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 Humoral
complement, interferon, TNF etc.

Specific Humoral Cellular
T cells; other effectors cells

Cellular
macrophages, neutrophils

antibodies

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 H

Factor I, Factor H Factor I, 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

• 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

Determining Ab Structure

• 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 H L + + Anti-Fc + -

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 • Log phase • Plateau phase • Decline phase
Ab Titer

LAG

LOG

PLATEAU

DECLINE

Ag

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
– 1 - IgM
o

– 2o - IgG, IgA or IgE

Ab Titer

1o Ag

2o Ag

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 Antihapten Abs

How to produce Abs to Hapten?

Antigen-Antibody Reaction

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

2. Secondary Reaction (Visible)

Forces binding Antigen to Antibody
Non-covalent forces
Electrostatic forces Hydrogen bonds

Origin

Attraction between opposite charges Hydrogen shared between electronegative atoms (N, O) Fluctuation in electron clouds around molecules oppositely polarize neighboring atoms Hydrophobic groups interact unfavorably with water and t end to pack together to exclu de water molecules. The attr action also involves van der Waal forces

van der Waal forces

Hydrophobic 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 Immunodiffusi on
Agar matrix

Reaction of Identity / Non-Identity / Partial Identity

Reaction of Identity, NonIdentity or Partial Identity
Ag A

Ag D

Ab

Ag B

Ag C

Immunoelectrophoresis (IEP) Protein electrophoresis in
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. NSS (mL)

1 0.5

2 0.5 0.5 0.5 1: 8 +

3 0.5 0.5 0.5 1: 16 +

4 0.5 0.5 0.5 1: 32 +

5 0.5 0.5 0.5 1: 64 +

6 0.5 0.5 0.5

7 0.5 0.5 0.5

8 0.5 0.5 0.5

9 0.5 0.5 0.5

10 0.5 0.5 -

Serum (mL) 0.5 Ag (mL) Dilution Aggltn

0.5 1: 4 +/-

1: 1: 1: 1: 128 256 512 1024 + -

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. NSS (mL)

1 0.9

2 0.5 0.5 0.5 1: ? +

3 0.5 0.5 0.5 1: ? +

4 0.5 0.5 0.5 1: ? +

5 0.5 0.5 0.5 1: ? +

6 0.5 0.5 0.5 1: ? +

7 0.5 0.5 0.5 1: ? -

8 0.5 0.5 0.5 1: ? -

9 0.5 0.5 0.5
1:

10 0.5 0.5 -

Serum (mL) 0.1 Ag (mL) Dilution n Agglt

0.5 1: ? +

? -

Ab Titer = ?

Dilution & Titer = ?
Serial Two-fold dilution
Tube no. NSS (mL)

1 0.9

2 0.5 0.5 0.1 1: ? +

3 0.5 0.5 0.1 1: ? +

4 0.5 0.5 0.1 1: ? +

5 0.5 0.5 0.1 1: ? +

6 0.5 0.5 0.1 1: ? +

7 0.5 0.5 0.1 1: ? -

8 0.5 0.5 0.1 1: ? -

9 0.5 0.5 0.1
1:

10 0.5 0.1 -

Serum (mL) 0.1 Ag (mL) Dilution n Agglt

0.1 1: ? +

? -

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

• Maternal blood

Antiglobulin test in Hemolytic Disease of Newborn

– 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 + EA Ag-Ab-C No hemolysis

No hemolysis = Positive test Hemolysis = Negative test

CF test Controls
1. Serum (Ab) control : Ab + C + EA  ? 2. Ag control 3. C control 4. RBC control : Ag + C + EA  ? : : C + EA  ? 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 Ab + Ag*(F) Ag*-Ab (B) Ag-Ab

B/F

Ag

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

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 Fluorescence

Side scatter 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 2 CD45 CD45 CD3 CD3 CD4 CD8

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

Fig. 1 Ungated CD45 vs SSC dot plot.

Fig. 2 Lymphocytegated CD3 vs CD4 dot 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
Enzyme conjugated Ab2

Color Product
Enzyme conjugated avidin

E E
Ag

E

B

Biotinylated anti-p24 Ab2
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 Product
Enzyme conjugated Ag

Color

P

IgM Ab

P

IgG Ab
Test Ag

Fourth generation Sandwich ELISA
Substrate Product Enzyme
conjugated Ag

Color
Enzyme-conjugated Ab

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

Ab against Ab1 =

Control band

Test band

= Ab2 coated band
= Dye-labeled Ab1 (mobile)

Dye labeled reagent

Sample flow

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 (Ab against Ag)
Test band (Ag coated band)

• Negative = control band only

Dye-labeled Ag (mobile)

Sample flow

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