The document discusses several topics related to the complement system and immunoassays: 1) It describes the complement system as part of the immune system that enhances the ability of antibodies and phagocytes to clear pathogens. It is activated via three pathways: classical, lectin, and alternative. 2) It explains the processes of complement activation, including the formation of C3 and C5 convertases that cleave complement proteins and amplification of the response. 3) It discusses two types of immunoassays - radioimmunoassay (RIA) and immunofluorescence. RIA uses radioactive labels on antigens or antibodies to measure concentrations via competition binding assays. Immunofluorescence utilizes fluorescent-labeled antibodies to detect target antigens

1. COMPLEMENT FIXATION,
RADIOIMMUNO ASSAY,
IMMUNOFLUORESCENCE

2. Complement system
• The complement system is a part of the immune system that
enhances (complements) the ability of antibodies and
phagocytic cells to clear microbes and damaged cells from an
organism, promotes inflammation, and attacks the pathogen's
plasma membrane
• Complement is a system of plasma proteins that can be
activated directly by pathogens or indirectly by pathogen-
bound antibody, leading to a cascade of reactions that occurs
on the surface of pathogens and generates active components
with various effector functions

3. Complement Activation Pathways
• Involves a “cascade” of successive components.
• Enhances a small initiating signal.
• Components are cleaved into activated fragments.
• Fragments induce intense inflammatory responses to
eliminate infectious agents.
Distinct recognition events for each pathway
• Classical - C-reactive protein
• Lectin – Mannose-binding lectin (MBL)
• Alternative – serum factors B, D, and P

4. C1 is a complex that is made from the
combination of 3 different proteins:
The first component of the classical pathway is
C1q, an 18-polypeptide chain component of
C1, with two molecules each of C1s and C1r
When C1 binds to the Ab in an antigen-
antibody complex it initiates the classical
pathway and becomes enzymatically active
and is referred to as C1s esterase
Activation of C1 occurs when the globular
head regions of the subunit C1q bind to the
Fc regions of either the IgM or two closely
spaced IgG molecules bound to the antigen

5. Classical pathway –as we can see from this diagram, the globular head regions
of the C1q subunits bind to C1q-specific receptors on the Fc region of the IgM or
two closely spaced IgG molecules, (as depicted in this illustration).
C1s esterase cleaves the next component, C4, into two fragments—(C4a =small
piece, remains in fluid and C4b which covalently binds to the surface of the
bacterium.)
C4b then binds C2 which is then cleaved by C1s

6. This diagram shows that several proteins regulate the classical pathway C3 convertase by
binding to C4b and displacing C2a from the complex:
•C4BP (C4 binding proteins) which is a serum protein.
•DAF; CD55 – decay-accelerating factor which are widely distributed cell-surface molecules
•MCP; CD46 – membrane cofactor protein
•CR1; CD35 – complement receptor 1
•Complement receptors (CRs) – are cell surface proteins on various cells that
recognize and bind complement proteins that have bound pathogens or other
antigens. CRs on phagocytes allow them to identify pathogens coated with
complement proteins for uptake and destruction.
•Factor 1 – serum protein that cleaves C4b on the cell surface after C2a has been displaced.
•Cofactors for factor 1-mediated cleavage (CR1, MCP, and C4BP), factor 1 cleavage of C4b
must have one or more of these molecules present.

7. Mannose-binding Lectin
Pathway
Mannose-binding lectin (MBL) pathway is structurally the same as C1q in the
classical pathway.
Activated by the terminal mannose residue of proteins and polysaccharides
located on the surface of bacteria.
MASP = (mannose associated serine protease)
•MASP-1 cleaves C4 and C2 sequentially
•Forms the classical pathway C3 convertase C4b2a

8. Alternative Pathway
In the alternative pathway:
•C3b binds foreign surface antigen on bacteria
•Can bind host cell as well – role of sialic acid
•Factor B binds C3b and exposes site cleaved by Factor D (Bb)
•Cleavage generates C3bBb binding protein (C3 convertase) that is bound by properdin
(factor P – a positive regulator that stabilizes C3b bind protein.)
•C3bBb – hydrolyzes C3 into C3a and C3b to amplify response (amplification loop) –
done in a short time
•During the activation of the alternative pathway both C3a and C5a are generated; both
are anaphylatoxins (anafhilatoxins) and cause degranulation of mast cells.

9. Factor H exclusively regulates the
alternative pathway
Factor H is a soluble protein that blocks
formation of C3 convertase by binding C3b;
cofactor of C3b by factor 1

10. C3 and C5 convertases of each pathway
Convertases…an enzyme component in the complement cascade that “converts”
the inactive form of the next component in the pathway into an active form by
cleaving it. C3 is cleaved into fragments C3a (released into the fluid phase) and C3b
(deposits on the cell surface) C5 is cleaved into C5a (released into fluid phase) and
C5b (deposits on the cell surface).

11. Formation of the membrane attack
complex (MAC)
(MAC) Formation Steps:
1st step in MAC formation---C6 binding to C5b on a cell surface.
2nd C7 then binds to C5b and C6 and undergoes a hydrophilic-amphiphilic structural
transition. Transition is required to bind phospholipid and inserts into the target cell
outer membrane.
3rd C8 binding to C5bC6C7 induces a hydrophilic-amphiphilic structural transition
creating a small pore that can lyse red blood cells (not nucleated cells). C5b-C8 acts as
a receptor on the cell membrane.

12. Formation of MAC
C5 is cleaved by the C5 convertase of either the Classical or Alternative Pathways. The C5 is
cleaved into C5a and C5b. The C5a fragment diffuses away, and the C5b fragment binds to the
surface of the target cell. This component is very labile and is inactivated within two minutes
unless C6 binds and stabilizes its activity.
•Once C7 binds, the complex undergoes a hydrophilic-amphiphilic transition that exposes
hydrophobic regions, which allow the complex to be inserted into the phospholipid bilayer.
•Binding of C8 induces a structural change in C8 exposing a hydrophobic region, which
interacts with the plasma membrane. A small pore is formed.
The final step in the sequence is the polymerization of C9 ( a perforin-like molecule). As many
as 10-16 C9s can be polymerized by a single C5bC6C7C8 complex.
Once bound, C9 undergoes a hydrophilic-amphiphilic transition, so that it too can be inserted
into the plasma membrane. This result in a hole formation. Even nucleated cells cannot
maintain osmotic stability and are lysed by an influx of water and loss of electrolytes.

15. RADIOIMMUNO ASSAY
• Radioimmunoassay (RIA) is a very
sensitive in vitro assay technique
used to measure concentrations
of antigens by use of antibodies.
HISTORY
• The technique was introduced in
1960 by Berson and Yalow as an
assay for the concentration of
insulin in plasma.
• It represented the first time that
hormone levels in the blood
could be detected by an invitro
assay.
Dr. Rosalyn Yalow became the
first
female to win a Nobel Prize with
her work on the
radioimmunoassay

16. LABELS IN IMMUNOASSAYS
• Immunoassays require the use of labeled materials in
order to measure the amount of antigen or antibody
present.
• A label is a molecule that will react as part of the
assay, and in doing so produce a signal that can be
measured in the solution. Examples of a label include
a radioactive compound, or an enzyme that causes a
change of color in a solution or its fluorescence

17. PRINCIPLE
• Radioimmunoassay (RIA) involves the
separation of a protein (from a mixture) using
the specificity of antibody - antigen binding
and quantitation using radioactivity.

18. Radioimmunoassay (RIAs) utilize
a radioactive label (usually 125I, 3H
or 14C), which emits radiation
that can be measured with a beta
or gamma counter.
Ag + Ag* + Ab  AgAb + Ag*Ab +
Ag + Ab*
◦ Unbound Ag* and Ag washed
out
◦ Radioactivity of bound residue
measured
◦ Ligand conc. is inversely related
to radioactivity
[Ag : ligand to be measured ; Ag*
radiolabelled ligand]
+
+P
P*
P*Q
Radioactive
tag
Analyte
Binding
agentFree Bound
Q
PQ

19. THE TECHNIQUE
• A mixture is prepared of
– radioactive antigen
• Because of the ease with which iodine atoms can be
introduced into tyrosine residues in a protein, the
radioactive isotopes 125I or 131I are often used.
– antibodies against that antigen.
• Known amounts of unlabeled ("cold") antigen are added to samples
of the mixture. These compete for the binding sites of the
antibodies.
•
• At increasing concentrations of unlabeled antigen, an increasing
amount of radioactive antigen is displaced from the antibody
molecules.
• The antibody-bound antigen is separated from the free antigen in
the supernatant fluid, and the radioactivity of each is measured.

22. * From these data, a standard binding
curve, like thee one shown in red, can be
drawn.
* The samples to be assayed (the
unknowns) are run in parallel.
After determining the ratio of bound to free
antigen in each unknown, the antigen
concentrations can be read directly from the
standard curve.
SEPARATING BOUND FROM FREE ANTIGEN
Precipitate the antigen-antibody complexes by adding a "second"
antibody directed against the first. For example, if a rabbit IgG is used to
bind the antigen, the complex can be precipitated by adding an
antirabbit-IgG antiserum (e.g., raised by immunizing a goat with rabbit
IgG).

23. ADVANTAGES
• Radioimmunoassay is widely-used because of its great sensitivity.
• Using antibodies of high affinity, it is possible to detect a few pictograms
(10−12 g) of antigen in the tube.
• The greater the specificity of the antiserum, the greater the specificity of
the assay
• RIA has become a major tool in the clinical laboratory where it is used to
assay .
• plasma levels of:
– most of our hormones;
– digitoxin or digoxin in patients receiving these drugs;
– certain abused drugs.
• Presence of hepatitis B surface antigen (HBsAg) in donated blood.
• Anti-DNA antibodies in systemic lupus erythematosus (SLE).

24. IMMUNOFLUORESCENCE
• Immunofluorescence : Immunofluorescence is a
powerful technique that utilizes fluorescent-labeled
antibodies to detect specific target antigens..
 Fluorescein is a dye which emits greenish fluorescence
under UV light. It can be tagged to immunoglobulin
molecules.
• This technique is sometimes used to make viral plaques
more readily visible to the human eye.
• Immunofluorescent labeled tissue sections are studied
using a fluorescence microscope.

25. Examples Of Fluorescent Dyes
Fluorescein Rhodamine

26. • There are two ways of doing IF staining
– Direct immunofluorescence
– Indirect immunofluorescence
1. Direct immunofluorescence
• It’s just a simple & a very common procedure
in this regard.
• Ag is fixed on the slide
• Fluorescein labeled Ab’s are layered over it
• Slide is washed to remove unattached Ab’s
• Examined under UV light in an fluorescent
microscope
• The site where the Ab attaches to its specific
Ag will show apple green fluorescence
• Use: Direct detection of Pathogens or their
Ag’s in tissues or in pathological samples.

27. 2. Indirect immunofluorescence:
• Indirect test is a double-layer technique
• The unlabelled antibody is applied directly to the tissue
substrate
• Treated with a fluorochrome-conjugated anti-
immunoglobulin serum.

28. What Immunoflouroscence Does
 Immunoflourescence is a Microscopic-based
technique, used clinically to diagnose certain
cutaneous diseases ( e.g; Lyme Disease) by the
detection of AG:AB Complexes.
 Techniques including DIF, IDIF & Salt-split Skin are
utilized depending on clinical scenario.
 DIF is performed on patient’s skin using flourophore-
labeled antibodies that directly bind to pathogenic
autoantibody-antigen complexes in the skin.

29. • IDIF techniques are used in Dermatology
primarily to detect circulating pathogenic
autoantibodies.
LIMITATIONS
• Fluorescence signals depend on the quality &
Concentration of antibodies, proper handling of
specimen & detection with appropriate
secondary antibodies.