immunity, types,Innate immunity and Adaptive Immunity, primary and secondary immune response, structure and functions of antibodies, immunoglobulins, hypergammaglobulinemia, multiple myeloma, bence jones protein, electrophoretic pattern of multiple myeloma.

1. Ms. Meghalatha T S
Assistant professor
Department of Biochemistry
ZMCH, Dahod
Immunochemistry

2. Introduction - immunology
• The Study Of Immune System
• Latin Word immunis=“exempt”
• Pasteur Was First To Successfully Apply Vaccination
• Emil von Behring introduced antibody therapy
• Paul Ehrlich awarded Nobel prize in 1908 for explaining
antibody production.
• Peter Doherty & Rolf Zinkernagel shared the Nobel prize
(1996) for specificity of cell mediated immune response

3. Terminology in immunology
• Antibody: a protein produced by the human immune
system to tag and destroy invasive microbes.
• Antibiotic: various chemicals produced by certain soil mi
crobes that are toxic to many bacteria. Some we use as
medicines.
• Antigen: any foreign substances which introduced paren
terally.

4. Definitions
*Immune system = cells, tissues, and molecules that mediate
resistance to infections
*Immunology = study of structure and function of the immune
system
*Immunity = resistance of a host to pathogens and their toxic
effects
*Immune response = collective and coordinated response to
the introduction of foreign substances in an individual
mediated by the cells and molecules of the immune system

5. Immunity
• Immunity is body's ability to resist or eliminate
potentially harmful foreign materials or abnormal
cells

6. • Immunity (immunis- Latin-exempt, state of protection from
infectious diseases)
• Immunity is body's ability to resist or eliminate potentially
harmful foreign materials or abnormal cells
• consists of following activities:
– Defense against invading pathogens (viruses & bacteria)
– Removal of 'worn-out' cells (e.g., old RBCs) & tissue debris (e.g., from
injury or disease)
– Identification & destruction of abnormal or mutant cells (primary
defense against cancer)
– Rejection of 'foreign' cells (e.g., organ transplant)
– Inappropriate responses:
• Allergies - response to normally harmless substances
• Autoimmune diseases

7. Overview of the Immune System

8. Innate immunity vs Adaptive Immunity
Innate Immunity
(first line of defence)
Adaptive Immunity
(second line of defence)
No time lag A lag period
Not antigen specific Antigen specific
No memory Development of memory

9. Structure and function of immune system
• It comprises a variety of organs which are responsible
for growth & development of immunocompetent cells.
• They are lymphoid organs & reticuloendothelial system
Lymphoreticular system
Lymphoid system Reticuloendothelial System
Lymphoid Cells Reticuloendothelial components
Lymphocytes & plasma cells Phagocytic cells
specific immune response Nonspecific immunity(Phagocytosis)

10. The lymphoid system
• Lymphocytes are the major immunological effector cells.
• They arise from stem cells found in liver & bone marrow
Lymphoid organs
Primary/Central Secondary/Peripheral
Thymus Lymph nodes
Bursa of Fabricius Spleen
Intestinal epithelium MALT (mucosa associated lymphoid tissue)

11. Central/Primary
• These are the lymphoid organs i
n which prolifer-ation and differe
ntiation of lymphocytes takes
place without antigenic
stimulation
• T-lymphocytes mature in thymus
• B-lymphocytes mature at bone
marrow
Peripheral/ Secondary
• These are the organs, which
receive and maintain functional
lymphocytes.
• They initiate & participate in
immune response to antigenic
stimuli.

12. Cells of the immune system
• B- lymphocytes
o Originate from precursor cells from the yolk sac, fetal liver and bo
ne marrow
o During maturation, the pre b-cell is programmed to produce only
one class/subclass of Ig after a switch from initial IgM production.
o On the basis of immunoglobulin, which is programmed to synthes
ize, B-lymphocytes can be subdivided into nine different subsets: I
gM, IgD, IgG1, IgG2,IgG3, IgG4, IgA1, IgA2 & IgE
o B-cells process specific receptors on their surfaces, known as
B-cell receptors.

13. T-Lymphocytes
• T-cells originate from precursor cells from yolk sac, fetal liver and
bone marrow and migrate to thymus and mature there.
• Several subsets of T-cells arise during maturation process each on
e is responsible for a specific function
• The earliest identifiable T-cells are the CD7+ pro T cells, which ac
quire CD2 on entering the thymus
• They synthesize CD3 in the cytoplasm and become pre T-cells
• They also synthesize T-cell receptor

14. Immune response
Humoral Or Cellular Immunity?
• Pasteur Did Not Know How Vaccination Worked
• Behring and Kitasato (1890) Proposed Serum Was
Responsible For Immunity
• Elvin Kabat (1930), gamma-globulin, Antibody
• Antibodies Were Present in Body Fluids=Humor
• Therefore: Humoral Immunity

15. • Cellular/cell mediated immunity:
T – lymphocytes can directly kill the target cells
The major activities of T-lymphocytes are: immunity against infections,
rejection of allograft, tumor cell destruction, helper functions &
suppressor function
• Humoral immunity:
Antibodies are produced by the plasma cells.
These are immunoglobulins.

16. Innate (Non-Specific) Immunity
• Innate Immunity Made Up Of 4 Forms
• Anatomical, physiological, phagocytic and inflammatory
• Anatomical: skin, epidermis (densely packed dead cells)
• Flow of Mucus Prevents Bacterial Entry By Washing
Them Away
• Normal Flora Colonize Epithelial Cells Of Mucosal
Surfaces, Pathogens Compete With Them For Attachment
Sites

18. • Physiologic Barriers
– pH (stomach)
– Temperature (fever)
– Soluble Factors (interferons, lysozyme)
• Phagocytic Barriers
– Specialized Cells Perform Most Of Phagocytosis
(macrophages, neutrophils)

20. Cell Mediated Immunity
• In 1883 Ellie Metchnikoff Showed That Cells
Responsible For Immune State
• Phagocytes More Active In Immune Animals
• She Hypothesized That Cells Responsible For
Immunity, Not Serum Components
• Controversy Developed But Humoral School
Prevailed Till 1940
• Merrill Chase Expt (1940) with Tuberculosis Infected
Animals, Immunity Thru White Blood Cell Transfers

21. Immunogens and antigens
• Immunogen / antigen: a substance that elicits
an immune response [i.e. a humoral (antibody
response) or cell-mediated immune response]
Immune response generator

22. What is an antibody?

23. What does an antibody look like ?
ConstantregionVariableregion
2 identical heavy chains
2 identical light chains
Each heavy chain – has a constant and a
variable region
Each light chain has a constant and a
variable region

24. Antibody: structure and function
• Fab – fragment antigen
binding
• Fc- Fragment constant

25. • digestion of an immunoglobulin by the
enzyme papain produces two antigen-binding
fragments (Fab) and one crystallizable
fragment (Fc), which is responsible for
functions of immunoglobulins other than
direct binding of antigens.

27. Antibody: Fab
Fab region
Variable region of the antibody
Tip of the antibody
Binds the antigen
Specificity of antigen binding determined
by
VH and VL

28. Antibodies exist in two forms
Antibodies occur in 2 forms
• Soluble Ab: secreted in blood and tissue
• Membrane-bound Ab: found on surface of
B-cell, also known as a B-cell receptor (BCR)

29. Antibody: Fc
Fc region
• Constant region
• Base of the antibody
• Can bind cell receptors and
complement proteins

30. • Both the heavy and light chains
contain relatively variable (V) and
constant (C) regions with regard
to their amino acid composition.
• VL and CL are the general terms
for these regions on the light chain
; while VH and CH specify variable
and constant regions on the heavy
chain

31. Different classes of immunoglobulins
Depending on the heavy chain make up, the immuno
globulins are differentiated into 5 major classes.
• 1. Immunoglobulin-G (lgG) is made up of heavy
chain γ(gamma)
• 2. lgM has µ (mu) heavy chain
• 3. lgA has α (alpha) heavy chain
• 4. lgD contains δ (delta)
• 5. lgE heavy chain is called ε (epsilon).

32. IgG [HEAVY CHAIN γ]
• Monomer
• Major class of Ig in serum -70% of total
• Main antibody in the secondary response
• Constitutes important defence against
bacteria and viruses
• Only antibody that crosses the placenta
• Maternal antibody that protects the foetus

33. IgA [HEAVY CHAIN α]
• Monomer or dimer
• IgA occurs in two forms
 Secretory IgA and [Dimer]
 Serum IgA [Monomer]
• Second most abundant class – 20% of serum Ig
• Major component of colostrum
• They are the secretory antibodies so present in saliva, tears and respiratory,
intestinal and genital secretions
• Protects mucous membrane from antigenic attack
• In dimer form the J-chain connects the monomers.

34. IgM [HEAVY CHAIN μ]
• Monomer or pentamer
• 8-10% of normal serum
immunoglobulins
• Produced early in the
primary response to an
antigen
• Activate complement,
promotes phagocytosis and
causes lysis of antigenic cells
(bacteria

35. IgD [HEAVY CHAIN δ]
• Monomer
• Less than 1% of serum Ig
• No known antibody
function
• May function as an
antigen receptor
• IgD is labile molecule

36. IgE [HEAVY CHAIN ε]
• IgE exists as a monomer.
• Trace amount in serum
0.004%
• Involved in allergic
reactions
• Binds very tightly
basophils and mast cells

38. Sequential IgM-IgG humoral response
•IgM
– produced as a first response to many antigens
– levels remain high transiently
•IgG
– produced after IgM
– higher levels persist in small amounts throughout life
– produced in large amounts during secondary response
• persistence of antigen sensitive ‘memory cells’
after primary response

40. Primary and Secondary Immune Responses
• When an antigen is injected, antibodies in blood appear
within about 10 days, reach a peak level within 20 days
and response declines by about 30 days. The lgM
molecules will be predominant in this primary response.
• When the same antigen is reinjected into the same animal
after a few months, the antibody response is quicker
(within 3 days), stronger (100 to 1000 times more quantit
y of antibody), more avid (lgG type) and more prolonged
(response lasts for months). This is the secondary
immune response, which is due to the memory cells
produced in the primary response

41. • Active immunity is induced by immunization with
toxoid, or killed or attenuated organisms.
Examples are diphtheria, pertussis, tetanus (DPT)
vaccine, oral polio vaccine and hepatitis B vaccine.
• In passive immunity, protection is given by
preformed antibodies. This is used in immunotherapy
against diphtheria, tetanus, snake bites, etc.

42. Primary and secondary immune responses
• Primary
• Appears in 10 days and reach peak in 20 days.
• IgM will be predominant
• Secondary
• If the same antigen attacks , Ab response is
quicker in 3 days
• Its stronger and more prolonged

43. hypergammaglobulinemia
• It is an uncommon condition that is usua
lly the result of an infection, autoimmune
disorder or malignancy such as multiple
myeloma.
• Its characterized by elevated levels of im
munoglobulins in blood.
•

44. Causes Hypergammaglobulinemia
 Chronic infections, where antibody production is high. Examples are
leprosy, tuberculosis, syphilis, malaria, kala-azar, subacute bacterial
endocarditis.
 Aberrant immune reactions, such as rheumatoid arthritis, collagen dis
orders, glomerulonephritis, and such autoimmune disorders where
cryoglobulins may also be present.
 Paraproteinemias such as in multiple myeloma and Waldenstrom’s
macroglobulinemia.
 Monoclonal Gammopathy of undetermined significance (MGUS) is
characterized by hypergammaglobulinemia and M band on electroph-
oresis. But these patients do not have any end organ disease like
anaemia or bone involvement. The plasma cells in bone marrow is less
than 10%. They, however, have a higher chance of progressing to
multiple myeloma.

45. Multiple Myeloma (Plasmacytoma)
• When Ig-secreting cells are transformed into malignant c
ells, one clone alone is enormously proliferated.
• Thus, Ig molecules of the very same type are produced i
n large quantities. This is seen on electrophoresis as the
myeloma band or monoclonal band or M band with a s
harp narrow spike.
• Multiple myeloma is characterized by paraproteinemia,
anemia, lytic bone lesions and proteinuria. Bone marro
w examination reveals large number of malignant plasm
a cells.

47. Cont…
• Raised beta-2-microglobulin (Mol.wt. 11,800 D) is another
feature of multiple myeloma
• Total Ig content may be very high; but useful antibodies
may be very low, so that general immunity is depressed
and recurrent infections are common.
• The prognosis is generally good, and average survival is
more than 5 years with adequate chemotherapy and local
irradiation.

48. Bence-Jones Proteinuria
• Henry Bence-Jones described it in 1848. This disorder is seen in
20% of patients with multiple myeloma.
• Monoclonal light chains are excreted in urine. This is due to
asynchronous production of H and L chains or due to deletion of
portions of L chains, so that they cannot combine with H chains.
• The Bence-Jones proteins have the special property of precipitation
when heated between 45oC and 60oC; but re-dissolving at higher
than 80oC and lower than 45oC.
• These proteins may block kidney tubules, leading to renal failure.
So, myeloma with Bence-Jones proteinuria has poor prognosis.

49. • Bradshaw’s test is also positive, when a
few mL of urine is layered over a few mL
of concentrated hydrochloric acid, a white
ring of precipitate is formed.

50. How useful is Electrophoresis of serum/plasma
proteins (pH 8.6)?
• Electrophoresis may be used to study protein a
bnormalities;
• Serum is a better choice for Electrophoresis, becau
se the Fibrinogen of Plasma gives a discrete band,
which can easily be mistaken for Paraproteins
• Shows order of migration along Horizontal Axis with
proteins of highest mobility closest to Anode,

51. • Height of the band along the Vertical Axis shows
the protein concentration,
• Location of some major proteins are indicated
underneath their Electrophoretic mobility peaks.
• Electrophoresis can also show gross deficiency or
excess of Immunoglobulins and whether
Para-proteins are present
• Quantitative measure of each protein class may be
obtained by scanning Electrophoretic strip

52. Electrophoresis of Serum proteins: (a) Normal pattern, (b) Presence of Paraproteins,
(c) Presence of Para proteins

53. Scan of an Electrophoretic strip (Gawet al 1999)