mother fucker

1. BCM 217
1

2. Course Outline
 Purpose of the Course
 The course is designed to introduce the students to basic and
contemporary concepts in immunology with the emphasis on
clinical applications and disease management.
 Expected Learning Outcomes
 At the end of the course the students should be able to:
1. Describe the basic principles of immunology, with emphasis on clinical
applications.
2. Identify and describe the common diseases associated with immune
deficiency, autoimmunity and hypersensitivity.
3. Describe the transplantation and immune reaction immunology in
relation to blood transfusion, tissue and organ transplantation and
malignancies.
4. Demonstrate understanding of the principles of immunology in disease
prevention
2

3. Course Content
 Introduction to clinical immunology and immunological applications in
diagnosis, management and control of human diseases;
 Immunoglobulins: Structure, functions, production and properties of
immunoglobulins;
 Reproductive immunology: Immunology of breast milk and breastfeeding;
 Immunopathology: Hypersensitivity reactions; Autoimmunity;
Immunodeficiency diseases; Transplantation and tumour immunology;
 Complement system: activation cascades, biological functions of activation
products and diseases associated with the complement system components;
 Immunity and immunopathology of parasitic, bacterial, viral and fungal
infections and their immune evasion mechanisms;
 Antigen-antibody reactions: immunodiagnostic tests; Immunization and
vaccination; Immunotherapy
3

4. Part One
4

5.  Immunoglobulins (Ig)
 Glycoproteins molecules produced by plasma cells in
response to an immunogen
 Function as abs
 Ig derive their name in that they migrate with globular
proteins when ab-containing serum is placed in an electric
field
 All are build from same basic units though they differ
structurally
 Structural differences dictate their specific biological and
physicochemical properties
 All Igs have four chain structure as the basic unit
 Are composed of 2 identical light chains (23kD) and 2
identical heavy chains (50-70kD)
5

6. 6

7. Antibody Structure
 Antibodies Are Made Up Of:
 2 Light Chains (identical) ~25 KDa
 2 Heavy Chains (identical) ~50 KDa
 Each Light chain bound to heavy chain by disulfide
bonds (H-L)
 Heavy Chain bound to heavy chain through hydrogen
bonds(H-H)
 First 100 a/a of amino terminal vary of both h and l
chain are variable
 Referred To As VL , VH, CH And CL
 CDR (Complementarity Determining Regions) sites
that bind the Ag
 Remaining regions are very similar within same class
7

8.  Held together by inter-chain and intra-chain disulphide bonds
 Have variable (V) and constant (C) regions on the two chains
 The V region has the hypervariable (HVR) or complementarity
determining regions (CDR)
 This is a region which binds the antigen
 There is the Hinge region at which the arms of the ab molecule
form a Y. there is some flexibility at this point
 Domains – the Ig molecule is folded into globular regions known
as domains, where we have intra-chain disulphide bonds.
 Digestion with papain breaks the Ig in the hinge region and yield
2 fragments
 Fab – fragment ag binding and
 Fc – fragment crystallisable
8

9. • Structural conservation and a capacity for infinite
variability in a single molecule is provided by a
DOMAIN structure.
• Ig domains are derived from a single ancestral gene
that has duplicated, diversified and been modified to
endow an assortment of functional qualities on a
common basic structure.
• Ig domains are not restricted to immunoglobulins.
• The most striking characteristic of the Ig domain is a
disulphide bond - linked structure of 110 amino acids
long.
Immunoglobulin domains
9

10. S
S
S
S
S
S
S
S
Fc Fab
F(ab)2
Domains are folded, compact, protease resistant structures
Domain Structure of Immunoglobulins
Pepsin cleavage sites - 1 x (Fab)2 & 1 x Fc
Papain cleavage sites - 2 x Fab 1 x Fc
Light chain C
domains
k or l
Heavy chain C
domains
a, d, e, g, or m

11. 11

12.  Fab contain the ag binding sites – paratope or idiotype
to bind the ag epitope
 The CDR is created by both VH and VL
 Fc is easily crystallized and is the region which
mediates ab effector functions. This normally require
prior ag binding though not always.
12

13. General functions of Igs
 Abs function in a variety of ways designed to eliminate the
ag which elicited their production
 Ag binding – each Ig binds to a specific Ag determinant
 Ag binding is the primary function of abs and results in
protection of the host
 Effector functions – ag binding generally has no
biological effects but significantly it is the consequence of
effector functions which include;
 Complement fixation – IgM and IgG
 Binding of various effector cell types like phagocytes,
lymphocytes, platelets, mast cells and basophils
 The effector cells bears specific ab receptors (FcRs) and are
activated the moment the receptor is bound.
13

14. Immunoglobulin Structure-Function
Relationship
 Cell surface antigen receptor on B cells
 Allows B cells to sense their antigenic environment
 Connects extracellular space with intracellular signalling
machinery
 Secreted antibody
 Neutralisation
 Arming/recruiting effector cells
 Complement fixation
14

15. Immunoglobulins are Bifunctional Proteins
• Immunoglobulins must interact with a small number of
specialised molecules -
• Fc receptors on cells
• Complement proteins
• Intracellular cell signalling molecules
• whilst simultaneously recognising an infinite array of
antigenic determinants.
15

16. Antibodies have 4 main mechanisms of action:
 Precipitation, Lysis, Agglutination, and Neutralization.
 Precipitation occurs when antibodies bind soluble
antigens into clumps. This increases the likelihood of
phagocytosis.
 Lysis occurs when antibodies activate complement. This
results in the formation of a membrane attack complex,
and bursting of the bacterial cell.
 Agglutination occurs when antibodies bind cell-bound
antigens into clumps. This increases the likelihood of
phagocytosis.
 Neutralization occurs when antibodies bind to and mask
the dangerous portions of antigens, toxins, and viruses.
16

17. TYPES OF ANTIBODY
 IgM, (Mu) µ - heavy chains, 3rd most common
 First immunoglobulin to appear after exposure to antigen.
 Indicates new infection.
 Activates Complement,
 Big, can not cross placenta, is a pentamer
 Naturally occurring IgMs against A and B blood antigens
 IgG, (gammaglobulin) ɣ - heavy chains, 75% of serum Ig
 Most common
 Small in molecular weight
 Only produced in large amount during the secondary immune response.
 (The secondary immune response is after immunological memory has
taken place)
 The more exposure to antigen, the more IgG.
 Crosses the placenta.
 Acts as an opsin (coats the bacteria so it is easily phagocytized.
 4 subclasses – IgG1, IgG2, IgG3 and IgG4
17

18.  IgA, (alpha) α – heavy chains, 2nd most common
 Found in secretions (mucous, milk, eyes, sweat, etc.).
 Offers primary protection in mucous membranes
 Is protected from digestive enzymes.
 Is proceeded by IgM.
 2 subclasses – IgA1 and IgA2
 IgE, (epsilon) Ɛ – heavy chains,
 Least common, Only a small amount in serum.
 Usually found in tissue attached to Mast cells.
 Mediates allergic reactions.
 Important in fighting parasites.
 Systemic release can cause anaphylactic shock.
 IgD, (delta) delta heavy chains
 Not released into serum or body fluids.
 Found on B-cell, is a B cell ag receptor on naïve B cells
 Helps the B cell become antigentically active.
18

19. 30 strongly neutralising McAb
60 strongly neutralising McAb Fab regions 60 weakly neutralising McAb Fab regions
Human Rhinovirus 14
- a common cold virus
30nm
Models of
Human
Rhinovirus 14
neutralised by
monoclonal
antibodies

20. Electron micrographs of Antibodies
and complement opsonising
Epstein Barr Virus (EBV)
Negatively stained EBV
EBV coated with a corona of
anti-EBV antibodies
EBV coated with antibodies and
activated complement components

21. Antibody + complement- mediated
damage to E. coli
Healthy E. coli
Electron micrographs of the effect of antibodies and
complement upon bacteria

22. Generation of Ab diversity
 The immune system has capacity to recognise and
respond to about 107 different ags
 This extreme diversity can be generated in two
possible ways
 Variable recombination during B cell development
 Mutation during B cell differentiation and development
22

23.  Immunize animal with antigen
 Multiple clones are generated, good for in vivo
protection
 For clinical diagnosis, research, one clone that
reacts to single epitope is preferred
 Solution by Kohler and Milstein
 Fuse a Myeloma Cell (Cancerous) with a normal
plasma cells
 Resulting clones can be cultured indefinitely
 Produces an antibody recognizing one epitope
Monoclonal Antibodies
23

24. 24

25. The types of mAb designed
A. Murine source mAbs: rodent mAbs with excellent
affinities and specificities, generated using
conventional hydrioma technology. Clinical efficacy
compromised by HAMA(human anti murine antibody)
response, which lead to allergic or immune complex
herpersensitivities.
B. Chimeric mAbs: chimers combine the human constant
regions with the intact rodent variable regions. Affinity
and specificity unchanged. Also cause human
antichimeric antibody response (30% murine resource)
C.Humanized mAbs: contain only the CDRs of the
rodent variable region grafted onto human variable
region framework
25

26. Monoclonal Antibody Production
26

27. Antibody
 An antibody is a protein used by the immune system to
identify and neutralize foreign objects like bacteria and
viruses. Each antibody recognizes a specific antigen unique
to its target.
 Monoclonal antibodies (mAb) are antibodies that are
identical because they were produced by one type of
immune cell, all clones of a single parent cell.
 Polyclonal antibodies are antibodies that are derived
from different cell lines.
 Isotypes – According to differences in their heavy chain
constant domains, immunoglobulins are grouped into five
classes, or isotypes: IgG, IgA, IgM, IgD, and IgE.
27

28. Monoclonal Antibodies
 Monoclonal antibodies (mAb) are antibodies that are
identical because they were produced by one type of
immune cell, all clones of a single parent cell
 Given (almost) any substance, it is possible to create
monoclonal antibodies that specifically bind to that
substance; they can then serve to detect or purify that
substance
 This has become an important tool in biochemistry,
molecular biology and medicine

29. The structure of antibodies
 http://www.path.cam.ac.uk/~mrc7/igs/mikeimages.html

31. History of Mab development
 1890 Von Behring and Kitasato discovered the serum of
vaccinated persons contained certain substances, termed
antibodies
 1900 Ehrlich proposed the “ side-chain theory”
 1955 Jerne postulated natural selection theory. Frank Macfarlane
Burnet expended.
 Almost the same time, Porter isolated fragment of antigen
binding (Fab) and fragment crystalline (Fc) from rabbit y-
globulin.
 1964 Littlefield developed a way to isolate hybrid cells from 2
parent cell lines using the hypoxanthine-aminopterin-thymidine
(HAT) selection media.
 1975 Kohler and Milstein provided the most outstanding proof
of the clonal selection theory by fusion of normal and malignant
cells.
 This resulted in the first monoclonal antibodies, for which they
received the Nobel Prize in 1984.
31

32. Hybridoma technology
 It is technique of producing hybrid cell lines called
“hybridomas” by the fusion of fusing a specific
antibody-producing lymphocyte B cell with a myeloma
cell that has an ability to grow in tissue culture
 Hydridoma produce antibodies produced that have
single specificity and are called monoclonal
antibodies.
 This technique was discovered Georges Kohler of West
Germany, Cesal Milstein of Argentina and Niels Jerne
of Denmark in 1975.
 They were awarded Nobel Prize for Physiology and
Medicine the 1984
32

33. Principal
 This is based on fusion between myeoma cell (malignant plasma
cell) and spleen cell from suitable immunised animal.
 Spleen can produce antibody but die in short period under culture
condition
 Myeloma cell are adapted to grow permanently in cell culture
 Myeloma cells cannot synthesize antibodies as they lack HGPRT
gene required for the synthesize the enzyme, hypoxanthine
guanine phosphoribosyl transferase (azaquinine resistant) or
thymidine kinase (bromodeoxy uridine resistant)
 Mutant myeloma cell can not grow in HAT (Hypoxanthine
Aminopterin Thymidine) medium
 Only hybrid cell can grow in HAT medium and produce
monoclonal antibody

34. Method
1. Immunization of a lab animal
2. Isolation of B cells from the
spleen
3. Cultivation of myeloma cells
4. Fusion of myeloma and B cells
5. Separation of cell lines
6. Screening of suitable cell lines
7. in vitro (a) or in vivo (b)
multiplication
8. Harvesting

35. Immunization of a lab animal
 Immunize a rabbit through repeated injection of a
specific antigen for the production of specific
antibody, facilitated due to proliferation of the desired
B cells
 Produce tumors in a mouse or a rabbit
 Culture separately the spleen cells that produce
specific antibodies and the myeloma cells that produce
tumors
35

36. 36

37. Fusion
 Fusion of spleen cells to myeloma cells is induced using
polyethylene glycol (PEG), to produce hybridoma
 Hybridomas are grown in selective hypoxanthine
aminopterin thymidine (HAT) medium.
 HAT medium contains a chemical, aminopterin that blocks
one pathway for nucleotide synthesis, making the cells
dependent on another pathway that needs HGPRT enzyme,
which is absent in myeloma cells.
 Myeloma cells that do not fuse with B cells will die.
 B cells that do not fuse with myeloma cells will also die
because they lack tumorigenic property of immortal
growth.
37

38. HAT selection is used to select for growth of hybrids and against
the growth of the parental myeloma.
38

39. Selection and Storage
 Select desired hybridoma for cloning and antibody
production
 Prepare single cell colonies that can grow and use them to
screen of antibody producing hybridomas
 Only one in several hundred cell hybrids will produce
antibodies of the desired specificity
 Culture selected hybridoma cells for the production of
monoclonal antibodies in large quantities
 Hybridoma cells can be frozen for future use or can be
injected in the body of an animal, so that antibodies will
be produced in the body and recovered later from the body
fluid.
39

40. 40

41. Applications of Polyclonal and Monoclonal antibodies
 Applications include simple qualitative and/or
quantitative analyses to ascertain the following:
(1) whether an epitope is present within a solution,
cell, tissue, or organism, and if so, where.
(2) methods to facilitate purification of an antigen,
antigen associated molecules, or cells expressing an
antigen
(3) techniques that use antibodies to mediate and/or
modulate physiological effects for research,
diagnostic, or therapeutic purposes.
41

42. Monoclonal Antibody Applications
 Diagnostic Tests
 Abs are capable to detect tiny amounts (pg/mL) of
molecules and highly specific
 E.g. Pregnancy hormones
 Diagnostic Imaging
 mAbs that recognize tumor antigens are radiolabeled
with iodine I-131
 Immunotoxins
 mAbs conjugated with toxins, therapeutic
 mAbs used to clear pathogens
 Therapeutic and prophylactic uses
42

43. Techniques that employ mAbs
 Radioimmune Assay (RIA)
 Enyzme Linked Immune Sorbant Assay (ELISA)
 Western blot
 Immunoprecipitation
 Flow cytometry
 Expression cloning
43

44. Conclusion
Antibodies have provided and will continue to provide
scientists and clinicians an extraordinarily powerful and
important tool for use in the research laboratory and
clinic. The unique molecular structure of the antibody
by which it bivalently binds to a broad array of antigenic
epitopes (on, e.g., proteins, carbohydrates, and nucleic
acids) serves as the foundation of its utility.
44

45. Part Two
45

46. Introduction
 The complement system is part of the innate immune
system (vs adaptive)
 It is named “complement system” because it was first
identified as a heat-labile component of serum that
“complemented” antibodies in the killing of bacteria
 Complement refers to a group of plasma proteins synthesized by
the liver.
 They’re normally found in the blood in an inactive state.
 They may be activated by interacting directly with a pathogen or
by members of the adaptive immune system.
 It is now known that it consists of over 30 proteins and
contributes 3 g/L to overall serum protein quantities
46

47. Discovery of Complement
 In 1984, Pfeiffer, demonstrated that some cholera vibrios
can be lysed by guinea pig anti-cholera serum.
 Heating of the serum at 56 0C for 30 minutes abolished
this activity but heating did not abolish the Ab activity,
because heated serum serum could still transfer immunity
to another guinea pig.
 Addition of normal fresh serum to the heat treated
antiserum restored its lytic activity.
 He concluded that antibodies to the bacilli, plus a heat
labile component present in immune as well as normal
serum, were necessary for the lysis of V. cholerae in vitro.
 Subsequently Bordet and Ehrlich confirmed that the same
result could be obtained in vitro, suspending vibrios in the
presence of fresh immune serum
47

48. History of Complement
 Jules Jean Baptiste Vincent Bordet • (Belgium) 13 June
1870 – 6 April 1961) won noble prize in 1919 for
discovery in immunity
48

49. Complement activation
 A system of plasma proteins that interact with
 Antigen/antibody complexes
 Pathogen surface motifs (alternative and lectin pathways
 Activation of complement results in
 Chemo-attraction of inflammatory cells
 Peptide mediators of inflammation (anaphylatoxins)
 Increased blood vessel permeability
 Smooth muscle contraction
 Mast cell degranulation
 Opsonization of pathogens (enhances phagocytosis)
 Killing of pathogens (membrane attack complex)
49

50. Overview of the Complement Cascade
50

51. Effector
Actions of
Complement
51

52. Activation of complement results in 4 effector roles:
1. Chemotaxis – activated complement proteins attract
WBCs.
2. Opsonization – binding of activated complement
proteins to bacteria increases the likelihood of their
phagocytosis.
3. Inflammation – activated complement proteins bind to
basophils and mast cells and stimulate histamine release.
This results in vasodilation and increased capillary
permeability and inflammation.
4. Lysis – activated complement proteins can form a
“membrane attack complex,” which is a tube that pierces
the bacterial cell membrane.
 This allows salt and water to flow in and results in cell lysis.
52

53.  The inflammatory response occurs whenever tissues
are damaged.
 It helps to prevent pathogen spread, disposes of
pathogens and debris, and sets the stage for repair.
 The signs of inflammation include heat, redness,
swelling, pain, and loss of function.
 It begins when damaged tissues release inflammatory
chemicals (histamine, prostaglandins, leukotrienes,
kinins, etc.)
53

54. These chemicals act to:
 Increase WBC count,
 Increase local capillary permeability,
 Cause local vasodilation,
 Attract WBCs to the injury site, and
 Stimulate pain-sensitive neurons.
 The increase in vasodilation and capillary permeability
yields an increase in blood flow and capillary fluid loss.
 This results in swelling, heat, redness, and increased
access to the injury site by WBCs, complement proteins,
antibodies, and clotting proteins.
54

55. Beneficial effects of complement activation
 Beneficial effects of complement activation
1. Trigger inflammation
2. Chemotactically attract phagocytes to the infection site;
3. Promote the attachment of antigens to phagocytes
(enhanced attachment or opsonization)
4. Cause lysis of gram-negative bacteria and human cells
displaying foreign epitopes
5. Plays a role in the activation of naive B-lymphocytes (via
C3d) and
6. Remove harmful immune complexes from the body.
55

56. Classical Pathway
 Begins with antibody binding to a cell surface and ends with
the lysis of the cell
 The proteins in this pathway are named C1-C9 (the order
they were discovered and not the order of the reaction)
 When complement is activated it is split into two parts
 a – smaller of the two
 B – larger part and usually the active part (except with factor 2)
 Remember 3 Key Words
 ACTIVATION
 AMPLIFICATION
 ATTACK
56

57. Classical Pathway
 ACTIVATION
 C1q portion of C1 attaches to the Fc portion of an
antibody
 Only IgG and IgM can activate complement
 Once activated C1s is eventually cleaved which activates
C4 and C2
 C4b & C2a come together to form the C4b2a which is
the C3 convertase
 C3 convertase activates C3 to C3a and C3b
57

58. Classical Pathway
 ACTIVATION
 C3a binds to receptors on basophils and mast cells
triggering them to release there vasoactive compounds
(enhances vasodilation and vasopermeability)
 C3a is called an anaphylatoxin
 C3b serves as an opsonin which facilitates immune
complex clearance
58

59. Classical Pathway
 AMPLIFICATION
 Each C1s creates many C4b and C2a fragments
 Each C4bC2a creates many C3b (activated C3)
 Each C3b goes on to create many Membrane Attack
Complexes
 Example
 1 C1S makes 100 C4bC2a
 100 C4bC2a makes 10,000 C3b
 10,000 C3b makes 1,000,000 MAC
59

60. Classical Pathway
 ATTACK
 Most C3b serves an opsonin function
 Some C3b binds to C4bC2a to form the C5 convertase
C4bC2aC3b
 C5 convertase cleaves C5 leading to the formation of the
Membrane attack Complex (C5-C6-C7-C8-C9)
 The MAC “punches holes” in cell walls resulting in lysis
60

61. C1q
C2
C4
2a
2b
4b
4a
C3-convertase
C3
C3a
C3b
C5-Convertase
C3a binds to receptors on
basophils and mast cells
triggering them to release
there vasoactive compounds
(enhances vasodilation and
vasopermeability) -
ANAPHYLATOXIN
C5
C5a
C5b
C5a is a:
1. Potent anaphylatoxin
2. Chemoattractant for
neutrophils
C6
C7
C8
C9
Classical
Pathway 61

62. Alternative Pathway
 Requires no specific recognition of antigen in order to
cause activation
62

63. Alternative Pathway
 ACTIVATION
 Spontaneous conversion from C3 to C3b occurs in body
 Normally, C3b is very short lived and quickly inactivated
by proteins on the surface of the body’s own cell
 However, bacteria or other foreign material may lack
these surface proteins allowing C3b to bind and stay
active
63

64. Alternative Pathway
 AMPLIFICATION
 Factor B binds to C3b
 Factor B is then cleaved by factor D into Ba and Bb
 C3bBb remains which acts as a C3 convertase (C3  C3a
and C3b)
 C3bBbC3b is formed which acts as a C5 convertase
64

65. Alternative Pathway
 ATTACK
 C5 is cleaved to C5a and C5b
 C5b then starts the assembly of the Membrane Attack
Complex
65

66. C3
C3b
C3a
Anaphylatoxin
B
D
Bb Ba
C3
C3a C3b
C5-Convertase
C3-Convertase
C5
C5a
C5b
Alternative
Pathway
C6
C7
C8
C9
66

67. The Lectin Pathway
 The lectin pathway is mediated by mannan-binding lectin
(also known as mannan-binding protein or MBP).
 MBP is a protein that binds to the mannose groups found
in many microbial carbohydrates but not usually found in
the carbohydrates carbohydrates of humans.
 The MBP is equivalent equivalent to C 1 q in the classical
complement pathway.
 Activation of the lectin pathway begins when mannan-
binding protein (MBP) binds to the mannose groups of
microbial carbohydrates.
 This forms an enzyme similar to C1 of the classical
complement pathway that is able to cleave C4 and C2 to
form C4bC2a, the C3 convertase capable of enzymatically
splitting hundreds of molecules of C3 into C3a and C3b
67

68. Lectin Pathway
 Mannan Binding Protein is structurally very similar to
C1q but it can combine directly to any mannose groups
on the bacterial cell wall without the need for specific
antibodies.
 MASP is a protease capable of reacting with C4 & C2
resulting in the formation of C4bC2b, the same C3
convertase that is formed by the action of the Classical
Pathway
68

69. Activation of Lectin Pathway
 Activation of the lectin pathway begins when mannan-
binding protein (MBP) binds to the mannose groups
of the carbohydrates on microorganisms.
 Two more lectin pathway proteins called MASP1 and
MASP2 (equivalent to C1r and C1s of the classical
pathway) now bind to the MBP.
 This forms an enzyme similar to C1 of the classical
complement pathway that is able to cleave C4 and C2
to form C4bC2a, the C3 convertase that is able to
enzymatically split hundreds of molecules of C3 into
C3a and C3b
69

70. Complement Pathways
Activation pathway Classic Alternative Lectin
Activator Ag–Ab Complex
spontaneous
hydrolysis of C3
MBL-Mannose
Complex
C3-convertase C4b2a C3bBb C4b2a
C5-convertase C4b2a3b C3bC3bBb C4b2a3b
MAC development C5b+C6+C7+C8+C9
70

71. Complement mediated diseases
 Deficiency of C5-C8 & MBL – Enhances susceptibility
to Neisseria infections
 Deficiency of C3- – Severe recurrent pyogenic and
respiratory tract infections.
 Inherited Deficiency of C1 esterase inhibitors –
Angioedema
 Acquired Deficiency of Decay accelerating factor –
Paroxysmal nocturnal haemoglobinuria
 Mismatched ABO transfusion – Haemolysis, Shock
 Immune complex mediated disease –
Glomerulonephritis
71

72. Other Diseases
 It is thought that the complement system might play a
role in many diseases with an immune component,
such as;
 Barraquer–Simons syndrome,
 asthma,
 lupus erythematosus,
 various forms of arthritis,
 autoimmune heart disease,
 multiple sclerosis,
 inflammatory bowel disease.
72

73. Complement based therapies
 Considerations in development of therapies
 An imbalance by overactive or attenuated complement
activation has severe pathological implications.
 Enhancement or inhibition of complement creates a
powerful strategy for therapeutic intervention.
 Synergy between complement and cell-mediated
effector functions impacts therapeutic efficacy.
 Antibody combination therapies, engineering
approaches and optimized treatment designs provide
exciting opportunities for new and improved therapies.
73

74. Complement Inhibitors in Clinical Trials for
Glomerular Diseases
 Defective complement action is a cause of several human
glomerular diseases including atypical hemolytic uremic
syndrome (aHUS), anti-neutrophil cytoplasmic antibody
mediated vasculitis (ANCA), C3 glomerulopathy, IgA
nephropathy, immune complex membranoproliferative
glomerulonephritis, ischemic reperfusion injury, lupus nephritis,
membranous nephropathy, and chronic transplant mediated
glomerulopathy.
 Pathology of these kidney disorders are caused or modified by;
i. genetic alterations in complement genes that lead to impaired
protein expression and/or function,
ii. by autoantibodies that target complement components or
regulators,
iii. by autoantibodies which recognize specific surface structures,
DNA and IgGs, and upon binding initiate complement
activation.
74

75. 75
• Antibody-based treatments can be employed to restore the balance in the
complement network in order to achieve therapeutic effects (Fig).
• Complement inhibition can be beneficial in pathologies where the system
is hyperactivated (e.g. sepsis, transplant rejection, ischemia and
reperfusion (I/R) injury) or where it is chronically activated and attacks or
damages healthy tissues (e.g. autoimmune disease).
• It is envisioned that such inhibition can be achieved by targeting key
components of the complement cascade mediating the initiation,
amplification, and/or termination steps of the system.

76. Examples of Therapies
 The complement inhibitors Eculizumab (Soliris),
Berinert, or Cinryze are currently approved by the Food
and Drug administration (FDA) in the US and the
European Medicines Agency (EMA).
 A new generation of complement inhibitors is currently
evaluated in clinical trials and new inhibitors are being
developed and tested in preclinical settings.
 Different types of inhibitors exist, including;
 humanized monoclonal antibodies,
 small proteins binding to specific complement components,
 recombinant proteins allowing substitution of defective or
absent proteins,
 small interfering RNAs.
76

77.  Eculizimab (anti-CD20) is indicated in the following;
 Atypical Hemolytic uremic syndrome (aHUS); Paroxysmal
nocturnal hemoglobinuria
 Antibody-associated vasculitis; Antiphospholipid syndrome; Budd
Chiari syndrome; Chronic hemolysis; Cold agglutinin disease; Dry age
related macular degeneration; Glomerulonephritis; Heart transplant
rejection; Kidney transplant rejection; Kidney transplantation;
Myasthenia gravis
 Mubodina (anti C5)- Hemolytic uremic syndrome; Membrano-
proliferative glomerulonephritis
 Ergidina (anti-C5) - Ischemia; Reperfusion injury
 Lampalizumab (anti Factor D) - Age-related macular
degeneration
 IFX-1, CaCP-29 (Anti C5a) - Severe sepsis; Septic shock
 Rituximab (anti CD 20) - B-cell lymphoma; Chronic lymphocytic
leukemia; Microscopic polyangiitis; Non-Hodgkin lymphoma;
Rheumatoid arthritis; Wegener granulomatosis
77

78. Complement based Diagnostic tools
 Diagnostic tools to measure complement activity
include the total complement activity test.
 The presence or absence of complement fixation upon
a challenge can indicate whether particular antigens or
antibodies are present in the blood.
 This is the principle of the complement fixation test
78

79. Modulation by infections
 Recent research has suggested that the complement
system is manipulated during HIV/AIDS, in a way that
further damages the body (Datta PK, Rappaport J
(2006). "HIV and Complement: hijacking an immune
defense". Biomedicine & Pharmacotherapy. 60 (9):
561–8).
 This could indicate evolution of a strategy to evade the
immune system
79

80. Summary
 Complement consists of serum proteins in inactive
form, which when activated they act in concert, in
orderly sequence to exert their biological effects.
 The biological effects of complement activation
include – Cytolysis of target – Opsonization – Immune
adherence – Release of mediators of inflammation –
Chemotaxis
 There 3 pathways for activation – Classical – Alternate
– Lectin mediated
 Deficiency of complement is associated with many
diseases like SLE, increased susceptibility to infections
( gonococcal, nephritis).
80

81. Part Three
81

82. HYPERSENSITIVITY REACTIONS
 Hypersensitivity refers to excessive, undesirable (damaging,
discomfort-producing and sometimes fatal) reactions produced
by the normal immune system. Hypersensitivity reactions require
a pre-sensitized (immune) state of the host.
 Hypersensitivity is a heightened secondary reaction to an
antigen (allergen) which in most cases is actually harmless.
82

83.  Hypersensitivity reactions can be divided into four types:
type I, type II, type III and type IV, based on the
mechanisms involved and time taken for the reaction.
Frequently, a particular clinical condition (disease) may
involve more than one type of reaction.
83

84. 84
TYPE I HYPERSENSITIVITY
 Type I hypersensitivity is also known as immediate or
anaphylactic hypersensitivity.
 The reaction may involve skin (urticaria and eczema), eyes
(conjunctivitis), nasopharynx (rhinorrhea, rhinitis),
bronchopulmonary tissues (asthma) and gastrointestinal tract
(gastroenteritis).
 The reaction may cause a range of symptoms from minor
inconvenience to death.
 The reaction usually takes 15 - 30 minutes from the time of
exposure to the antigen, although sometimes it may have a
delayed onset (10 - 12 hours).

85.  Immediate hypersensitivity is mediated by IgE. The primary
cellular component in this hypersensitivity is the mast cell or
basophil.
 The reaction is amplified and/or modified by platelets,
neutrophils and eosinophils.
 A biopsy of the reaction site demonstrates mainly mast cells and
eosinophils.
85

86.  The mechanism of reaction involves preferential production of
IgE, in response to certain antigens (often called allergens).
 The precise mechanism as to why some individuals are more
prone to type-I hypersensitivity is not clear.
 However, it has been shown that such individuals preferentially
produce more of TH2 cells that secrete IL-4, IL-5 and IL-13
which in turn favour IgE class switch.
 IgE has very high affinity for its receptor (Fcε; CD23) on mast
cells and basophils.
86

87.  A subsequent exposure to the same allergen cross links the cell-
bound IgE and triggers the release of various pharmacologically
active substances.
 Cross-linking of IgE Fc-receptor is important in mast cell
triggering.
 Mast cell degranulation is preceded by increased Ca++ influx,
which is a crucial process; ionophores which increase
cytoplasmic Ca++ also promote degranulation, whereas, agents
which deplete cytoplasmic Ca++ suppress degranulation.
87

88. Slide 7.9
Activation of mast
cells in type I
hypersensitivity and
release of their
mediators. ECF,
eosinophil chemotactic
factor; NCF, neutrophil
chemotactic factor;
PAF, platelet-activating
factor. (From Robbins
Basic Pathology ,2003）
88

89. Pharmacologic Mediators of Immediate Hypersensitivity
MEDIATOR
Preformed mediators in granules
Histamine Bronchoconstriction, mucus secretion, vasodilatation, vascular permeability
Tryptase Proteolysis
Kininogenase Kinins and vasodilatation, vascular permeability, Oedema
ECF-A
(tetrapeptides)
Attract eosinophil and neutrophils
Newly formed mediators
Leukotriene B4 basophil attractant
Leukotriene C4, D4 same as histamine but 1000x more potent
Prostaglandins D2 edema and pain
PAF platelet aggregation and heparin release: microthrombi
89

90.  The reaction is amplified by PAF (platelet activation factor) which
causes platelet aggregation and release of histamine, heparin
and vasoactive amines.
 Eosinophil chemotactic factor of anaphylaxis (ECF-A) and
neutrophil chemotactic factors attract eosinophils and
neutrophils, respectively, which release various hydrolytic
enzymes that cause necrosis.
 Eosinophils may also control the local reaction by releasing
arylsulphatase, histaminase, phospholipase-D and
prostaglandin-E, although this role of eosinophils is now in
question.
90

91.  Diagnostic tests for immediate hypersensitivity include skin (prick
and intradermal) tests, measurement of total IgE and specific
IgE antibodies against the suspected allergens.
 Total IgE and specific IgE antibodies are measured by a
modification of enzyme immunoassay (ELISA).
 Increased IgE levels are indicative of an atopic condition,
although IgE may be elevated in some non-atopic diseases
(e.g., myelomas, helminthic infection, etc.).
91

92.  Symptomatic treatment is achieved with anti-histamines which block
histamine receptors. Chromolyn sodium inhibits mast cell
degranulation, probably, by inhibiting Ca++ influx.
 Epinephrine and corticosteroids are also used
 Late onset allergic symptoms, particularly bronchoconstriction which
is mediated by leukotrienes, are treated with leukotriene receptor
blockers (Singulair, Accolate) or inhibitors of the cyclooxygenase
pathway (Zileutoin).
 Symptomatic, although short term, relief from bronchoconstriction is
provided by bronchodilators (inhalants) such as isoproterenol
derivatives (Terbutaline, Albuterol).
 Thophylline elevates cAMP by inhibiting cAMP-phosphodiesterase
and inhibits intracellular Ca++ release is also used to relieve
bronchopulmonary symptoms.
92

93.  The use of IgG antibodies against the Fc portions of IgE that
binds to mast cells has been approved for treatment of certain
allergies, as it can block mast cell sensitization.
 Hyposensitization (immunotherapy or desensitization) is another
treatment modality which is successful in a number of allergies,
particularly to insect venoms and, to some extent, pollens.
 The mechanism is not clear, but there is a correlation between
appearance of IgG (blocking) antibodies and relief from
symptoms.
 Suppressor T cells that specifically inhibit IgE antibodies may
play a role.
93

94. TYPE II HYPERSENSITIVITY
 Type II hypersensitivity is also known as cytotoxic
hypersensitivity and may affect a variety of organs and tissues.
 The antigens are normally endogenous, although exogenous
chemicals (haptens) which can attach to cell membranes can
also lead to type II hypersensitivity.
 Drug-induced haemolytic anaemia, granulocytopenia and
thrombocytopenia are such examples. The reaction time is
minutes to hours.
 Type II hypersensitivity is primarily mediated by antibodies of the
IgM or IgG classes and complement.
 Phagocytes and K cells may also play a role
94

95. Examples
 Autoimmune haemolytic anaemia
 Pernicious anaemia
 Immune thrombocytopenia
 Transfusion reactions
 Hashimoto's thyroiditis
 Graves' disease
 Myasthenia gravis
 Farmer's Lung
 Hemolytic disease of the newborn
95

96. Schematic illustration of three different mechanisms of antibody-
mediated injury in type II hypersensitivity. A, Complement-dependent
reactions that lead to lysis of cells or render them susceptible to
phagocytosis. (From Robbins Basic Pathology ,2003）
96

97. Antibody-dependent cell-mediated cytotoxicity (ADCC). IgG-coated target
cells are killed by cells that bear Fc receptors for IgG (e.g., NK cells,
macrophages). (From Robbins Basic Pathology ,2003）
97

98. Anti-receptor antibodies disturb the normal function of receptors. In this
example, acetylcholine receptor antibodies impair neuromuscular transmission
in myasthenia gravis. (From Robbins Basic Pathology ,2003）
98

99.  The lesion contains antibody, complement and neutrophils.
 Diagnostic tests include detection of circulating antibody against
the tissues involved and the presence of antibody and
complement in the lesion (biopsy) by immunofluorescence.
 The staining pattern is normally smooth and linear, such as that
seen in Goodpasture's nephritis (renal and lung basement
membrane) and pemphigus (skin intercellular protein,
desmosome)
 Treatment involves anti-inflammatory and immunosuppressive
agents.
99

100. TYPE III HYPERSENSITIVITY
 Type III hypersensitivity is also known as immune complex
hypersensitivity.
 The reaction may be general (e.g., serum sickness) or may
involve individual organs including skin (e.g., systemic lupus
erythematosus, Arthus reaction), kidneys (e.g., lupus nephritis),
lungs (e.g., aspergillosis), blood vessels (e.g., polyarteritis),
joints (e.g., rheumatoid arthritis) or other organs.
 This reaction may be the pathogenic mechanism of diseases
caused by many microorganisms.
100

101.  The reaction may take 3 - 10 hours after exposure to the antigen
(as in Arthus reaction).
 It is mediated by soluble immune complexes.
 They are mostly of the IgG class, although IgM may also be
involved.
 The antigen may be exogenous (chronic bacterial, viral or
parasitic infections), or endogenous (non-organ specific
autoimmunity: e.g., systemic lupus erythematosus, SLE).
101

102.  The antigen is soluble and not attached to the organ involved.
 Primary components are soluble immune complexes and
complement (C3a, 4a and 5a).
 The damage is caused by platelets and neutrophils.
 The lesion contains primarily neutrophils and deposits of
immune complexes and complement.
 Macrophages infiltrating in later stages may be involved in the
healing process.
 The affinity of antibody and size of immune complexes are
important in production of disease and determining the tissue
involved.
102

103. Schematic illustration of the three sequential phases in the
induction of systemic type Ⅲ (immune complex) hypersensitivity.
(From Robbins Basic Pathology ,2003）
103

104. Schematic representation of the pathogenesis of immune complex-mediated
tissue injury. The morphologic consequences are depicted as boxed areas.
(From Robbins Basic Pathology ,2003）
104

105. Immune complex vasculitis. The necrotic vessel wall is replaced by
smudgy, pink “fibrinoid” (From Robbins Basic Pathology ,2003）
105

106.  Diagnosis involves examination of tissue biopsies for deposits of
immunoglobulin and complement by immunofluorescence
microscopy.
 The immunofluorescent staining in type III hypersensitivity is
granular (as opposed to linear in type II such as seen in
Goodpasture's syndrome).
 The presence of immune complexes in serum and depletion in
the level of complement can also be used to diagnose.
 Polyethylene glycol-mediated turbidity (nephelometry) binding of
C1q and Raji cell test are utilized to detect immune complexes.
 Treatment includes use of anti-inflammatory agents.
106

107. TYPE IV HYPERSENSITIVITY
 Type IV hypersensitivity is also known as cell mediated or
delayed type hypersensitivity.
 The classical example of this hypersensitivity is tuberculin
(Montoux) reaction which peaks at 48 hours after the injection of
antigen (PPD or old tuberculin).
 The lesion is characterized by induration and erythema.
107

108.  Type IV hypersensitivity is involved in the pathogenesis of many
autoimmune and infectious diseases (tuberculosis, leprosy,
blastomycosis, histoplasmosis, toxoplasmosis, leishmaniasis,
etc.) and granulomas due to infections and foreign antigens.
 Another form of delayed hypersensitivity is contact dermatitis
(poison ivy, chemicals, heavy metals in wrist watches, belts,
earings etc.) in which the lesions are more papular (A small,
solid, usually inflammatory elevation of the skin that does not
contain pus).
 Type IV hypersensitivity can be classified into three categories
depending on the time of onset and clinical and histological
presentation (Table 3)
108

109. Table 3 - Delayed hypersensitivity reactions
Type Reaction time Clinical appearance Histology Antigen and site
contact 48-72 hr eczema
lymphocytes, followed by
macrophages; edema of
epidermis
epidermal ( organic chemicals,
poison ivy, heavy metals, etc.)
tuberculin 48-72 hr local induration
lymphocytes, monocytes,
macrophages
intradermal (tuberculin, lepromin,
etc.)
granuloma 21-28 days hardening
macrophages, epitheloid and
giant cells, fibrosis
persistent antigen or foreign body
presence (tuberculosis, leprosy,
etc.)
109

110.  Mechanisms of damage in delayed hypersensitivity include T
lymphocytes and monocytes and/or macrophages.
 Cytotoxic T cells (Tc) cause direct damage whereas helper T
(TH1) cells secrete cytokines which activate cytotoxic T cells and
recruit and activate monocytes and macrophages, which cause
the bulk of the damage.
 The delayed hypersensitivity lesions mainly contain
monocytes and a few T cells.
 Major lymphokines involved in delayed hypersensitivity reaction
include monocyte chemotactic factor, interleukin-2, interferon-
gamma, TNF alpha/beta, etc.
110

111. Schematic illustration of
the events that give rise to
the formation of
granuloma in type Ⅳ
hypersensitivity reactions.
Note the role played by T
cell-derived cytokines.
(From Robbins Basic
Pathology ,2003）
111

112.  Diagnostic tests in vivo include delayed cutaneous reaction (e.g.
Montoux test ) and patch test (for contact dermatitis).
 In vitro tests for delayed hypersensitivity include mitogenic
response, lympho-cytotoxicity and IL-2 production.
 Treatment of Type IV involves use of Corticosteroids and other
immunosuppressive agents.
112

113. 113
Table 5 - Comparison of Different Types of hypersensitivity
characteristics
type-I
(anaphylactic)
type-II
(cytotoxic)
type-III
(immune complex)
type-IV
(delayed type)
antibody IgE IgG, IgM IgG, IgM None
antigen exogenous cell surface soluble tissues & organs
response time 15-30 minutes minutes-hours 3-8 hours 48-72 hours
appearance weal & flare lysis and necrosis
erythema and edema,
necrosis
erythema and
induration
histology basophils and eosinophil antibody and complement
complement and
neutrophils
monocytes and
lymphocytes
transferred with antibody antibody antibody T-cells
examples allergic asthma, hay fever
erythroblastosis
fetalis, Goodpasture's
nephritis
SLE, farmer's lung
disease
tuberculin test, poison
ivy, granuloma

114. The Hypersensitivity Reactions
114

115. Part Four
115

116. Immunodeficiency
 Immunodeficiency refers to defects in the immune system
characterised by recurrent, persistent, severe or unusual
infections
 It is the absence or failure of normal function of one or more
elements of the immune system
 In any encounter with a microorganism the host resistance must
be balanced against the virulence of the pathogen and the
parasitaemia
116

117.  Infections like Listeria monocytogenes or Pneumonia carinii are
almost unknown other than in patients with underlying
immunodeficiency while some infections like Cytomegalovirus
have potent immunosuppressive effects
 Environmental factors influence host defence for example
malnutrition predispose the individual to infections which further
aggravate the malnutrition
117

118. Classification of Immunodeficiency
 Primary immunodeficiency and Secondary immunodeficiency
 Primary Immunodeficiency (PID) are the congenital or inherited
defects and could affect specific immunity or innate immunity
 Can be specific or non specific immune mechanisms
 Specific = Abnormalities of B & T cells
 Non specific = Abnormalities of non specific components
 Secondary immunodeficiency are acquired and normally has
underlying an cause especially infections which could affect the
immune system directly and could affect specific or innate
mechanism
118

119. Primary Immunodeficiency Diseases
 The primary immunodeficiency diseases are a group of
disorders in which the primary defect appears to be
intrinsic to one or more components of the immune
system
119

120. The immune system functional
compartments
 The B-lymphocyte system
 The T-lymphocyte system
 The Phagocytic system
 The Complement system
120

121. Frequency of the Primary
Immunodeficiency Diseases
 The primary immunodeficiency diseases were
originally thought to be quite rare.
 some of the primary immunodeficiency diseases are
relatively common.
 For example, Selective IgA deficiency occurs in as
many as 1/500-1/1000 individuals.
121

122. Frequency of the Primary
Immunodeficiency Diseases
 Other primary immunodeficiency diseases are much
less common and occur with a frequency of between
1/10,000 and 1/100,000.
 Because there are so many primary immunodeficiency
diseases, when taken together as a group of disorders,
they become a significant health problem, occurring
with a frequency comparable to leukaemia and
lymphoma in children and four times as frequently as
cystic fibrosis.
122

123. Causes of the Primary
Immunodeficiency Diseases
 Many of the primary immunodeficiency diseases are
genetically determined.
 Some of these are inherited as autosomal recessive
traits, some as X-linked recessive traits, and at least
one is inherited as an autosomal dominant trait.
123

124. Causes of the Primary
Immunodeficiency Diseases
 Others are not inherited as single gene defects. In fact,
two of the most common primary immunodeficiency
diseases, Common Variable Immunodeficiency
(CVID) and Selective IgA Deficiency, usually occur
sporadically and do not appear to be due to single gene
defects in most cases.
 However, there are even some rare cases of Common
Variable Immunodeficiency Disease and Selective IgA
Deficiency that occur in a familial setting.
124

125. Clinical Manifestations of the Primary
Immunodeficiency Diseases
 Infectious diseases
 Autoimmune and rheumatic diseases
 Gastrointestinal disease
 Hematologic diseases
125

126. Infectious Diseases
 An increased susceptibility to infection is the hallmark
of the primary immunodeficiency diseases.
 In most patients, this is manifested by recurrent
infections.
 Typically, the infections do not occur only in a single
anatomic site, but usually involve multiple organs or
multiple sites within the same organ.
 Recurrent otitis media in association with recurrent
sinusitis and/or pneumonia, while other patients may
have recurrent pneumonia, with episodes occurring in
different lobes.
126

127. Infectious Diseases
 Recurrent sinopulmonary infections, such as otitis,
sinusitis, bronchitis, and pneumonia, are the most
common presenting manifestations of the primary
immunodeficiency diseases
 Also recurrent systemic infections such as bacteremia
and meningitis are also seen.
 Chronic changes of the lungs and sinuses are not
unusual.
 unusually severe, leads to an unexpected or unusual
complication, or is caused by an organism of relatively
low virulence (i.e. an opportunistic organism).
127

128. Infectious Diseases
 Some patients the first infection may be so severe or
unusual that it raises the question of an underlying
immunodeficiency.
 The type of infectious agent and the location of the
infection may give valuable insight into the nature of
the immunologic defect.
 For example, individuals who have B-cell deficiencies
characteristically have an increased susceptibility to
infection with encapsulated pyogenic bacteria, such as
the pneumococcus and H. influenzae, and to
enteroviruses.
128

129. Infectious Diseases
 Patients who are deficient in T-cells may have
infections with a variety of microorganisms but appear
especially susceptible to fungi, viruses and
Pneumocystis.
 Patients with complement deficiencies often present
with blood-borne infections, such as bacteremia and
meningitis, caused by encapsulated bacteria.
 Patients with phagocytic disorders characteristically
have infections of the skin and reticuloendothelial
system.
129

130. Autoimmune and Rheumatic Diseases
 Rheumatoid arthritis, systemic lupus erythematosus,
and/or dermatomyositis.
 Autoimmune and rheumatic diseases are more
commonly seen in some of the primary
immunodeficiency diseases than in others.
 For example, they are relatively common in Selective
IgA Deficiency, Common Variable Immunodeficiency
and deficiencies of the complement system
 Relatively uncommon in X-linked
agammaglobulinemia.
130

131. Gastrointestinal Diseases
 Chronic diarrhea, malabsorption and even malnutrition
may be important manifestations of primary
immunodeficiency diseases, especially in infants and
young children.
 Infectious. Chronic giardiasis, rotavirus and
cryptosporidium, among other infections, have each been
significant problems in patients with primary
immunodeficiency diseases.
 Non infectious etiology includes inflammatory bowel
disease, gluten-sensitive enteropathy, atrophic gastritis
with pernicious anaemia and nodular lymphoid
hyperplasia.
131

132. Haematologic Diseases
 Anaemia, thrombocytopenia, or leukopenia are seen
frequently in patients with primary immunodeficiency
diseases.
 For example, the Wiskott-Aldrich Syndrome is
characterized by variable defects in B-lymphocyte and
T-lymphocyte function.
 These patients also have intrinsic abnormalities of
their platelets which result in small platelets and
significant thrombocytopenia.
132

133. Haematologic Diseases
 Haematologic abnormalities in consequence of the
autoimmune diseases that are seen in patients with
primary immunodeficiency.
 For example, a significant proportion of patients with
autoimmune haemolytic anaemia or idiopathic
thrombocytopenic purpura (ITP)
 Autoimmune haemolytic anaemia, and/or
thrombocytopenia, and/or neutropenia are often seen
in patients with Common Variable Immunodeficiency
or Selective IgA Deficiency, and the hyper IgM
Syndrome
133

134. Examples
Primary defects affecting specific immunity
 Are mainly as a result primary antibody deficiency and
are defects in antibody synthesis in qualitative or
quantitative like involving all Ab classes i.e.
Panhypogammaglobulinemia higher or lower
production or can involve only a class or subclass of Ab
= selective deficiency
134

135. 1. Transient hypogammaglobulinemia – after birth only
placental transferred IgG is there and other Ab classes are
lacking Ab synthesis yet to pick up. Due to delay in in IgG
synthesis approximately up to 36 months. So infants are
susceptible to infections
2. Infantile sex-linked hypogammaglobulinemia
(Bruton’s diseases) – Boys between 4 m – 2 years present
with recurrent pyogenic infections.
 No circulating B cells and no plasma cells in lymphoid organs and
tissues
 Few or no B cells in blood
 Very small lymph nodes and tonsils
 No Igs
 Small amount of Ig G in early age
135

136. 3. Common variable immunodeficiency (CVID)
 Is a heterogenous group of disorders affecting B-cell
differentiation, do not develop due to lacking T-cell help due
to defects in T cell signaling to B cells
 Low IgG levels, low IgA levels, abnormally in CMI
 Acquired agammaglobulinemia in the 2nd or 3rd decade of
life
 May follow viral infection
 Pyogenic infection
 80% of patients have B cells that are not functioning
 B cells are not defective. They fail to receive signalling from T
lymphocytes
 High chances of A.D in this group
136

137. 4. Selective IgA deficiency – commonest form
affecting specific immunity.
 Susceptible to pyogenic infection.
 Result from failure in terminal differentiation of B cells.
 Patients tend to develop immune complex disease
5. 5. IgG subclass deficiencies – About 20% lack
IgG2and IgG4.
 Not serious, there are high levels of IgG1 so masks
deficiencies in other subclasses.
 Mainly presents as infections of the respiratory system
137

138. Complications of Ab deficiency
Predisposes to a wide range of infections including:
Respiratory system – 70%, otitis media, chronic
sepsis of RT, sinusitis etc.
GI system – 19%, pernicious anaemia, diarrhoea and
several infections
Rheumatic similar to arthritis – 12%
Haematological – 9%
Malignancy – 7%, 10-200X increase in incidences of
malignancy with humoral/CMI defects
CNS – 3%, esp. viral infections
138

139. Management of Ab deficiency
Are aimed at preventing infections and
incidences of complications and include
1. Ig replacement therapy – IVIG mainly
IgG
2. Improve diagnosis and recognition of
new infections
139

140. Other primary defects affecting specific immunity
 Defects of T-cell function – depressed T-cell immunity
accompanied by B-cell function abnormalities e.g.
1. Digeorge syndrome
 Also known as congenital thymic aplasia/hypoplasia
 Thymic hypoplasia so low nos. of functional T cells, impaired CMI
and Ab production.
 It the most understood T-cell immunodeficiency
 Associated with hypoparathyroidism, congenital heart disease, fish
shaped mouth.
 Defects results from abnormal development of foetus during 6th-
10th week of gestation when parathyroid, thymus, lips, ears and
aortic arch are being formed
140

141. 2. Ataxia telangiectasia –
 Associated with a lack of coordination of movement (ataxis) and
dilation of small blood vessels of the facial area (telangiectasis).
 cerebellar ataxia with progressive neurological deterioration caused by
abnormal DNA repair affecting TCR genes. The defects arise from a
breakage in chromosome 14 at the site of TCR and Ig heavy chain genes
 impaired CMI and Ab production
 T-cells and their functions are reduced to various degrees.
 B cell numbers and IgM concentrations are normal to low.
 IgG is often reduced
 IgA is considerably reduced (in 70% of the cases).
 There is a high incidence of malignancy, particularly leukaemia in
these patients.
141

142. 3. Wiskott-Aldrich syndrome –
 X-linked membrane defect of T-cells and platelets –
thrombocytopaenia, bleeding, malignant diseases
 Associated with normal T cell numbers with reduced
functions, which get progressively worse.
 IgM concentrations are reduced but IgG levels are
normal
 Both IgA and IgE levels are elevated.
 Boys with this syndrome develop severe eczema.
 They respond poorly to polysaccharide antigens and
are prone to pyogenic infection.
142

143. MHC Deficiency -
Bare leukocyte syndrome:
 Due to defect in the MHC class II transactivator
(CIITA) protein gene, which results in a lack of class-II
MHC molecule on APC.
 Patients have fewer CD4 cells and are infection prone
 There are also individuals who have a defect in their
transport associated protein (TAP) gene and hence do
not express the class-I MHC molecules and
consequently are deficient in CD8+ T cells.
143

144. Severe Combined Immunodeficiency (SCID)
 failure of both B & T cell function, caused by
autosomal recessive adenosine deaminase deficiency
or purine nucleoside phosphorilase deficiency or MHC
class I or II deficiency – common in infants.
 Presents early in life with chronic diarrhoea and failure
to thrive, leucopaenia and impaired CMI, low or
absent abs, undeveloped secondary lymphoid organs.
 In about 50% of SCID patients the immunodeficiency
is x-linked whereas in the other half the deficiency is
autosomal
144

145.  The x-linked SCID is due to a defect in gamma-chain of IL-
2 also shared by IL-4, -7, -11 and 15, all involved in
lymphocyte proliferation and/or differentiation.
 The autosomal SCIDs arise primarily from defects in
adenosine deaminase (ADA) or purine nucleoside
phosphorylase (PNP) genes which results is accumulation
of deoxyadenosine triphosphate (dATP) or deoxyguanosine
triphosphate (dGTP), respectively, and cause toxicity to
lymphoid stem cells hence apoptosis of lymphoid cells
causing immunodeficiency.
 Patients with SCID are susceptible to a variety of bacterial,
viral, mycotic and protozoan infections
145

146. Management of defects in CMI
1. General measures
 Genetic counselling
 Prenatal diagnosis
 Minimize using live vaccines
2. Grafting measures
 BM transplants
 Foetal thymus grafts
 Foetal liver transplants
3. Replace missing factors
 Cell extracts
 Thymic hormones
 Enzyme replacement
 Gene therapy e.g. for SCID
146

147. Primary defects in non-specific immunity
 Non-specific immunity depends on Ab synthesis and
effector functions to eliminate bound ags
Include:
 Defects in Neutrophil functions – qualitative (neutrophil
dysfunction) or quantitative (neutropenia)
1. Cyclic Neutropenia
 It is marked by low numbers of circulating neutrophil
approximately every three weeks.
 The neutropenia lasts about a week during which the
patients are susceptible to infection.
 The defect appears to be due to poor regulation of
neutrophil production
147

148. NB. Neutropenia is a frequent side effect of chemotherapy,
 normal values 1.5 X 109/L
 and life-threatening infections when it falls to 0.5 X 109/L
148

149. 2. Chronic granulomatous disease (CGD):
 CGD is characterized by marked lymphadenopathy,
hepato-splenomegaly and chronic draining lymph
nodes.
 In majority of patients with CGD, the deficiency is due
to a defect in nicotinamide adenine dinucleotide
phosphade hydrogen (NADPH) oxidase that
participate in phagocytic respiratory burst.
149

150. 3. Leukocyte Adhesion Deficiency:
 Leukocytes lack the complement receptor CR3 due to a
defect in CD11 or CD18 peptides and consequently they
cannot respond to C3b opsonin
 Alternatively there may a defect in integrin molecules,
Lymphocyte function associated-1 (LFA-1) or major
adhesion molecule (MAC-1) arising from defective
CD11a or CD11b peptides, respectively
 These molecules are involved in diapedesis and hence
defective neutrophils cannot respond effectively to
chemotactic signals
150

151. 4. Chediak-Higashi syndrome:
 This syndrome is marked by reduced (slower rate)
intracellular killing and chemotactic movement
accompanied by inability of phagosome and lysosome
fusion and proteinase deficiency.
 Respiratory burst is normal.
 Associated with NK cell defect, platelet and
neurological disorders
151

152. Primary defects Presents with:
Recurrent and prolonged infections
Minimal clinical features despite severe infection
Poor response to antibiotics
Staphylococcal infections
Infections on skin and mucous membranes
152

153. Complement deficiency
 Complement abnormalities also lead to increased susceptibility
to infections.
 There are genetic deficiencies of various components of
complement system, which lead to increased infections
 Is usually secondary to complement consuming infections via CP
or AP e.g. SLE which predisposes to failure to solubilize IC and
reduced capacity to neutralize or lyse viruses.
 The most serious among these is the C3 deficiency which may
arise from low C3 synthesis or deficiency in factor I or factor H
153

154.  Inherited Complement component deficiencies are
associated with characteristic syndromes
 Deficiencies in C1, 2 & 4 associated with persistent
viral infections
 C3 deficiencies present with increased susceptibility to
recurrent bacterial infections; common life-
threatening infections like pneumonia, septicaemia
and meningitis
 C5, 6, 7 or 8 deficiencies – recurrent Neisserial
infections, gonococcal infections esp. septicaemia or
meningococcal meningitis.
 C1 deficiency is associated with hereditary angioedema
154

155. Warning signs for PIDs
 8 or more otitis media infections per year
 2 or more serious sinus infections per year
 2 or more pneumonias per year
 recurrent deep infections or infections in unusual
areas
 infections with opportunistic pathogens
 persistent thrush in patients older than 1 year
 family history of PID
 family history of early childhood deaths
155

156. Determining defects in cellular
immunity
 Often present with:
 Opportunistic infections [Pneumocystis jirovecii (previously called
Pneumocystis carinii (Fungal pneumonia)), Cryptococcus
(Cryptococcal meningitis), Candida spp.]
 Disseminated viral infections (CMV, EBV, VZV - Varicella Zoster
Virus chicken pox)
 Failure to thrive, chronic diarrhoea, persistent thrush
 Clinical evaluations:
 Complete blood count (CBC) with differential, lymphocyte subsets
 Vaccine titres (e.g. tetanus, diphtheria)
 Ig levels (IgA, IgE, IgM, IgG)
 T cell proliferation assays, (e.g., PHA, Concanavalin A (ConA),
PMA/ionomycin)
 Skin testing (e.g. Candida protein)
156

157. Determining defects in humoral
immunity
 Often present with:
 Recurrent sinopulmonary infections
 Encapsulated bacteria (Haemophilus influenza,
Pneumococcus spp., etc..)
 Parasitic infections (Giardia)
 Some virus infections (enteroviruses, papillomavirus)
 Chronic diarrhoea, poor growth
 Clinical evaluations:
 Vaccine titers (e.g. tetanus, diphtheria)
 Ig levels (IgA, IgE, IgM, IgG)
 B cell subset analysis (e.g. naïve, memory, etc..)
157

158. Determining defects in phagocytosis
 Often present with:
 Recurrent abscesses, abscesses in unusual areas
 Recurrent oral ulcers
 Severe pneumonias
 Catalase+ organisms (e.g. Staph. aureus, Serratia, etc..)
 Clinical evaluations:
 CBC with differential
 nitro blue tetrazolium (NBT) test for production of oxygen
radicals
158

159. Secondary Immunodeficiency
 Low levels reflects either depressed production or increased
catabolism
 Decreased production;
 Malnutrition
 Lymphoproliferative disorders like cancers
 Drugs; causing immunosuppression
 Infections
 Minerals and vitamins
 Obesity
159

160. Immunodeficiency due to drugs
1. CORTICOSTEROIDS
 Cause changes in circulating leukocytes
 Depletion of CD4 cells
 Monocytopenia
 Decreased in circulating eosinophils and basophils
 Inhibition of T cell activation and B cell maturation
 Inhibit cytokine synthesis
160

161. 2. METHOTREXATE
 Structural analogue of folic acid
 Blocks folic acid dependent synthetic pathways essential
for DNA synthesis
 Prolonged use for treatment reduces immunoglobulin
synthesis
3. CYCOLOSPORIN
 Have severe effects on T cell signalling and functions
 It binds to immunophilins which are believed to have a
critical role in signal transduction
 Also inhibit IL 2 dependent signal transduction
161

162. Increased loss or catabolism
 Nephritic syndrome – proteinuria, losing proteins through the
kidney – predispose to hypogammaglobulinemia and
hypoproteinamia
 Protein losing enteropathy – predisposes to
hypogammaglobulinemia and hypoproteinamia
 Burns
162

163.  Proteins can be lost from the gut in a variety of inflammatory
diseases like Crohn’s disease, ulcerative colitis and coelac
disease
 Also in intestinal lymhangiectasia dilated lymphatics leak
lymphocytes and proteins
 Protein energy malnutrition causes impaired synthesis hence
increased disease incidences, with impaired ab synthesis and
defects in CMI, low phagocyte function and complement activity.
163

164.  Metabolic diseases like diabetes mellitus, renal failure, cirrhosis
and Cushing syndrome affect CMI and innate immunity
 Lymphoproliferative diseases predisposes to infections and
opportunistic infections are common in malignancy
 Leukemias suppress functioning of leucocytes which could be
myeloid, lymphoid, monocytic or myelomonocytic.
164

165.  Non-hodgkins lymphoma is associated with defects of both
humoral and CMI
 Multiple myeloma patients have depressed levels of polyclonal
abs
 Immunosuppressive drugs affect many aspects of cell function
with impaired lymphocyte and neutrophil function
165

166.  Patients taking anti-cancer drugs or receiving treatment to
prevent organ transplant may develop unusual or opportunistic
infections
 Some pathogens suppress rather than stimulate the immune
system
 Many viral infections like CMV, measles, rubella and viral hepatitis
impair CMI
 Also immunosuppression noted in bacterial infections like TB,
brucellosis, leprosy and syphilis.
166

167.  Currently the clearest example of acquired immunodeficiency is
HIV causing AIDS;
 Contribution of opportunistic diseases
 AIDS and poverty in Africa
 AIDS and lifestyles
 Targets for management of HIV
 Emergence of discordant couples
167

168. Management of secondary
immunodeficiency
1. Treatment of the primary disease e.g. TB
2. Removal of complicating or exacerbating factors
3. Immune restoration e.g. cell and tissue grafting like BM, thymic
transplants, immune enhancing treatment e.g. IVIG, cytokines
and use of vaccines, use of anti oxidants
168

169. Part Five
169

170. Antigen-Antibody Reactions
 Specific Objectives: THE STUDENT SHOULD BE ABLE TO
 1. Discuss immunoglobulin variability (i.e.. the variable region)
 2. Describe bonds between the variable region and the antigenic
determinant
 3. Define antibody affinity and antibody avidity
 4. Describe a precipitin curve and discuss lattice formation involving
proteins verses carbohydrate antigens and be able to define "zone of
equivalence".
 5. Understand immunodiffusion in agar gels.
 (identity, nonidentity and partial identity)
 6. Have a conceptual understanding of immunoelectrophoresis, Fluorescent
antibody techniques and ELISA (enzyme-linked immunoassay)
 7. Define "agglutination" and understand the functional differences between
monomeric Ab (ie. IgG) and polymeric Ab (ie. IgM and S-IgA)
170

171. Definitions:
 1. The "antibody affinity" of an antibody-antigen
reaction is related to the strength of attractiveness
between an antibody (Fab region) and its antigenic
determinant.
 2. The "antibody avidity" is the total strength of
binding of the Fab regions of the population of
antibodies evoked to an antigen, and involves the
reaction with all the antigenic determinates. Thus it is
the total strength of the binding of antibodies to
antigens.
 3. Immune Complex = Antigen-Antibody Complex
[the size depends on the ratio of antigen to antibody].
171

172. The affinity:
 is the strength of the reaction between a single
antigenic determinant and a single combining site on
the antibody
or it is the association constant for binding (KA)
KA= k1/k2
Valence: the number of epitopes
Avidity: is the collective affinity of multiple
binding sites(affinity+ Valence)
172

173. Antibody Avidity
 Multiple interactions between antigen binding
sites and epitopes
 Greater than additive
 More relevant to biologic systems than affinity
173

174. Also the student should be prepared to
answer and discuss the following:
 1. List and describe the possible bonds between the
immunoglobulin variable region and an antigenic
determinant. Then draw and explain a precipitin curve
and "lattice formation" involving protein antigens and
polyclonal Ab.
 2. What is meant by "hypervariable regions" on
immunoglobulins? How do B cell clones differ in regard
to the hypervariable regions of the immunoglobulins on
their surface? At the level of the gene, explain what is
believed to account for these clonal diversities.
 3. Can two different classes of immunoglobulins have
identical variable regions? In your answer include a
discussion of the switch mechanism.
174

175.  Note That -
Antigenic Determinants Interact
With Specific Antibody
175

176. Binding of the epitope in the
antigen binding site
176
POOR FIT
GOOD FIT
antibody combining site
antigen determinant
high attraction
low repulsion
high repulsion
low attraction

177. Effect of multivalent
interactions
177
antibody Fab IgG IgG IgM
effective antibody valence 1 1 2 up to 10
antigen valence 1 1 n n
equilibrium constant (L/M) 104 104 107 1011
advantage of multi-valence - - 103-fold 107-fold
definition of bindng affinity affinity avidity avidity
intrinsic affinity functional affinity

178. Biological Consequences of
Antibody Affinity/Avidity
 Neutralization of toxins
 Complement activation
 Immune elimination of antigen
 Virus neutralization
 More intense immune complex disease in animals
 higher levels of circulating antigen-antibody
complexes
 more intense localization of immune complexes on
basement membranes.
 more severe impairment of organ function
178

179. Consequences of Antibody Binding
179

180. Antigen-Antibody Interactions
Quality and quantity are important in
resolution of disease
May contribute to pathology
Useful in immunological assays
180

181. Types of Antigen-Antibody reactions:
 Precipitation
 Agglutination
 Neutralization (Antitoxins)
 Opsonization
 Antibody-dependent cell-mediated cytotoxicity
 The complement activation Membrane attack
complex
181

182. Factors affecting measurement of Ag/Ab
reactions
 The only way that one knows that an antigen-antibody
reaction has occurred is to have some means of directly or
indirectly detecting the complexes formed between the
antigen and antibody.
 The ease with which one can detect antigen-antibody
reactions will depend on a number of factors.
1. Affinity - The higher the affinity of the antibody for the
antigen, the more stable will be the interaction. Thus,
the ease with which one can detect the interaction is
enhanced.
2. Avidity - Reactions between multivalent antigens and
multivalent antibodies are more stable and thus easier
to detect.
182

183. 3. Ag:Ab ratio - The ratio between the antigen and antibody
influences the detection of Ag/Ab complexes because the
sizes of the complexes formed is related to the
concentration of the antigen and antibody. (Fig 6)
4. Physical form of the antigen - The physical form of the
antigen influences how one detects its reaction with an
antibody. If the antigen is a particulate, one generally
looks for agglutination of the antigen by the antibody. If
the antigen is soluble one generally looks for the
precipitation of the antigen after the production of large
insoluble Ag/Ab complexes.
183

184. 184

185. CH2 CH3
CH2 CH3
IgG has a Valence of 2
TWO Identical ANTIGEN BINDING SITES
185

186. Movement
at the
Hinge
Region
CH2 CH3
CH2 CH3
IgG
Surface
of an
Antigen
i.e.
bacterial
cell surface
186

187. Non-Covalent Interactions
Ball in glove fit
Antigenic
Determinant
VL
VH
187

188. -
Gene rearrangements
and
Mutational Hot Spots
Charge-Charge Interactions
Hydrophobic Interactions - And good fit
+
-
VL
VH
+
188

189. The Ag-Ab
interaction is
due to lots of
non-covalent
interactions- lock
and key!
189

190. Antigen-antibody interactions:
Are reversible specific non-covalent biochemical
reactions:
 Hydrogen bonds - A chemical bond in which a hydrogen atom of one
molecule is attracted to an electronegative atom of another molecule
 Electrostatic bonds - A valence bond in which two atoms, attracted by
electrostatic forces, transfer one or more electrons between atoms
 Van der Waal forces - forces acting between non bonded atoms or
molecules
 Hydrophobic bonds - The attractive force between molecules due to
the close positioning of non-hydrophilic portions of the two molecules
Can be represented by the formula:
K1=constant of association
K2=constant of dissociation
Ag + Ab Ag Ab
K1
K2
Forces of Attraction
190

191. Y
Antibody Affinity
-
+
-
VL
VH
+
191

192. Antigenic determinant 1
Antigenic determinant 2
Antigenic determinant 3
Antigenic determinant 4
PROTEIN
ANTIGEN
192

193. Y
Y
Y
MUST HAVE POLYCLONAL ANTIBODY
and at least two different antigenic determinants
TO CROSS-LINK PROTEIN ANTIGENS
Immune
Complexes 193

194. Y
Y
Y
Y
ANTIBODY
EXCESS
NO CROSSLINKS
NO Precipitate
Y
Y
Y
Y
Y Y
Y
Y
Excess Antibody
194

195. Y
Excess Antigen = Not enough
Cross-links to cause a
Precipitation
195

196. Y
Y
More cross-links, and higher individual
affinities
= higher AVIDITY of the Immune Complexes
Y
196

197. Ab CONC
ANTIBODY
EXCESS
ANTIGEN
EXCESS
ZONE of
Equivalence
No Soluble Ag or Ab
197

198. 198

199. 202
Consequences of Antibody Binding

200. Immune Precipitation
203
Antigen
Antibody
• Is the reaction of soluble Ag with soluble Ab.
• The reaction results in the formation of Ag-Ab complexes
(lattices)

201. Antigen Antibody
DOUBLE DIFFUSION
Immune Complex
204

202. Antigen
Antibody
Antigen
Immune Complexes
Zone of Equivalence
205

203. Rabbit Serum
as antigens
1:4 1:20
Goat anti-rabbit serum
(Antibodies to rabbit serum)
206

204. Non-Identity
Antigen #1 Antigen #2
207

205. No Shared Antigenic
Determinants
Antigen #1 Antigen #2
208

206. OUCHTERLONY ANALYSIS
Diffusion of Antigens and Polyclonal Antibodies
Antigen 1
(Molecule #1)
Antibodies to both antigens
The same Animal was injected with
antigen 1 and with antigen 2
Antigen 2
(Molecule #2)
Non-Identity
209

207. OUCHTERLONY ANALYSIS
Antigen 3
is a part
of antigen 4
Antibody
Antigen 4
Partial - Identity
Remember
that Protein
Antigens have
different
antigenic
determinants
Also remember that
this antibody is a
poly-clonal antibody
such as an anti-
serum to an antigenic
preparation
This animal
was only
injected
with
Antigen #4
210

208. OUCHTERLONY ANALYSIS
Antigen 3
Antibody
Antigen 4
Partial - Identity
211

209. Antigen 3
Antibodies
polyclonal antibody
Antigen 4
Partial - Identity
Antibodies to determinants c and d are only on Antigen 3
and they pass by antigen 4
212

210. OUCHTERLONY ANALYSIS
Antigen 5
Antibody
Antigen 6
is Antigen 5
Reaction of Identity
These two Antigens are the Same Molecule
No spikes were formed
because:
Antigenic determinants on
Antigen 5 captured all the
antibodies to Antigen 6 and
antigenic determinants on
Antigen 6 captured all the
antibodies to Antigen 5
213

211. 214

212. Immunoelectrophoresis
The antigens are
electrophoresed in agarose,
then the antibody applied.
215

213. AGGLUTINATION
• Abs can bind and cross-link cells or particles 
aggregate formation
• Entrap microbial invaders
• IgM & IgA are the most suitable (IgG in sufficient
amounts can agglutinate cells)
216

214. Agglutination
217
RBC
RBC
RBC
RBC
IgM Antibody
IgG Antibody
RBC
RBC
RBC
RBC RBC
RBC
RBC

215. Applications of Agglutination
1. Agglutination/Hemagglutination:
a. Qualitative agglutination test
Determination of blood types or antibodies to blood group Ags
b. Quantitative agglutination test
Agglutination tests can also be used to measure the level of
antibodies to particulate antigens.(titration)
2. Passive hemagglutination: erythrocytes are
coated with a soluble antigen (e.g. viral antigen, a
polysaccharide or a hapten) and use the coated red
blood cells in an agglutination test for antibody to the
soluble antigen
218

216. Agglutination- clumping of
RBC’s, or other particles
219
3. Coomb's Test (Antiglobulin Test)

217. Old pregnancy
test. It also
illustrates
agglutination
inhibition
220

218. Immuno Fluorescence
Double layer
Sandwich
UV Light
Antigens
221

219. Antigens on Cells or on Tissue Sections
UV Light
Fluorescence
222

220. Indirect immunofluorescence
223

221. Detects cell
component as
cytoplasmic, rather
than nuclear
224

222. FACS machine
Fluorescence-Activated
Cell Sorter..
Rapid communication
between computer and
deflection plates. If
both dyes- deflect
right; one or the other-
deflect left. No dye-
no deflection. Cells are
individually counted.
225

223. Using flow cytometer to diagnose acute lymphocytic
leukemia
226

224. 227
Ag
Peroxidase Enzyme is permanently
attached to the Antibody Probe
Microtiter ELISA
Antigens are immobilized to the plastic surface of a
Microtiter Plate
Enzyme Linked Immuno-Sorbant Assay
ELISA
Ag
Substrate that
turns from clear
to green
Secondary
Ab
Primary
Ab

225. Capture ELISA -- using pre-
immobilized mouse monoclonal ab to
capture the specific antigen and a second
probe monoclonal antibody against a
different antigenic determinant
Ag Ag
228

226. Detecting Ab’s against HIV- HIV
coat protein is the Ag
229

227. Elispot- how many
cells are making a
particular
cytokine??
230

228. Terms to Remember
 Affinity
 Agglutination
 Avidity
 Complement
 Classical pathway
 Alternative (properdin)
pathway
 Cross-reaction
 Epitope/Antigenic
Determinant
 Precipitation
 Second antibody
Specificity
 Valence
231