Immunotechnology

1. The Complement System
By Dr. B.P. Nayak

2. Innate Immune Response
• Inflammatory response enhances phagocytosis through
acute phase proteins
• Mannose-binding lectin (MBL)
• Binds to bacterial surface with particular spatial arrangement of
mannose or fucose
• C-reactive protein (CRP)
• Binds to phosphorylcholine on bacterial surface
• Complement
• Set of proteins which bind to bacterial surface
• Inflammatory response
• Accumulation of fluid and cells at infection site
(swelling, redness, heat and pain)

3. Adaptive Immune Response
• Creates millions of different B and T cells for specific
antibody-mediated and cell-mediated immunity
• Antibody-Mediated Immunity (AMI)
• Involves B lymphocytes, plasma cells and antibodies
• Humoral immunity
• Name derives from antibodies found in body fluids (humors - old medical term)
• Cell-Mediated Immunity (CMI)
• Involves T lymphocytes, antigen-presenting cells and MHC (major
histocompatibility complex) molecules
• Cellular immunity

4. Antibody binds
antigens on the
surface of target cells
Fc receptors on
NK cells recognize
bound antibody
Cross-linking of Fc
receptors signals the
NK cell to kill
the target cell
Target cell dies by
apoptosis and
membrane damage
ADCC
Around 1960-70, it was found that, ADCC is possible without the help of NKs
Then, HOW ?

5. The complement system
 Complements act as a bridge between innate and
adaptive immunity.
 It not only initiates a robust innate immunity (innate
detection and elimination of pathogenic infections), it can
effectively modulate the adaptive immune responses
through regulation of interplay between B andT cells

6. The complement system
 Complement refers to a complex set of 14 distinct serum
proteins (nine components) + 12 surface proteins that are
involved in three separate pathways of activation.
 Components numbered in order of discovery.
 Sequence of activation is not in numerical order.
 Components circulate in inactive precursor form, develop activity
upon activation.
 Complement proteins designated by “C” followed by
numbers and letters.

7. General Properties

8. The complement system
• A defensive system consisting of over 30 proteins
produced by the liver and found in circulating blood serum.
• Complement kills microbes in three different ways
1. Opsonization
2. Inflammation
3. Cytolysis

9. •The complement works as a cascade system.
• Cascade is when one reaction triggers another reaction
which trigger others and so on.
• These types of systems can grow exponentially very fast.
A Cascade system

10. • Complement proteins are often designated by an uppercase
letter C and are inactive until they are split into products.
• Example: C1
• When the products are split they become active. The active
products are usually designated with a lower case a or b.
• Example: C1a and C1b
Cascade activation

11. • The complement pathway can be activated by either of three
different pathways.
THREE Pathways

12. The Classical Pathway

13. The building of a C3 activation complex

14. C3 Activation complex

15. C3b
• Many C3b molecules are produced by the C3 activation
complex.
• The C3b bind to and coat the surface of the bacteria.
• C3b is an opsonin
• Opsonins are molecules that bind both to
bacteria and phagocytes
• Opsonization increases phagocytosis by 1,000
fold.
Opsonins

16. C3a
C3a increases the inflammatory response by binding to mast
cells and causing them to release histamine
C3a

17. Building the C5 activation complex

18. • Eventually, enough C3b is cleaved that the surface of the
bacteria begins to become saturated with it.
• C2b and C4b which make up the C3 activation complex has a
slight affinity forC3b and C3b binds to them
• When C3b binds to C2b and C4b it forms a new complex
referred to as the C5 activation complex
Building the C5 activation complex

19. The C5 activation complex
• The C5 activation complex (C2b, C4b, C3b) activates C5
proteins by cleaving them into C5a and C5b
• Many C5b proteins are produced by the C5activation
complex.
• These C5b begin to coat the surface of the bacteria.
C5 activation complex

20. C5a
•C5a disperses away from the bacteria.
• Binds to mast cells and increases inflammation.
• Most powerful chemotactic factor known for leukocytes

21. Building the Membrane Attack complex

22. Building the Membrane Attack complex
• C5b on the surface of bacteria binds to C6
• The binding of C6 to C5b activates C6 so that it can bind to
C7
• C7 binds to C8 which in turn binds to many C9’s
• Together these proteins form a circular complex called the
Membrane attack complex (MAC)

23. Membrane Attack complex
• The MAC causes Cytolysis.
• The circular membrane attack complex acts as a channel in
which cytoplasm can rush out of and water rushes in.
• The cells inner integrity is compromised and it dies

24. Overview

25. The alternative pathway
 Cleavage of C3 and activation of the remainder of the
complement cascade occurs independently of antibody.
 Triggers for the alternative pathway include
 Bacterial cell walls
 Bacterial lipopolysaccharide
 Fungal cell walls
 Virally infected cells
 Rabbit erythrocytes

26.  Molecules of C3 undergo cleavage at continuous
low level in normal plasma.
 At least 4 serum proteins (factor B, factor D,
properdin (P), and initiating factor (IF) function in
this pathway.
 C3b attaches to appropriate site (activating
surface) which is actually a protective surface
 Action by the 4 serum proteins on C3b proceeds
to the C3 activator stage without participation of
C1, C4 or C2.
 Activation sequence:
C3, C5, C6, C7, C8, C9.
The alternative pathway
The alternative pathway

27. • C3 contains in unstable thioester bond.
• This unstable bond makesC3 subject to
slow spontaneous hydrolysis to C3b and
C3a
• The C3b is able to bind to foreign surface
antigens.
Initiation: The alternative pathway
Mammalian cells contain sialic acid
which inactivates C3b

28. Factor B
• C3b on the surface of a foreign cells
binds to another plasma protein
called factor B

29. Factor D
• The binding of C3b to factor B
allows a protein enzyme called
Factor D to cleave Factor B to Ba
and Bb.
• Factor Bb remains bound to C3b
while Ba and Factor D disperse
away.
Factor D

30. The C3 activation complex
• Properdin, also called factor P, binds to the C3bBb complex to
stabilize it.
• C3bBbP make up the C3 activation complex for the alternative
pathway

31. The C3 activation Complex
• The C3 activation complex causes the
production of more C3b.
• This allows the initial steps of this
pathway to be repeated and amplified
• 2X106 molecules can be generated in 5
minutes
The C3 activation complex

32. C5 activation complex
• When an additional C3b binds to
the C3 activation complex it
converts it into a C5 activation
complex.
• The C5 activation complex cleaves
C5 into C5a and C5b.
• C5b begins the production of the
MAC.
C5 activation complex

33. Overview

34. Lectin Pathway
• Activation of the lectin pathway begins when mannose-binding
protein (MBP) binds to the mannose groups of microbial
carbohydrates.
• Two more lectin pathway proteins called MASP1 and MASP2
(equivalent to C1r and C1s of the classical pathway) 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 C4bC2b, the C3
convertase capable of enzymatically splitting hundreds of
molecules of C3 into C3a and C3b

35. LECTIN PATHWAY
 The beneficial results are the same as in the classical
complement pathway:
Trigger inflammation (C5a>C3a>C4a);
Chemotactically attract phagocytes to the infection site (C5a);
Promote the attachment of antigens to phagocytes via enhanced
attachment or opsonization (C3b>C4b);
Serves as a second signal for the activation of naive B-lymphocytes
(C3d);
Cause lysis of gram-negative bacteria and human cells displaying
foreign epitopes (MAC).
 And remove harmful immune complexes from the body
(C3b>C4b).
Lectin Pathway

36. Regulation: Complement Cascade
 Modulating mechanisms are necessary to regulate complement
activation and control production of biologically active split
products
 First type of control is extreme lability of activated complement
If activated complement does not combine within milliseconds the
activity is lost or decreased.
Active fragments rapidly cleared from the body.
 Second type of control involves specific control proteins
C1 inhibitor blocks activity of C1r and C1s.
Factor H and I, activators in the presence of certain cofactors
inactivates C3b and C4b.
A number of proteins act to control membrane attack unit

37. Regulation: Complement Cascade

38. Complement Deficiencies
 Major Pathway components
 Hereditary deficiency of any component except C9 results in
increased susceptibility to infection and delayed clearance of
complexes.
 Deficiency of MBL important during infant transition to making
their own antibody, associated with pneumonia, sepsis and
meningococcal disease.
 C3 deficiency most serious, key mediator in ALL pathways. Prone
to serious life threatening infections and immune complex disease.
 Deficiency of terminal components causes increased susceptibility
to Neisseria infections.

39. Complement Deficiencies
•Regulatory Factor components
• PNH – RBCs deficient in DAF, results in hemolysis due to
“bystander” affect.
• Hereditary angiodema results in excess cleavage of C4
and C2,
• keeps classical pathway going
• Results in increased vascular permeability causing edema.
• Usually spontaneously subsides but can be life threatening –
oropharynx
• Inhibitors of factors H and I produces constant turnover
of C3 to depletion – recurrent bacterial infections occur.
Complement Deficiencies

40. Tests for Complement
To test the classical pathway
• Hemolytic titration (CH50) assay,
• Lytic activity,
• ELISA
To test the alternative pathway
• AH50
• ELISA test.
Other tests
• QuantitativeTests
• Test all three pathways using test strips coated with
reagents specific for each pathway.
• Use complement fixation test to detect viral, fungal and
rickettsial antibodies, hemolysis of RBCs is a positive result.
Tests for Complements

41. CH50
• Hemolytic titration (CH50) assay
• Serial dilutions of the sample are made and incubated with
antibody‐sensitized sheep erythrocytes.
• The results are reported as the reciprocal of the dilution at
which 50% hemolysis is seen (CH50).
CH50

42. AH50
• A similar assay, using rabbit (or guinea pig) erythrocytes, which
specifically activate the alternative pathway by virtue of their
decreased sialic acid content.
• Additionally, activation of the classical pathway is further blocked by
chelation of calcium by EGTA.
• The results are again reported as the reciprocal dilution at which
50% hemolysis is seen (AH50).
• A second method to quantitate classical pathway activation is to
measure the neoantigen formed when the MAC complex (C7, 8, 9) is
generated.
AH50

43. • Quantification of the majority of individual complement
components can be performed through standard
immunochemical assays, including immunodiffusion,
nephelometry, and turbidometry.
• The most commonly used methods for quantification of C3 and
C4 measures intact proteins as well as the major soluble
fragments formed during activation.
• The presence of cell‐bound regulatory proteins such as CD55,
CD59, and CD35 (CR1) can be assessed using flow cytometry.
Quantitative Tests