Complement,

Immunoglobulins, Antigen,
Hapten,
Antigen-Antibody Reaction
Sakchai Dettrairat
Division of Clinical Immunology
Department of Medical Technology
Faculty of Associated Medical Sciences
Chiang Mai University

03/06/2009
 Components of the Immune System

Immune System
Nonspecific Specific

Humoral Cellular Humoral Cellular

complement,
macrophages, T cells; other
interferon, antibodies
neutrophils effectors cells
TNF etc.
Immune Response
 Complement System
• Serum globulins
• Zymogens or proenzyme
• >20 components
– C1q, C1r, C1s, C4, C2, C3, C5, C6, C7, C8, C9
– Factor B, Factor D, Properdin,
– etc.
• Heat-labile (56 C, 30 min)
• React with Ag-Ab complexes (IgG, IgM
Abs)
• Causing lysis of some Ab sensitized cells
• Non-species specific
• More active in fresh serum (Guinea pig)
Complement Activation
Pathway
Function of Complement
• Opsonization of cellular
(bacterial) antigen
• Provoke inflammation
• Poke holes in membranes
leading to lysis of bacteria
• Clear immune complexes
• Activates antigen-specific B
cell
Function of Complement
Regulation of Complement
Activation
C1 inhibitor (C1INH)
DAF

Factor I, C4bp Factor I,

Factor H
Factor I,
Factor H Factor H
Protein S,
SP40,40

DAF
 Immunoglobulins (Igs)

Antigen
(Ag)
specifical
ly react

Antibody
(Ab)
Antibody  Serum Protein

Protein Electrophoresis
 Immune serum

Ag adsorbed
serum or
normal serum

Glycoprotein molecules that are produced by plasma

cells in response to an immunogen and which
function as antibodies
Protein Structure: Polypeptide chain
Protein Structure
Immunoglobulin Structure
• Ig Subunits
• Ig Fragments
• Relation of Ig Subunits and
Fragments
 Determining Ab
Structure
• Work in 1950s and 60s using biochemical
techniques
• Rodney Porter used partial proteolysis with papain -
2 identical antigen-binding fragments (Fab) and
one “tail” fragment (Fc)
• Alfred Nisinoff used pepsin - one fragment with
divalent antigen binding (F(ab2)’)
• Gary Edelman used b-ME to reduce Ig, resolving
heavy (H) and light (L) chains
• Rodney Porter probed H and L chains with anti- Fab
and anti-Fc antibodies: anti-Fab detected both H &
L, but anti-Fc detected only H
• Combined work earned Porter and Edelman
1972 Nobel Prize
Immunoglobulin Subunits
Ig  Reduction 
Subunits
Immunoglobulin Subunits

Heavy chain (H)

Light chain
(L)
Immunoglobulin fragments
Ig  papain digestion 
fragments
Immunoglobulin fragments
(Fab and Fc)
Relation of Fab and Fc to H and L chains

•rabbit Fab  goat  goat anti-Fab

•Rabbit Fc  goat  goat anti-FC
•anti-Fab, anti-Fc react with H and L chains

Anti-Fab Anti-Fc
H + +
L + -
 Immunoglobulin Structure

• Subunits:
– 2H+2L
• Fragments:
– 2 Fab + 1 Fc
• Relation of Fab to
H and L chains
and Fc to H chai
n
Fab-Fragment antigen binding
Fc-Fragment crystallizable
Fv-Fragment variable

Fig. 3.3 The Y-shaped

immunoglobulin molecule
can be dissected by partial
digestion with proteases.
 Structure of the Variable
Region
• Hypervariable (HVR) or complimentarity
determining regions (CDR)
Domains
Human Immunoglobulin Classes
Heavy Chain Types
• IgG - Gamma heavy chains
• IgM - Mu heavy chains
• IgA - Alpha heavy chains
• IgD - Delta heavy chains
• IgE - Epsilon heavy chains
Light Chain Types
• Kappa
• Lambda
 IgG
• Structure
– Monomer (7S)
• Properties
– Major serum Ig
– Major Ig in extravascular spaces
– Placental transfer – Does not require Ag
binding
– Fixes complement
– Binds to Fc receptors
• Phagocytes - opsonization
• K cells - ADCC
 IgM
• Structure
– Pentamer (19S)
– Extra domain (CH4)
– J chain
• Properties
– 3rd highest serum Ig
– First Ig made by fetus and B cells
– Fixes complement
– Agglutinating Ig
– Binds to Fc receptors
– B cell surface Ig
 IgA
• Structure
– Serum - monomer
– Secretions (sIgA)
• Dimer (11S)
• J chain
• Secretory component
• Properties
– 2nd highest serum Ig
– Major secretory Ig (Mucosal or Local Immunity)
• Tears, saliva, gastric and pulmonary secretions
– Does not fix complement (unless aggregated)
– Binds to Fc receptors on some cells
Secretory IgA
(SIgA)
 IgD
• Structure
– Monomer
– Tail piece
• Properties
– 4th highest serum Ig
– B cell surface Ig
– Does not bind complement
 IgE
• Structure
– Monomer
– Extra domain (CH4)
• Properties
– Least common serum Ig
• Binds to basophils and mast cells (Does not
require Ag binding)
– Allergic reactions
– Parasitic infections (Helminths)
• Binds to Fc receptor on eosinophils
– Does not fix complement
Ab formation
 Kinetics of the Ab Response
T-dependent Ag; 1o Response

• Lag phase LAG LOG PLATEAU DECLINE

Ab Titer
• Log phase
• Plateau phase
Ag
• Decline phase
Days After Immunization
Kinetics of the Ab Response
T-dependent Ag; 2o Response
Ab Titer

1o Ag 2o Ag

Days After Immunization

 Qualitative Ab Changes during
1o and 2o Responses
Total Ab IgM Ab IgG Ab

• Class variation
1o Ag 2o Ag
– 1 - IgM Ab Titer
o

– 2o - IgG, IgA or IgE

Days After Immunization

Monoclonal Antibody
 Antigen & Hapten
• What is an antigen?
– a substance that can induce a specific
immune response
• An immunogen induces a humoral
(B-cell) or cell-mediated (T-cell)
response
• Haptens are too small to induce an
IR unless coupled to a carrier (an
antigen)
 Antigen
• Foreigness
• High Moleclular Weight
– >10,000 Da
– <1000 Da are not usually immunogenic
• Chemical Complexity
– Proteins are often good immunogens.
– Homopolymers are not good
immunogens
• Induces Immune Response
 Hapten
• Low MW Chemicals
• Does not induce IR by itself
• Reacts specifically with Anti-
hapten Abs
How to
produce
Abs to
Hapten?
Antigen-Antibody
Reaction
Antigen-Antibody Reaction

Ag + Ab Ag-Ab
complex
1. Primary Reaction
(Invisible)

2. Secondary Reaction (Visible)

 Forces binding Antigen to
Antibody
Non-covalent forces Origin
Attraction between
Electrostatic forces opposite charges

Hydrogen shared between

Hydrogen bonds electronegative atoms
(N, O)

Fluctuation in electron
clouds around molecules
van der Waal forces oppositely polarize
neighboring atoms

Hydrophobic groups interact

unfavorably with water and t
Hydrophobic forces end to pack together to exclu
de water molecules. The attr
action also involves van der
Waal forces
Forces binding Antigen to Antibody
 Factors Affecting Ag-Ab
Reaction

• Temperature : 4 - 40 oC (RT, 37
o
C)
• pH : 7.2 - 7.4
• Ionic strength : 0.15 M NaCl
 Precipitation

• Soluble Ag + specific Ab
• Precipitation in Liquid Media
• Precipitation in Semi-solid
media
 Precipitation in Liquid Media

• Constant
amount of Ab

……....
• Varied
amount of Ag

• Amount of
Precipitate ? ? ? ? ? ? ……….. ?
 Quantitative Precipitation Curve

• Constant
amount of Ab
• Varied
amount of Ag
• Amount of
Precipitate
• Ab excess zone (Prozone)
• Equivalence zone
• Antigen excess zone (Post zone)

(Lattice formation)
Precipitation in Semi-solid media (Gel)
• Single diffusion in one dimension

• Double diffusion in one dimension

in Agar

in Agar
 Single diffusion in two
dimensions(Mancini’ s technique)
• Radial immunodiffusion
• Precipitin ring
• Diameter ~> Ag concentration
• Quantitation of Ag concentration

2
 Double diffusion in two
dimensions (Ouchterlony’ s techni
que)Antibody Antigen

Double
Agar matrix
Immunodiffusi
on
Reaction of Identity / Non-Identity / Partial
Identity
Reaction of Identity, Non-
Identity or Partial Identity
Ag A

Ag D Ab Ag B

Ag C
 Immunoelectrophoresis
(IEP) in
Protein electrophoresis
Gel + Double
immunodiffusion
 Counter
immunoelectrophoresis (CIE)

Anode Cathode
Electroimmunodiffusion (EID)
• Ag Quantitation

Anode (+)

Ab-containing
gel

Ag well

Cathode (-)
 Agglutination

• Particulate Ag + specific Ab
• RBC Ag + Ab --> Hemagglutination
• Reaction in liquid media
• Direct Agglutination
• Indirect (or Passive) Agglutination
• Reverse Passive Agglutination
• Agglutination Inhibition
• Antiglobulin Test (Coombs’ test)
Direct Agglutination
• Ag or Ab assay
• Bacterial Agglutination (Bacterial
typing)
• RBC Agglutination (Blood grouping)
• Slide agglutination / Tube
agglutination
Dilution & Titer
Serial Two-fold dilution
Tube no. 1 2 3 4 5 6 7 8 9 10
NSS (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Serum (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
Ag (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Dilution 1: 1: 1: 1: 1: 1: 1: 1: 1: -
4 8 16 32 64 128 256 512 1024
Aggltn +/- + + + + + - - - -

Final positive serum dilution = 1:128 (or 1/128)

Ab Titer = 128
Hemagglutination in microtiter
plate

A
B
C
D
E
F
G
H
Dilution & Titer = ?
Serial Two-fold dilution
Tube no. 1 2 3 4 5 6 7 8 9 10
NSS (mL) 0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Serum (mL) 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
Ag (mL) 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Dilution 1: 1: 1: 1: 1: 1: 1: 1: 1: -
? ? ? ? ? ? ? ? ?
n
Agglt + + + + + + - - - -

Ab Titer = ?
Dilution & Titer = ?
Serial Two-fold dilution
Tube no. 1 2 3 4 5 6 7 8 9 10
NSS (mL) 0.9 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5
Serum (mL) 0.1 0.5 0.5 0.5 0.5 0.5 0.5 0.5 0.5 -
Ag (mL) 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1
Dilution 1: 1: 1: 1: 1: 1: 1: 1: 1: -
? ? ? ? ? ? ? ? ?
n
Agglt + + + + + + - - - -

Ab Titer = ?
Indirect (or Passive)
Agglutination
• Test Ag --> Soluble Ag
• Ag coated inert particle
• Inert particles : latex particle, gelatin
particle, human gr. O RBC, sheep RBC
(particles that do not react with test
serum.)
• Ab detection
Reverse Passive Agglutination
• Ab coated inert particle
• Ag detection
Agglutination Inhibition
• Ag coated inert particle + limit amount of
Ab
• Ag detection : eg. HCG in urine
 Antiglobulin test (Coombs’
Test)
Direct Coombs’ Test
• Detect Ab sensitized patient’s
RBC
Indirect Coombs’ Test
• Detect & identify free Ab in patient’s
serum
 Antiglobulin test in
Hemolytic Disease of
Newborn
• Maternal blood
– Direct Antiglobulin test = +ve or –ve?
– Indirect Antiglobulin test = +ve or –
ve?
• Fetal blood
– Direct Antiglobulin test = +ve or –ve?
– Indirect Antiglobulin test = +ve or –
ve?
• What is the blood group of RBC
used in Indirect Antiglobulin test?
Complement Activation

Membrane attack pathway

(Common or Terminal
pathway)
 Function of Complement

• Opsonization of cellular (bacterial)

antigen
• Provoke inflammation
• Poke holes in membranes leading
to lysis of bacteria
• Clear immune complexes
• Activates antigen-specific B cell
Complememt Fixation (CF) test
• Detection of Ag or Ab
• Use Complement (C) and
• Ab sensitized SRBC or EA

Ag+ Ab +C Ag-Ab-C
+ EA No hemolysis

No hemolysis = Positive test

Hemolysis = Negative test
CF test Controls

1. Serum (Ab) control : Ab + C + EA  ?

2. Ag control : Ag + C + EA  ?
3. C control : C + EA  ?
4. RBC control : EA  ?

? = Hemolysis or No hemolysis
CF Test
 Labeled Ag-Ab Reaction
(Labeled Immunoassay)
• Radioimmunoassay
• Immunofluorescence or
Fluorescence
Immunoassay
• Enzyme immunoassay
 Radioimmunoassay (RIA)
• Use radioisotope : 125I, 131I
• Radioiotope-labeled Ag and limit
amount of specific antibody
• Ag detection : eg. hormones
• High sensitivity  ng/ml, pg/ml
• Competitive binding format
• Separation of free Ag* (Free form, F)
from Ag*-Ab complex (Bound form,
B)
• Detect by gamma counting
Radioimmunoassay (RIA)

Ag Ag-Ab
Ab +
Ag*(F) Ag*-Ab (B)

B/F Ag
Radioimmunoassay (RIA)

Ag Ag-Ab
Ab +
Ag*(F) Ag*-Ab (B)

B/F Ag
RIA Standard curve
Separation of Free form (F) from
Bound form (B)
1. Salt precipitation
of B form
2. Precipitation of
B form by
Antiglobulin
3.F form precipitation
by Dextran or
charcoal
4. Ab coatiing on
solid phase
 Immunofluorescence
• Fluorochromes :
• Fluorescein isothiocyanate (FITC)
• Rhodamine isothiocyanate (RITC)
• Fluorochrome labeled Ab
(or Fluorochrome labeled protein A)
• Ag -> cells or particulate Ag
• Direct method (Ag detection)
• Indirect method (Ab detection)
Immunofluoresce
nce
Flow Cytometric
CD4+ T cell count
 The parameters of
flow cytometric analysis

Laser beam Forward light scatter

Side scatter
Fluorescence
granularity

Forward angle light scatter, 90° light

scatter and Fluorescence
Properties of cells measured
by Flow Cytometry
• Its relative size
– Forward Scatter (FSC)
• Its relative granularity or
internal complexity
– Side Scatter (SSC)
• Its relative fluorescence
intensity
– FL1, FL2, FL3 and FL4
Example
 Three-color
monoclonal antibody
panel
Tube No. FL3-mAb FL1-mAb FL2-mAb

1 CD45 CD3 CD4

2 CD45 CD3 CD8

 CD4 Count using B-D TriTEST
CD3 FITC/ CD4 PE/CD45 PerCP
Reagent

Fig. 1 Ungated Fig. 2 Lymphocyte-

CD45 vs SSC dot gated CD3 vs CD4 dot
plot. plot.
 Enzyme Immunoassay
• Enzyme labeled Ag or Ab
– Alkaline Phosphatase (AP)
– Horse radish peroxidase (HRP, Px)
• Enzyme-Linked ImmunoSorbent Assay
(ELISA)
• Ab or Ag coated on solid phase
• Separation of Free from Bound form
(Washing)
ELISA format
• Ag assay
– Sandwich format
• Ab assay
– Indirect format
– Competitive format
– Sandwich format
 Double Antibody Sandwich or
Two-Site ELISA

Ag
assay
 Double Ab Sandwich ELISA
for detecting antigen
Substrate Color Product
Enzyme conjugated Enzyme conjugated avidin
Ab2 E
Biotinylated
E E anti-p24 Ab2
B
Ag Ag

Ab1 Ab1
Double Antibody Sandwich or
Two-Site ELISA
ELISA for Ab assay:
• Indirect ELISA
• Competitive ELISA
• Double Ag Sandwich
ELISA
 Double Ag Sandwich
ELISA for Ab detection
Third generation Double
Antigen Sandwich ELISA

Substrate Color
Product
Enzyme
conjugated P IgM Ab
P
Ag

IgG Ab

Test Ag
Fourth generation Sandwich
ELISA
Substrate Color
Product
Enzyme Enzyme-conjugated
conjugated Ab
Ag
P P

Ag
Ab

Ag Ab

for detecting HIV Abs and Ag

simultaneously
Substrate of ELISA
• Alkaline Phosphatase (AP)
– p-Nitrophenyl phosphate (pNPP)
– OD405 nm
• Horse radish peroxidase (HRP, Px)
– H2O2 + chromogen
• H2O2 + o-phenylelne diamine (OPD)
stop reaction with H2SO4 -> OD492 nm.
• H2O2 + Tetramethyl benzidine (TMB)
stop reaction with H2SO4 -> OD450 nm.
• Absorbance or Optical Density (OD)
– 0.000 - 2.000 / 3.000 OD.
Immunoblot (Western blot)
 Immunochromatographic assay
(Strip test)
Ag assay (Double Antibody sandwich assay)
• Positive = test and control bands
• Negative = control band only

Control
Ab against Ab1 = band Test
band
= Ab2 coated band

Dye
labeled = Dye-labeled Ab1
reagent
Sample flow (mobile)
 Ag Test Strip

Ab against Ab1
Ab2 coated band

Dye-labeled Ab1
(mobile)
 Ab Assay:
Double Antigen sandwich assay

• Positive
= test and control bands
Control band • Negative
(Ab against Ag) = control band only
Test band (Ag
coated band)

Dye-labeled Ag
(mobile)
Sample flow