Complement System |

While C3b binds to the bacteria. Then the phagocyte will recognize the C3b (opsonin)
because it has receptors that are expressed on the phagocyte’s membrane. The pathogen will now be
engulfed by the phagocyte with the receptor Receptor-Mediated Endocytosis.
Part of the Immune System since it helps in eliminating pathogens or agents that can cause
Once the bacteria is engulfed, it is contained in the phagosome/phagocytic vacuole. The
illness to humans
phagosome and lysosome will fuse = phagolysosome.
These are composed of more than 30 proteins that reinforce the complement system in the
Adaptive Immune System. If the bacteria or pathogens can breach the first line of defense, we
It will be digested into peptide because the contents of the lysosome will be released into
have the second line of defense and complement to help the body eliminate the pathogen.
the lumen of the phagosome. The digested peptide will now be presented to the APCs through MHC II
because the pathogen is an exogenous bacteria. CD4+ will recognize this then the B cell will get
If the pathogen breaches the tissue or blood stream, when the complement binds to the
activated, proliferate, differenciate into plasma cell and memory B cell.
pathogen, the complement system will be triggered via the 3 pathways depending on the kind of
pathogen that triggers it ultimately leading to the formation of MAC.
Memory B cell has an amnestic response. It can remember what kind of pathogen has entered the
o Classical Pathway, Alternative Pathway and the Lectin Pathway (MBL)
body in the previous exposure. During the second exposure, it can easily kill the pathogen already. It
will then differentiate into plasma cell to produce more antibodies to eliminate it.
If the pathogen can’t be killed in the classical pathway, there is still the alternative pathway
and MBL to kill it.
The antibody will attach or coat the bacteria as opsonins = opsonization. It will then be
engulfed. Opsonins can either be antibodies or complement proteins.
‘
 The complement system is a complex series of more than 30 proteins that play a significant
part in amplifying the inflammatory response to destroy and clear foreign antigens—hence,
their name ‘complement.’
3. Inflammation—complement proteins serve as an important link between the innate and
 Complement proteins are part of the innate immune system and are composed of soluble and
adaptive immune responses (C3a, C4a, C5a):
cell-bound proteins.
 Ability to increase vascular permeability.
 Cofactors; metal ions like calcium and magnesium are needed in the complement to be
 Recruit monocytes and neutrophils to the area of antigen concentration.
activated
 Trigger the secretion of immunoregulatory molecules that amplify the immune
 Complement is considered an acute-phase reactant because levels rise during an infection
response
- complement proteins are APRs because the proteins are produced by the liver. They APR
 C5a is the most potent
concentration in the serum or plasma will increase during infection or inflammation.
e.g., Glomerulonephritis
- take note that not all proteins are produced by the liver such as immunoglobulins which
are produced by the plasma cells
o Anaphylatoxin
- participates in the inflammatory response (C3a, C4a, C5a)
 It also has the unique property of being easily inactivated (i.e., heat-labile) by heating serum to
- is a small peptide or proteins that causes increased vascular permeability,
56°C for 30 minutes. contraction of smooth muscle, degranulation and release of histamine from
basophils and mast cells resulting in the cardinal signs of inflammation
 They have the following essential roles:
1. Lysis— or membrane attack lyse foreign cells (main actor – MAC ) when smooth muscles contract, there will be increased capillary permeability so that the
MAC will form pores on the pathogen’s membrane then the pores will allow the entry of phagocytes and neutrophils can perform their actions outside the blood vessel. We have a circulating
water and ions into the cell thus bursting the cell because of osmotic lysis. There is an pool and marginating pool; they will squeeze through the small gaps called Diapedesis or
imbalance of osmotic pressure inside and outside the cell = burst Extravasacion.
The neutrophils will now infiltrate the tissue that has been infected (Second line of
2. Phagocytosis—opsonize and tag the invaders for clearance. C1q and C3b are the major defense). If there are too much bacteria, the neutrophils will release chemical messengers
proteins. C3b has the most important opsonizing activity – bounds to phagocytes (chemokines - chemoattractant molecules) and call more back up. They will recruit more neutrophils
because of the chemokines
There are fragments formed during the formation of MAC. E.g., when C3 complement
protein breaks down, it will be broken down into C3a and C3b fragment. Decreased permeability so that the neutrophils can do diapedesis and goes to the site of
infection then engulf the bacteria/ pathogen
Once C3a is released from the intact C3 molecule it will release into the blood stream and
function as an Anaphyla toxin.

Immunoserology : ‘23 | 1

 Chronic activation can lead to inflammation and tissue damage to the host
 Because of its potential for far-reaching effects, complement activation needs to be carefully
regulated.
- if not properly regulated, it is always activated. Hence they circulate in the blood stream
in an inactive form. They will just activate when the need arises or when there is a
pathogen that enters the body. They will only activate then to kill the pathogen or
eliminate them from the body. It will be destroyed by the humoral and cellular immunity
Complement System |
Paul Ehrlich
- Complement was originally recognized in the 1890s
- replaced the term alexin and coined the term complement because the substance
‘complements’ the action of sensitizers/ antibodies in destroying microorganisms
- antibodies also function in the immune system by sensitizing or bonding to foreign agents
inorder for immune cells to recognize or tag them.

 Any deficiencies to the complement system can result in increased susceptibility to infection or Complement Proteins Year Discovered Discoverers
the accumulation of immune complexes resulting in possible autoimmune disorders. C1, C2 1907 Ferrata, A. & Brand, E.
C3 1914 Coca, A.F.
C4 1963 Muller-Eberhard, H.J. et al
C5 1965 Nilsson, U.R., & Muller-Eberhard, H.J.
C6, C7 1967 Nilsson, U.R
C8 1969 Manni, J.A. & Muller-Eberhard, H.J.
Ellie Metchnikoff C9 1969 Hadding, U. & Muller-Eberhard, H.J.
- discovery in animals of amoebalike cells that engulf foreign antibodies such as
bacteria – a phenomenon known as phagocytosis and a fundamental part.
- Theory of Metchnikoff proposed that phagocytes in the blood were capable of
ingesting and destroying the invading bacteria, then providing the basis of Innate Most of the soluble proteins circulate in functionality inactive forms as proenzymes (zymogens)
Cellular Immunity The activation of complement can take place on the surface of pathogen or damaged/ infected
- Screening Test for phagocytic engulfment – blood sample is collected through EDTA. cells by thee pathways.
Centrifuge, extract the buffy coat layer (granulocytes) then combine with
bacterial broth culture. Incubate at 37 C. Neutrophils will engulf the bacteria. All pathways are initiated by multiple stimuli independently from each other and subsequently
Then prepare bacterial smear, stained with Wright’s Stain. When the bacteria is the proteolytic cascades converge toward the activation of the major complement C3 resulting In
engulfed, it doesn’t mean that the bacteria is killed because we just stained it. the assembly with the Membrane-Attack Complex (MAC)
- Chronic Granulomatous Disease – defective killing or the microbicidal action because
there is deficiency in the lysosomes or enzymes, etc. Tested using Nitro Blue All triggers can be activated simultaneously especially in multiple infections.
Tetrazolium Test (NBT)

Hans Buchner Classical Pathway

- discovered naturally occurring substance in the blood – now known as Complement Proteins
that is capable of destroying bacteria
- Buchner and colleagues (1891) found a heat-labile factor (56 C for 30 mins) in the blood that
was capable of killing bacteria and named it Alexin (Greek; “to ward off”)
Jules Bordet
- was awarded the Nobel Prize in 1919 for his role in elucidating the nature of the complement.
- supported his “Humoral Theory” (immunity conferred due to antitoxic and bactericidal
substances in the body fluids by demonstrating that immune lysis required the presence
of two factors:
1. Heat-labile Factor – Lytic Factor (similar to Alexin)
2. Heat-stable Factor – Sensitizer
- he linked Buchner’s theory with his own humoral theory and came up with another theory
that the Alexin which is a heat-labile component will interact with this sensitizer
- he termed sensitizer which we now known as antibody
 Complement-fixing antibodies - IgM, IgG1 , IgG2 and IgG3 except IgG4
o they bound to their specific antigens on the cell membrane
o antibodies cannot bind without their specific antigen; therefore, immune complex
needs to form first in order for the antibody to bind to the cell during opsonization
o Immune Mediated Red Blood Cell Destruction – presence of antibodies in the
complement; disruption of RBC membrane by formation of MAC = intravascular
hemolysis
o Intravascular Hemolysis or Hemolytic Anemia – increased RBC destruction
 C-Reactive Protein
 Some viruses
 Mycoplasma spp.
 Some protozoans
 Gram-negative bacteria (such as Escherichia coli)

Immunoserology : ‘23 | 2
 Complement System |
Alternative Pathway 
 Antibody-directed mechanism for triggering the complement activation.
 Gram-negative bacteria membranes containing endotoxic lipopolysaccharide (LPS)
 The classical pathway, so-called because it was discovered first, using a plasma protein called
 Bacterial, Fungal/ yeast cell walls
C1q to detect antibodies bound to the surface of a microbe or other structure.
 Classical Pathway (via C3b generation)
 However, not all immunoglobulins are able to activate this pathway
 Virally-infected or virally-transformed host cells
 Tumor cell lines
 Some protozoans, especially Trypanosoma spp.
 Aggregated immunoglobulins (including IgA and IgE)
 Proteases (i.e., enhanced by C3b generation), released by PMNs, bacteria, damaged organs/
tissues, fibrinolysis (plasmin)
o Immunoglobulin M (IgM)
 Most efficient in complement fixation because of its multiple binding sites
Lectin Pathway  It takes one IgM molecule attached to two adjacent antigenic determinants to initiate the
classical cascade of activation
 Binding of mannose-binding lectin (MBL) in a calcium-dependent manner to carbohydrates
on pathogens and apoptotic/ tumor cells (e.g., D-mannose and L-fucose residues) o Immunoglobulin G (IgG)
 IgG1, IgG2, and IgG3, but not IgG4.
MBL has been shown to bind to:  Two (2) IgG molecules must attach to antigen within 30 nm to 40 nm of each other before
 Yeasts (Candida albicans) complement can bind; it may take at least 1,000 IgG molecules to ensure that there
 Viruses (HIV, SARS-CoV-2 and Influenza A, etc.) are two, close enough to initiate such binding.
 Bacteria (salmonella, Streptococci)  Some epitopes, notably the Rh group, are too far apart on the cell for this to occur;
 Protozoans (Leishmania) therefore, they are unable to fix complement.
 Within the IgG group, IgG3 is the most effective, followed by IgG1 and then IgG2.

When there is Transfusion Reaction with incompatible blood type, E.g.,

- when an O recipient receives blood from an AB donor, the anti-A and anti-B which are not
Complement proteins were named as they were isolated before the sequence of activation was
surely occurring in the type O recipient, will bind to the RBCs.
known — hence the irregularity in the numbering system
- Rh is IgG, ABO is IgM. What if IgG is the opsonin? We’ll need atleast 1 IgM and 2 IgG that are
close to each other.
The complement components C1-C4 were initially assigned names in order of their discovered,
- What if a patient has formed an Alloanti-D? Alloanti-D is IgG meaning, the patient had
and not according to their role in the activation sequence.
undergone a blood transfusion with incompatible antigen that results the patient to be
alloimmunized.
In 1968, the WHO Committee modified these nomenclatures being in order of activation and not
- After RBC sensitizing, it is recognized by the C1.
according to the year of discovery.

 Triggering substances: In addition to antibodies, a few substances can bind complement

directly to initiate the classical cascade. These include:
o Creactive protein (CRP)
o Several Viruses
o Mycoplasmas
o Some protozoa
o Certain Gramnegative bacteria such as Escherichia coli

The complement system can be activated in 3 different ways: Hemolytic Transfusion Reaction – RBCs are destroyed via classical pathway because there is
1. Classical Pathway involvement in the antigen-antibody which triggers the classical pathway. Antigens are in the RBCs
2. Alternative Pathway and antibodies can bind to those missing antigens then activates the complement = complement
3. Lectin Pathway fixation

Immunoserology : ‘23 | 3

Classical Pathway of Complement Activation
Complement System |
Proteins of the Classical Pathway
1. Recognition Unit
 Molecular weight: 740,000 Daltons
 Consists of three subunits—C1q, C1r, and C1s
 Calcium-dependent (to maintain its structure)
 It is composed of one 1 C1q subunit and 2 each of C1r and C1s
subunits
 Molecular weight: 410,000 Daltons
 Concentration in serum: 150 µg/mL
 protein; activates when it binds to an immune complex
 Chemoattractant for dendritic cells in the tissue. Pathogens coated with C1q are
attracted by dendritic cells via receptor-mediated endocytosis
- C1q and C3b play a role in phagocytosis
 Biggest out of the 3 components
C1q  Structure: composed of six strands that form six globular heads with a collagen-like
tail portion. This structure has been likened to a bouquet of tulips with six
blossoms extending outward.
- 2 out of the 6 globular heads will interact with the Fc portion of the 2 IgG molecules
but only 1 for IgM
 Function: C1q “recognizes” the Fc region of two adjacent antibody molecules, but at
least two of the globular heads of C1q must be bound to initiate the classical
pathway
 Molecular weight: 85,000 Daltons
 Concentration in serum: 50 µg/mL
C1r  A serine protease enzyme, initially an inactive proenzyme—becomes activated once
C1q is bound to the antibody molecules
- this is an enzyme that will catalyze a reaction. It activates C1s subunit
 Activated C1r is extremely specific—its only known substrate is the C1s subunit.
 Molecular weight: 85,000 Daltons
 Concentration in serum: 50 µg/mL
C1s  A serine protease enzyme, initially an inactive proenzyme—becomes activated once
C1q is bound to the antibody molecules.
 C1s subunit has a limited specificity, with its only substrates being C4 and C2.
 Once the C1s subunit is activated, the recognition stage ends.
Immunoserology : ‘23 | 4
 Complement System |
2. Activation Unit  The major and central constituent of the complement system—the key
Phase two, the formation of the activation unit, begins when C1s cleaves C4 and ends with the intermediate or shared in all three pathways of complement activation
production of the enzyme C5 convertase  People with C3 deficiency are susceptible to bacterial infection. MW: 190,000
Daltons
 Second most abundant complement protein - without C3, there is a lack in protein that forms the C5 convertase thus not forming
 Molecular weight: 205,000 Daltons MAC
 Concentration in serum: 300-600 µg/mL  Concentration in serum: 1,200 µg/mL
 The cleavage of C4 represents the first amplification step in the cascade because,  The cleavage of C3 to C3b represents the most significant step in the entire process
for every C1 attached, approximately 30 molecules of C4 are split and attached. of complement activation.
 Cleaved by C1s into 2 fragments:  The cleavage of C3 represents a second and major amplification step because about
200 molecules of C3 are split for every molecule of C3 convertase (𝑪𝟒𝒃𝟐𝒂
̅̅̅̅̅̅̅̅̅).
o C4b – bigger C4 fragment C3  Cleaved by the C3 convertase into 2 fragments:
 MW: 190,000 Daltons
C4  Interacts with the C2a fragment to form the C3 convertase (𝑪𝟒𝒃𝟐𝒂
̅̅̅̅̅̅̅̅̅) of o C3b – bigger C3 fragment
the classical pathway that activates C3
 remains bound in the cell membrane  MW: 180,000 Daltons
 Must bind to protein or carbohydrate within a few seconds or it will  C3b is potent in:
react with water molecules to form iC4b, which is rapidly degraded.  opsonization: tagging pathogens
 immune complexes (antigen-antibody), and
o C4a – smaller C4 fragment  apoptotic cells for clearance or phagocytosis
 MW: 9,000 Daltons
 Consists of 77 amino acids.  if bound to 𝐶4𝑏2𝑎
̅̅̅̅̅̅̅̅̅, creates new enzymes known as the C5 convertase
 Anaphylatoxin (𝐶4𝑏2𝑎
̅̅̅̅̅̅̅̅̅ 3𝑏).
 Macrophages have specific receptors for it.
 Closely related to Factor B of the alternative pathway.  Glycoprotein composed of the modified C3-α chain (α′) (MW:105,000)
 Molecular weight: 102,000 Daltons and the intact C3-β chain (MW: 75,000).
 Concentration in serum: 25 µg/mL  Chemoattractant for dendritic cells in the tissue. Pathogens coated
 When combined with C4b in the presence of magnesium ions (Mg2+), C2 is cleaved with C1q are attracted by dendritic cells via receptor-mediated
by C1s into two (2) fragments; an exception to the rule of complement protein endocytosis
nomenclature—C2a and C2b—the bigger and smaller C2 fragments, respectively.
o C3a – smaller C3 fragment
o C2b – smaller C2 fragment
 MW: 10,000 Daltons
 MW: 34,000 Daltons  An anaphylatoxin
 Accelerates decay-dissociation of the C3 convertase  C3a has a regulatory process and a structure homologous to that of
 Acts as a feedback inhibitor on the activation of the classical pathway of complement component C5a.
the complement system  C3a formation is triggered by pathogenic infection.
 induces chemotaxis of eosinophil, basophil and mast cells
C2 o C2a – bigger C2 fragment  has regulatory process and structure homologous to that of C5a

 MW: 70,000 Daltons

 C2a interacts with the C4b fragment to form the C3 convertase (𝐶4𝑏2𝑎
̅̅̅̅̅̅̅̅̅)
of the classical pathway.
 Provides the catalytic activity to the C3 and C5 convertases of classical
and lectin pathways.

 C3 Convertase (𝑪𝟒𝒃𝟒𝒂
̅̅̅̅̅̅̅̅̅):
 The C3 convertase complex (𝐶4𝑏2𝑎
̅̅̅̅̅̅̅̅̅) is not very stable.
 The half-life is estimated to be between 15 seconds and 3 minutes, so C3
must be bound quickly.
 If the binding does occur, C3 is cleaved by the C3 convertase into two
fragments, C3a and C3b.

Immunoserology : ‘23 | 5
 Complement System |
3. The Membrane Attack Complex (MAC)  Molecular weight: 150,000 Daltons
C8  Concentration serum: 55 µg/mL
The membrane attack complex (MAC) “punches” a hole through the plasma membrane of the  Function: binds to C5bC67 in MAC; starts the pore formation on the membrane
target cell, killing the pathogen in the process. It is composed of the following complement  MAC lyses gram-neg organisms but cannot pierce the S. pneumoniae cell wall.
components:  Pneumococci are not killed by serum alone and defects in these components do not
predispose to the infection
 Molecular weight: 190,000 Daltons  Molecular weight: 70,000 Daltons
 Concentration in serum: 80 µg/mL  Concentration in serum: 60 µg/mL
 Function: initiates the membrane attack complex (MAC).  Function: multiple C9 molecules bind to the 𝑪𝟓𝒃𝑪𝟔𝟕𝟖
̅̅̅̅̅̅̅̅̅̅̅̅̅ complex and polymerize to
o The cleaving of C5 with deposition of C5b at another site on the cell C9 form a transmembrane channel, the membrane attack complex (MAC), which
membrane constitutes the beginning of the MAC. form pores then the water and ions will get in the cell and causes cell lysis
 Structure: C5 consists of two polypeptide chains, α and β, which are linked by  If the complex is soluble in circulation, it is known as sC5b–9.
disulfide bonds.  Measurement of the level of sC5b–9 is an indicator of the amount of terminal
pathway activation that is occurring
 C5 convertase (𝑪𝟒𝒃𝟐𝒂𝟑𝒃
̅̅̅̅̅̅̅̅̅̅̅̅̅) cleaves C5 into two (2) fragments:
C5
o C5b – bigger C3 fragment
 MW: 75,000 Daltons 
 C5b attaches to the bacterial cell membrane, forming the beginning of
 First described by Pillemer and his associates in the early 1950s.
the MAC.
 C5b is extremely labile and rapidly inactivated unless binding to C6  It was originally named for the protein properdin, a constituent of normal serum with a
occurs. concentration of approximately 5 to 15 μg/mL.
 Once C6 is bound to C5b, subsequent binding involves C7,  In addition to properdin, the serum proteins Factor B and Factor D are unique to this pathway.
 C8, and C9. None of these proteins has enzymatic activity.  C3 is a key component of this pathway as well as the two other pathways (classical and lectin).
The conversion of C3 is the first step in this pathway
o C5a – smaller C2 fragment
 MW: 10,400 Daltons  Triggering substances for the alternative pathway include:
 Released into the circulation (as an anaphylatoxin – most potent) o bacterial cell walls, especially those containing teichoic acid
 chemoaatractant for neutrophils and macrophage o zymosan,
o lipopolysaccharide (LPS)
- Acute Infection – neutrophils (first responders)
 fungal cell walls
- Chronic Infection – mononuclear cells (monocytes and lymphocytes)
 yeast; viruses
 virally infected cellstumor cell lines
 causes smooth muscle contraction, increase in capillary permeability,
vasodilation and anaphylactic shock if systemically generated or  some parasites, especially trypanosomes
applied due to the activation of anaphylatoxin receptors on mast
cells and basophils leads to their degradation and release of
histamine and other vasolative or inflammatory mediators such as: Proteins of the Alternative Pathway
arachinodic acid metabolites Factor B  Molecular weight: 93,000 Daltons
 after the formation of C5, C6-C9 will bind and start the membrane attack  Concentration in serum: 200 μg/mL
complex  Function: Bb fragment binds to C3b to form the C3 convertase (C3bBb).
 Once bound to C3b, Factor B can be cleaved by Factor D.
 Molecular weight: 110,000 Daltons  The role of Factor B is thus analogous to that of C2 in the classical pathway
C6  Concentration in serum: 45 µg/mL because it forms an integral part of a C3 convertase
 Function: binds to C5b in MAC
 normally participates in the terminal by the lytic phase of complement activation,
being an integral member of MAC  Molecular weight: 24,000 Daltons
 Concentration in serum: 2 μg/mL (lowest concentration in the plasma)
 Molecular weight: 100,000 Daltons Factor D  Function: a serine protease that cleaves its only substrate, Factor B, into
C7  Concentration in serum: 90 µg/mL two (2) fragments—Ba and Bb
 Function: binds to C5bC6 in MAC

Immunoserology : ‘23 | 6
 Complement System |
o Bb Alternative Pathway of Complement Activation
 MW: 60,000 Daltons 1. C3 is hydrolyzed by water to produce C3b, which binds Factor B and together they attach to
 Bb remains attached to C3b, forming the initial C3 convertase of the the target cell surface.
alternative pathway (𝑪𝟑𝒃𝑩𝒃
̅̅̅̅̅̅̅̅̅̅) 2. B is cleaved by Factor D into the fragments Ba and Bb. Bb combines with C3b to form C3bBb,
 Bb is rapidly inactivated unless it becomes bound to a site on one of an enzyme with C3 convertase activity.
the triggering cellular antigens. 3. More C3 is cleaved, forming more C3bBb. This enzyme is stabilized by properdin, and it
 Some C3b attaches to cellular surfaces and acts as a binding site for continues to cleave additional C3.
more Factor B, resulting in an amplification loop
4. If a molecule of C3 remains attached to the C3bBbP enzyme, the convertase now has the
 C3 from the classical pathway is shared in the alternative pathway.
capability to cleave C5. The C5 convertase thus consists of C3bBbP3b. After C5 is cleaved,
 However, the breakdown is not the same as in the classical pathway
the pathway is identical to the classical pathway
this is because of hydrolysis. There is a small amount of C3 that
has broken down to C3b
 To make the reaction faster, we need enzymes to make the reaction
faster. Hence, we need the C3 convertase of the alternative
pathway

o Ba – MW: 33,000 Da
- will be released
Properdin  MW: 55,000 Daltons
 Concentration in serum: 15-25 g/mL
 Protein
 Function: Properdin (P) stabilizes the C3 convertase. The binding of
properdin increases the half-life of̅̅̅̅̅̅̅̅̅̅
𝐶3𝑏𝐵𝑏 from 90 seconds to several
minutes

C3 Convertase  As the alternative pathway convertase, C3bBb is then capable of cleaving

(𝑪𝟑𝒃𝑩𝒃
̅̅̅̅̅̅̅̅̅̅) additional C3 into C3a and C3b.
̅̅̅̅̅̅̅̅̅̅ can also cleave C5, but it is much more efficient at cleaving C3
 𝑪𝟑𝒃𝑩𝒃

C5 Convertase  If, however, some of the C3b produced remains bound to the C3
(𝑪𝟑𝒃𝑩𝒃𝟑𝒃𝑷
̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅) convertase, the enzyme is altered to form ̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
𝑪𝟑𝒃𝑩𝒃𝟑𝒃𝑷, which has a
high affinity for C5 and exhibits C5 convertase activity

Immunoserology : ‘23 | 7
 Complement System |

Proteins of the Lectin Pathway
 other name: Mannose-Binding Lectin or Mannan –Binding Lectin
Mannose-Binding  Another term: mannan-binding lectin
 Structure is the same with C1 molecule but they have different triggers
Lectin (MBL)  Molecular weight: 200,000 to 60,000 Daltons
 Instead of activation through antibody binding, the lectin pathway is activated by recognition of
 Concentration in serum: 0.0002 to 10 μg/mL
surface moieties that are found on pathogens  Analogous to C1q
 Binds to mannose/ carbohydrates to related sugars (non-selective or non-specific binding) in a  Function:
calcium-dependent manner to imitate this pathway o As the name implies, it binds to mannose or related sugars in a
o Binds to bacterias that have polysaccharides in their capsule – they cannot die in the calcium-dependent manner to initiate this pathway.
classical pathway since they have capsules that are anti-phagocytic. o It is considered an acute-phase protein because it is produced in
the liver and is normally present in the serum but increases
 Magnesium and Calcium in the complement  cofactor to enhance its activation; enzymes during the initial inflammatory response
won’t work without its cofactors
The enzymatic role played by C1r and C1s in the classical pathway is
o This pathway is initiated by the binding of : - played in the lectin pathway by serine proteases called MBL-associated
serine proteases (MASPs).
 mannose-binding lectin (MBL)
 collectin 11 (CL-K1)
MASP-1  Molecular weight: 93,000 Daltons
 ficolins (Ficolin-1, Ficolin-2, and Ficolin-3)
 Concentration in serum: 1.5 to 1.2 μg/mL
o to microbial surface oligosaccharides and acetylates residues, respectively  Analogous to C1r
 Function:
 It involves non-specific (non-selective) recognition of carbohydrates that are common
constituents of microbial cell walls and o MASP-1 is synthesized as a zymogen (an inactive form of the
that are distinct from those found on enzyme)
human cell surfaces. o Activated when it complexes with the pathogen recognition
 The lectin pathway plays an important role molecules of the lectin pathway, the mannose-binding
as a defense mechanism in infancy lectin, and the ficolins.
(innate immune response), during the o This protein is not directly involved in complement activation
interval between the loss of maternal but may play a role as an amplifier of complement activation
by:
antibody and the acquisition of a
fullfledged antibody response to
 cleaving complement C2 or
pathogens.
 by activating another complement serine protease MASP-2.
 Once C4 and C2 are cleaved, the rest of the
pathway is identical to the classical MASP-2  Molecular weight: 76,000 Daltons
pathway.  Analogous to C1s – cleaves C4 and C2 = C3 convertase
 Concentration in serum: unknown
 Function:
o MASP-2 is very similar to the C1s molecule, of the classical
complement pathway—it cleaves C4 and C2
 When the carbohydrate-recognizing heads of MBL bind to specifically
arranged mannose residues on the surface of a pathogen, MASP-2 is
activated to cleave complement components C4 and C2 into C4a,
C4b, C2a, and C2b, respectively.

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 Complement System |
Activation of complement could cause tissue damage and have devastating systemic effects if it
were allowed to proceed uncontrolled.

C1 Inhibitor  Molecular weight: 105,000 Daltons

(C1-INH)  Concentration in serum: 240 μg/mL
 Function:
o C1 inhibitor (C1-INH) inhibits activation at the first stages of both the
classical and lectin pathways.
o Its main role is to inactivate C1 by binding to the active sites of C1r and
C1s.
o C1-INH also inactivates MASP-2 binding to the MBL-MASP complex,
thus halting the lectin pathway.
o Cannot activate serine protease thus C4 and C2 won’t get activated

Further formation of C3 convertase in the classical and lectin pathways is

inhibited by four main regulators:
o soluble C4-binding protein (C4BP)
o three cell-bound receptors, complement receptor type 1 (CR1)
o membrane cofactor protein (MCP)
o decayaccelerating factor (DAF).

 Molecular weight: 88,000 Dalton

 Concentration in serum: 35 μg/mL
 Function:
o Cleaves or inactivates C3b and C4b
Factor I o For the regulation of the classical and lectin pathways
o All of the C3 convertase regulators mentioned above act in concert with
Factor I, a serine protease that inactivates C3b and C4b when bound
to one of these regulators.


 Molecular weight: 150,000 Daltons
 Concentration in serum: 300 to 450 μg/mL
 Function:
o Principal soluble regulator of the alternative pathway.
Factor H o It acts by binding to C3b, preventing the binding of Factor B.
o Factor H also accelerates the dissociation of the ̅̅̅̅̅̅̅̅̅̅
𝐶3𝑏𝐵𝑏 complex on
cell surfaces.
o When Factor H binds to ̅̅̅̅̅̅̅̅̅̅
𝐶3𝑏𝐵𝑏, Bb becomes displaced.
o Well-balanced complement response to endogenous ligands  Additionally, Factor H acts as a cofactor that allows Factor I to break down
- we need to increase the complement activation and regulate it to balance. C3b
- allow complement to act but also need to regulate so that they will not over react  Molecular weight: 520,000 Daltons
o Allowing opsonization, but preventing inflammation and organ damage  Concentration in serum: 250 μg/mL
o A beneficial imbalance to combat the invading pathogenic microorganism C4-Binding  It is abundant in plasma
o Full complement activation including lysis, inflammation and activating the immune system Protein  Function:
o An unbeneficial imbalance will result in immune pathology or extensive autoreactivity = cells (C4BP) o Acts as a cofactor with Factor I to inactivate C4b.
will be destroyed / autoimmunity o If C4BP attaches to cell-bound C4b, it can dissociate it from 𝑪𝟒𝒃𝟐𝒂
̅̅̅̅̅̅̅̅̅
complexes (classical pathway C3 convertase), causing the cessation
of the classical pathway
o If C3 and C5 convertase is inhibited the reaction will become slower

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 Complement System |
 Molecular weight: 84,000 Daltons  Ligand: C3b and C4b
 Concentration in serum: 500 μg/mL  Function:
 Function: o DAF is capable of dissociating both classical and alternative
o S protein is a soluble control protein that acts at a deeper level of pathway C3 convertases
S Protein complement activation (the terminal components). o It can bind to both C3b and C4b in a manner similar to CR1.
(Vitronectin) o Also known as vitronectin, S protein interacts with the C5b67 o The presence of DAF on host cells protects them from
complex as it forms in the fluid phase and prevents it from bystander lysis. It is one of the main mechanisms used in
binding to cell membranes. discrimination of self from non-self because foreign cells do
o Binding of C8 and C9 still proceeds, but polymerization of C9 does not possess this substance
not occur; therefore, the complex is unable to insert itself into
the cell membrane or to produce lysis. To prevent uninfected or normal cells from compliment activation, we
also need to inhibit the lysis of healthy cells.

 Also known as CD59

 Ligand: C8
Membrane  MIRL also acts to block formation of the MAC.
Inhibitor of  MIRL is widely distributed on the cell membranes of all circulating blood
This helps balance the action of complement system. This is to ensure that our complement Reactive Lysis cells, including RBCs, and on endothelial, epithelial, and many other
system will not over react and to ward autoimmune disorders and organ damage. (MIRL) types of cells
 Interferes with the binding of C5b67 complex to C8-9 and the formation
 Also known as CD35 of MAC in the terminal stage
 Ligand: C3b, iC3b, and C4b
 Molecular weight: 165,000–280,000 Daltons
 It is found mainly on peripheral blood cells, including neutrophils,
monocytes, macrophages, erythrocytes, eosinophils, B lymphocytes,
Complement some T lymphocytes, and follicular dendritic cells. Deficiencies of complement components can place an individual at risk for certain infections
Receptor 1 (CR1)  Function: because of the lack of production of complement proteins. Missing or deficient regulators are the
o A main function of CR1 is as a receptor on platelets and red
cause of diseases such as paroxysmal nocturnal hemoglobinuria and hereditary angioedema
blood cells (RBCs), which helps to mediate transport of C3b-
coated immune complexes to the liver and spleen
How do you measure complement or complement activity?
o It binds C3b and C4b but has the greatest affinity for C3b.
 Once bound to CR1, both C4b and C3b can then be degraded by Factor I  Eg., Complement Fixation
= cannot form C3 convertase  E.g., Radial Immunodiffusion

Agarose Gel with incorporated antibodies to C3 if we want to test C3. In the sample well, serum
 Also known as CD46 sample is added because complement proteins are found in the serum. Incubation time at room
 Ligand: C3b and C4b temperature, the antigens (compliment protein) since they have antigenic determinants that will be
 Molecular weight: 50,000–70,000 Daltons recognized by anti-C3 antibodies.
 It is found on virtually all epithelial and endothelial cells, except
Membrane erythrocytes (RBCs). They will form a complex then the zone of equivalence, the concentration of antigens and antibodies
Cofactor  MCP is the most efficient cofactor for Factor I mediated cleavage of C3b. are equal, they will form a zone of precipitation. It looks like a ring then the diameter will be
Protein (MCP)  It can serve as a cofactor for cleavage of C4b, but it is not as effective as
measured. The diameter will be compared and convert to a chart which is the basis for the
C4BP.
concentration.
 MCP also helps to control the alternative pathway because of the
binding of Factor B to C3b is inhibited/ stopped
To determine the concentration, the dilution of the standard curve from the standard.

 Also known as CD55 Why do we need to dilute the sample?

Decay  Molecular weight: 70,000 Daltons to detect the strength of the antibody, to know the concentration that is called titration.
Accelerating  Third main receptor and has a wide tissue distribution.
Factor (DAF)  It is found on peripheral blood cells, on endothelial cells and fibroblasts,
and on numerous types of epithelial cells.

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 Complement System |

 Hemolytic Titration (CH50) Assay:

 This assay measures the amount of patient serum required to lyse 50% of a standardized
concentration of antibody-sensitized SHEEP Erythrocytes.
 Because all proteins from C1 to C9 are necessary for this to occur, absence of any one
component will result in an abnormal CH50, essentially reducing this number to zero
 The titer is expressed in CH50 units, which is the reciprocal of the dilution that is able to
lyse 50% of the sensitized cells.
 The 50% point is used because this is when the change in lytic activity per unit change in
complement is at maximum.

 Hemolytic Titration (AH50) Assay

 An AH50 can be performed in the same manner as the CH50, except magnesium chloride
and ethylene glycol tetraacetic acid (EGTA) are added to the buffer and calcium is left
out.
 This buffer chelates calcium, which blocks classical pathway activation.
 RABBIT RBCs are used as the indicator because they provide an ideal surface for alternative
pathway activation

 Enzyme-Linked Immunosorbent Assay

 An additional means of testing for alternative pathway function is via ELISA.

Techniques to determine complement abnormalities generally fall into two (2) categories:  One such test can detect C3bBbP or C3bP complexes in very small quantities.
 Microtiter wells are typically coated with bacterial polysaccharide to trigger activation of
1. Measurement of components as antigens in serum the alternative pathway.
 Measurement of C3 and C4 levels – routinely measured by nephelometry or turbidimetry
 Measurement of C1-INH, C3bBbP, or C3bP –measured by enzyme-linked immunosorbent assay
(ELISA)
 Radial immunodiffusion (RID)
o uses agarose gel into which specific antibody is
incorporated. Serum serves as the antigen
and is placed in wells that are cut in the gel.
Diffusion of the antigen from the well occurs
in a circular pattern. The radius of the
resulting circle can be related to antigen
concentration

2. Measurement of functional activity

 The hemolytic titration or CH50 assay is a measure of lysis, the end point of complement
activation in the classical pathway. The AH50 assay is a similar test for measuring the
activity of the alternative pathway

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Complement System |

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