. Tumour cells destruction: This is predominantly by T-cell activity
• 4. Helper function: T helper cells(a subgroup of cells that carry CD4
determinants on the cell surface) are necessary for optimal antibody
production by plasma cells and for generation of cytotoxic T-cells.
• 5. Suppressor function: T-suppressor cells are CD8 positive cells. They
downregulate the activities of both T and B cells

8. Humoral Immunity
• Plasma (B-lymphocytes) cells produce antibodies. These are called
Immunoglobulins
• The antigen-antibody reactions leads to activation of the Complement
system, which destroys the foreign cells
• IMMUNOGLOBULINS(Ig) react specifically with antigens. Their affinity
is based on the complementary nature of the 3-dimensional
structureof antigen and antibody

9. IMMUNOGLOBULINS
• Immunoglobulin(Ig) is made up of 2 heavy chains (H) and 2 light chains (L),
combined together through disulfide brigdes
• For example in IgG: H chains has 440 amino acids. L chains has 214 amino
acids
• Ig are differentiated into 5 major classes based on the make up of the H
chains
• IgG is made up of γ(gamma) heavy chains
• IgM has µ(miu) heavy chains
• IgA has α(alpha) heavy chains
• IgD has δ(delta) heavy chains
• IgE has ε(epsilon) heavy chains
• The L chains are either κ(kappa) or λ(lambda) in all the classes. E. g.
• IgG may consist ot either γ2κ2 or γ2λ2
• In humans 60% of L chains are κ variety, 40% are λ type.

10. • Both the H and L chains
contain relatively
variable and constant
regions, with respect to
the amino acid
composition.
• The find the first 108 aa
in L chain and the first
118aa in gamma H
chains constitute the
variable regions

11. • Fab and Fc Portions
• Papain cleaves Ig into 2 Fab and 1 Fc portions. Antibody binding
part is in the Fab fragment.
• The cleavage takes place in the hinge region. Carbohydrates groups
of the Ig are also found at the hinge region
• The area for complement binding is in the Fc portion
• Pepsin cleaves Ig to yield F(ab)2. i.e. 2 Fab combined for stronger
antigen binding

12. CLASSES OF IMMUNOGLOBULINS
• 1. Immunoglobulin G (IgG)
• Its heavy chain is of gamma type, and sometimes referred to as 7S Ig due to its sedimentation
coefficient
• It’s the major antibody, about 70-80% of total Ig in circulation. It’s the antibody seen in
secondary immune response
• It can pass from vascular compartment to interstitial space. It can cross placenta barrier and
protect the newborn from infection (Maternal antibodies are seen in neonatal circulation up to
2-4 months)
• Placenta crossing of IgG explains the Rh iso-immunization
• No placenta crossing in cows. But the colostrium (1st
day milk) contains good quantities of
antibodies which can be absorbed by newborn calves. Human colosstrium contains less
antibodies

13. • 2. Immunoglobulin M (IgM)
• These are macroglobulins or 19S Ig.
• They have 5 subunits, each having 4 peptide chains (10 H chains and 10L chains)
joined together by a J-chain polypeptide
• They can combine with 5 antigens simultaneously, thus very effective in bacteria
agglutination
• They can not come out of vascular space, being a large molecule. It’s the
predominant class in primary immune response,
• Natural antibodies are IgM in nature. They are produced without any known
antigenic stimulation and hence called natural antibodies. E. g. a person having
blood group A antigen will have ant-B antibodies in his circulation)
• They are IgM, thus can not cross placenta. The fetus even with incompartible
antigen, is protected from natural antibodies of the mother

14. • 3. Immunoglobulin A (IgA)
• They are usually dimers (4 H and 4 L chains) joined by J-chain
• They are the secretory antibodies seen in seromucous secretions of GIT,
nasopharyngeal tract, urogenital tract, tears, saliva, sweat etc
• IgA dimers are stabilized against proteolytic enzymes by the secretory piece.
Secretory piece is produced in the liver, it reaches the intestinal mucosa cells and
combine with IgA dimers to form the secretory IgA which is then released.

15. • 4. Immunoglobulin E (IgE)
• These are cytophilic antibodies. They mediate allergy, hypersensitivity and
anaphylaxis
• They are able to fix on mast cells and basophils
• When certain antigens e.g. penicillin are injected a few times, IgE antibodies are
produced, which fix on mast cells. When the sane chemical is injected next time, the
antigen fix on the antibodies causing mast cell degranulation and release of histamine
and slow reacting substances
• This leads to vasodilation, hypotension and bronchiolar constriction. This is the basis
for penicillin anaphylaxis, hay fever by fungus, asthma by pollen untricaria by
absorbed food elements. These are called immediate type hypersensitivity, because
the reaction peak is about 30 mins.
• IgE level in serum is increased markedly in helminthic infections

16. • 5. Immunoglobulin D (IgD)
• IgD immunological functions are not known
• Its found on lymphocyte surface
• 0.2% of total Ig
• NOTE: Paraproteinemias characterized by increased Ig production, such as Multiple
myeloma, Bence-Jones proteinuria, macroglobulinemia (increased IgM) and hyper
gamma globulinemia are seen in certain disease conditions and infections

17. Functions of Immunoglobulins
IgG IgA IgM IgD IgE
• Placenta transfer + _ _ _ _
• Complement fixation + + ++ _ _
• Agglutination + ++ ++ _ _
• Binding to macrophages + _ _ _ _
• Fixing to Mast cells _ _ _ _ +
• Primary response antibody _ _ + _ _
• External Secretions _ + _ _ _
• Natural antibodies _ _ + _ _

18. Complement System
• Complement factors are proteins present in serum, and they form the complement
system thet mediate cell lysis by antibody.
• There are 9 components , designated as C1 to C9
• Most of the components are thermolabile. Complement activity is abolished in serum
incubated at 56oC for 30mins
• Half-life of the system at room temperature is a few hours.
• Complement Activation
• When Ig is bound to the bacteria, it fixes the first complement components C1. C1 has 3
subunits: C1q, C1r and C1s stabilized by Ca++
• C1q binds with the antiboby. C1q has 18 peptide chains forming 6 bramches which can fix
antibody subunit chains.
• C1r and C1s are bound. C1s (C1 esterase) when activated becomes proteolytic in action. It
cleaves the next component C4

20. Complement Activation contd
• A multiple enzyme cascade system is thus activated leading to chemical
amplification of tne original message
• When the final component is activated, it creates microscopic holes in the
target cell membrane. Water enters by osmosis through this pores and cause
lysis of the target cells
• Breakdown products C3a and C5a activate phargocytosis, chemotaxis and
immune adherence while increasing capillary permeabiliyy
• This is the classical pathway of activation
• Microbial polysacharides can activate the properdin system which bypasses the
initial complement components. This is the Alternate pathway. It acts as the
first line of defense against microorganisms.
• Another pathway of activation is the Lectin pathway. Ita activation is antibody-
independent. It occurs when Mannose binding Lectin (MBL) binds to mannose,
fucose or N-acetylglucosamine on bacteria, yeast or viruses

21. Types of Immune Responses
• A. Primary response: when an antigen is injected, antibodies in blood
appear within 10 days, and reach a peak within 20 days. Response declines
in 30 days. IgM molecules are predominant in this primary response.
• B. Secondary Response: When the same antigen is injected into the same
animal after a few months, the antibody response is
• Quicker (within 3 days)
• Stronger (100 to 1000 times more antibody)
• More avid (IgG types)
• More prolonged (response lasting for months)
• This is the secondary immune response. It is due to memory cells produced
in the primary response, and it is the basis of immunisation

22. • C. Active Immunity: It is induced by immunization with toxoid or killed
or attenuated organisms e.g. Diphteria pertussis tetanus (DPT)
vaccine, oral polio vaccine and hepatitis B vaccine.
• D. Passive Immunity: Protection is given by preformed antibodies. This
is used in immunotherapy against diphtheria, tetanus, snake bites etc.