Complement, Complement, Immunoglobulins, Antigen, Immunoglobulins, Antigen,

Hapten, Hapten, - Antigen Antibody Reaction- Antigen Antibody Reaction

Sakchai Dettrairat Sakchai DettrairatDivision of Division of Clinical Immunology Clinical Immunology

Department of Department of Medical TechnologyMedical Technology Faculty of Associated Medical Sciences Faculty of Associated Medical Sciences Chiang Mai University Chiang Mai University

03/06/2009

macrophages, neutrophils

complement, interferon, TNF etc.

T cells; other effectors cells

antibodies

Components of the Immune SystemComponents of the Immune System

Immune System

Nonspecific Specific

Humoral Cellular Humoral Cellular

Immune Response

Complement Complement SystemSystem• Serum globulins• Z ymogens or proenzyme• >20 components

– 1 1 1 4 2 3 5 6 7C q, C r, C s, C , C , C , C , C , C , C 8 , 9C

– DD DDDDDDDDDD ,, ,– etc.

• HH - eat labile (56 C, 30 min)- eat labile (56 C, 30 min)• RDDDD DDDD DD-DD DDDDDDDDD DDDDD DDD DDDD• D ausing lysis of some Ab sensitized cells• NDD-DDDDDDD DDDDDDDD• M ore active in fresh serum D Guinea pig)

ComplementComplement Activation Activation PathwayPathway

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)

Factor I, Factor H

Factor I 4, C bp

Protein S, SP40,40

DAF

Factor I, Factor H

Factor H

DAF

Antigen (Ag)

Antibody (Ab)

specifically react

Immunoglobulins (Igs)

Antibody Serum Protein

Protein Electrophoresis

Glycoprotein molecules that are produced by plasma cells in response to an immunogen and which function as antibodies

Immune serum

Ag adsorbed serum or normal serum

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

Ig Reduction Subunits

Immunoglobulin Subunits

Light chain (L)

Immunoglobulin Subunits

Heavy chain (H)

Ig papain digestion fragments

Immunoglobulin fragments

Immunoglobulin fragments (Fab and Fc)

Relation of Fab and Fc to H and L chains

Anti-Fab Anti-Fc

H + +

L + -

•rabbit Fab goat goat anti-Fab

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

Immunoglobulin StructureImmunoglobulin Structure

• Subunits:– 2 2H+ L

• Fragments:– 2 1Fab + Fc

• Rel at i on of Fab t o H and L chains

and Fc to H chain

Fig. 3.3 The Y-shaped immunoglobulin molecule can be dissected by partial digestion with proteases.

Fab-Fragment antigen bindingFc-Fragment crystallizableFv-Fragment variable

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 ResponseT-dependent Ag; 1o Response

• Lag phase

• Log phase

• Plateau phase

• Decline phaseAg

D a y s A f t e r I m m u n i z a t i o n

A b

T i

t e

r

LAG LOG DECLINEPLATEAU

Kinetics of the Ab ResponseT-dependent Ag; 2o Response

1o Ag 2o Ag

D a y s A f t e r I m m u n i z a t i o n

A b

T i

t e

r

Qualitative Ab Changes during 1o and 2o Responses

• Class variation

– 1o - IgM

– 2o - IgG, IgA or IgE

1o Ag

2o Ag

Total Ab

IgM Ab

IgG Ab

D a y s A f t e r I m m u n i z a t i o n

A b

T i t

e r

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 Reacti- Antigen Antibody Reactionon

Antigen-Antibody ReactionAntigen-Antibody Reaction

Ag + Ab - Ag Ab complex

1. Primary Reaction

(Invisible)

2. Secondary Reaction (Visible)

Forces binding Antigen to Forces binding Antigen to AntibodyAntibody

Non-covalent forces Origin

Electrostatic forces Attraction between opposite charges

Hydrogen bondsHydrogen shared between electronegative atoms (N, O)

van der Waal forces

Fluctuation in electron clouds around molecules oppositely polarize neighboring atoms

Hydrophobic forces

Hydrophobic groups interact unfavorably with water and tend to pack together to exclude water molecules. The attraction also involves van der Waal forces

Forces binding Antigen to AntibodyForces binding Antigen to Antibody

- Factors Affecting Ag Ab React - Factors Affecting Ag Ab Reactionion

• Temperature Temperature :: - 4 40o C (RT,

37oC)• pHpH : - 72 74 • Ionic strength Ionic strength : 0 .1 5 M NaCl

PrecipitationPrecipitation

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

diadia

Precipitation in Liquid MediaPrecipitation in Liquid Media

• Constant amo Constant amo unt of Ab unt of Ab

• Varied amoun Varied amoun

t of Ag t of Ag

• Amount of Pre Amount of Precipitatecipitate

……....

? ? ? ? ? ? ……….. ?

Quantitative Precipitation Curve Quantitative Precipitation Curve

• Constant amo unt of Ab

• Varied amoun t of Ag

• Amount of Precipitate

• Ab excess zone (Prozone)

• Equivalence zone

• Antigen excess zone (Post zone)

(Lattice formation)

Precipitation in Semi-solid media (Gel)Precipitation in Semi-solid media (Gel)

• Double diffusion in one dimensionDouble diffusion in one dimension

• Single diffusion in one dimensionSingle diffusion in one dimension

in Agar

in Agar

Single diffusion in two dimensions Single diffusion in two dimensions((MaMa ncini’ s technique) ncini’ s technique)

2

• Radial immunodiffusion Radial immunodiffusion • Precipitin ring Precipitin ring• Diameter ~> Ag concentration Diameter ~> Ag concentration• Quantitation of Ag concentration Quantitation of Ag concentration

Antibody Antigen

Agar matrix

Double diffusion in two dimensio Double diffusion in two dimensionsns (( Ouchterlony’ s technique) Ouchterlony’ s technique)

Double Immunodiffusion

Reaction of Identity / Non-Identity / Partial Identity

Reaction of Identity, - Non Identityor Partial Identity

Ag A

Ag C

Ag BAg D Ab

Immunoelectrophoresis (I Immunoelectrophoresis (IEP)EP) Protein electrophoresis in

Gel + Double immunodiffusion

Counter immunoelectrophoresi Counter immunoelectrophoresi s (CIE) s (CIE)

Anode Cathode

Electroimmunodiffusion (EID) Electroimmunodiffusion (EID)

• Ag Quantitation

Ag well

Cathode (-)

Anode (+)

- Ab containing gel

AgglutinationAgglutination

• Particulate Ag + specific Ab

• -- RBC Ag + Ab > HemagglutinationHemagglutination

• Reaction in liquid media• Direct Agglutination

• Indirect (or Passive) Agglutination

• Reverse Passive Agglutination

• Agglutination Inhibition

• Antiglobulin Test (Coombs’ test)

Direct AgglutinationDirect Agglutination

• Ag or Ab assay

• Bacterial Agglutination (Bacterial typing)

• RBC Agglutination (Blood grouping)

• Slide agglutination / Tube agglutination

Dilution & TiterDilution & Titer

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

Ab Titer = 128

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 1:4

1:8

1:16

1:32

1:64

1:128

1:256

1:512

1:1024

-

Aggltn +/- + + + + + - - - -

Serial Two-fold dilutionSerial Two-fold dilution

A

B

C

D

E

F

G

H

Hemagglutination in microtiter Hemagglutination in microtiterplateplate

Dilution & Titer = ?

Ab Titer = ?

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:?

-

Aggltn + + + + + + - - - -

Serial Two-fold dilutionSerial Two-fold dilution

Dilution & Titer = ?

Ab Titer = ?

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:?

-

Aggltn + + + + + + - - - -

Serial Two-fold dilutionSerial Two-fold dilution

Indirect (or Passive) Agglutinatio Indirect (or Passive) Agglutinationn• 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 AgglutinationReverse Passive Agglutination

• Ab coated inert particle

• Ag detection

Agglutination InhibitionAgglutination Inhibition• Ag coated inert particle + limit amount ofAb

• Ag detection : eg. HCG in urine

Direct Coombs’ Test Direct Coombs’ Test

• Detect Ab sensitized patient’s RBC

Antiglobulin test (Coombs’ T Antiglobulin test (Coombs’ Test)est)

Indirect Coombs’ Test Indirect Coombs’ Test• Detect & identify free Ab in Detect & identify free Ab in patient’s patient’s serserumum

Antiglobulin test in Hemolytic Disease of

Newborn• Maternal bloodMaternal blood

– Direct Antiglobulin test = +ve or –ve?– Indirect Antiglobulin test = +ve or –ve?

• Fetal bloodFetal 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 ActivationComplement 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) testComplememt Fixation (CF) test

• Detection of Ag or AbDetection of Ag or Ab

• Use Complement (C) and Use Complement (C) and

• Ab sensitized SRBC or EAAb sensitized SRBC or EA

Ag+ Ab +C Ag-Ab-C

+ EA No hemolysis

No hemolysisNo hemolysis = Positive test = Positive test

HemolysisHemolysis = Negative test = Negative test

CF test ControlsCF test Controls

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

2. Ag control 2. Ag control : Ag + C + EA : Ag + C + EA ??

3. C control : C + EA 3. C control : C + EA ??

4. RBC control : EA 4. RBC control : EA ??

? = Hemolysis or No hemolysis

CF TestCF Test

Labeled Ag-Ab ReactionLabeled Ag-Ab Reaction(Labeled Immunoassay)(Labeled Immunoassay)

• RadioimmunoassayRadioimmunoassay

• Immunofluorescence or Immunofluorescence or Fluorescence Fluorescence ImmunoassayImmunoassay

• Enzyme immunoassayEnzyme immunoassay

Radioimmunoassay (RIA)Radioimmunoassay (RIA)

• Use r adioisotope : 125 I , 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)

AgAg Ag-Ab Ag-Ab

Ab +Ab +

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

B/F Ag

Radioimmunoassay (RIA)

B/F Ag

AgAg Ag-Ab Ag-Ab

Ab +Ab +

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

RIA Standard curveRIA Standard curve

Separation of Free form (F) from Separation of Free form (F) from Bound form (B)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

ImmunofluorescenceImmunofluorescence• Fluorochromes :Fluorochromes :

• Fluorescein isothiocyanate (FITC)Fluorescein isothiocyanate (FITC)

• Rhodamine isothiocyanateRhodamine isothiocyanate (RITC)(RITC)

• Fluorochrome labeled Ab Fluorochrome labeled Ab

(or Fluorochrome labeled protein A)(or Fluorochrome labeled protein A)

• Ag -> cells or particulate AgAg -> cells or particulate Ag

• Direct method Direct method (Ag detection)(Ag detection)

• Indirect method Indirect method (Ab detection)(Ab detection)

ImmunofluoresceImmunofluorescencence

Flow Cytometric Flow Cytometric CD4+ T cell countCD4+ T cell count

The parameters of flow cytometric analysis

Laser beam Forward light scatter

FluorescenceSide scatter granularity

Forward angle light scatter, 90° light Forward angle light scatter, 90° light scatter and scatter and FluFluoresorescencecence

Properties of cells measured Properties of cells measured by Flow Cytometryby Flow Cytometry

• Its relative size Its relative size – Forward Scatter (FSC)Forward Scatter (FSC)

• Its relative granularity or Its relative granularity or internal complexity internal complexity – Side Scatter (SSC)Side Scatter (SSC)

• Its relative fluorescence Its relative fluorescence intensityintensity– FL1, FL2, FL3 and FL4FL1, 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 CD4 Count using B-D TriTEST

CD3 FITC/CD3 FITC/ CD4 PECD4 PE//CD45 PerCPCD45 PerCP ReagentReagent

Fig. 1 Ungated CD45 vs SSC dot plot.

Fig. 2 Lymphocyte-gated 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

Ag Ag assayassay

Double Antibody Sandwich or Double Antibody Sandwich or Two-Site ELISATwo-Site ELISA

Double Ab S andwich ELISAfor detecting antigen

EE

Enzyme conjugated Enzyme conjugated Ab2Ab2

AgAg

Ab1Ab1

BB

E

E Biotinylated Biotinylated anti-p24 Abanti-p24 Ab22

Enzyme conjugated avidinEnzyme conjugated avidin

Substrate Color Product

Ab1Ab1

AgAg

Double Antibody Sandwich or Double Antibody Sandwich or Two-Site ELISATwo-Site ELISA

ELISA for Ab assay:ELISA for Ab assay:• Indirect ELISAIndirect ELISA

• Competitive ELISACompetitive ELISA

• Double Ag Sandwich Double Ag Sandwich

ELISA ELISA

Double Ag Sandwich Double Ag Sandwich ELISA for Ab detectionELISA for Ab detection

Third generation Double Antig Third generation Double Antig en Sandwich ELISA en Sandwich ELISA

PP

Test Ag

Enzyme conjugated Ag

IgGIgG AbAb

PPIgM AbIgM Ab

Substrate Color Product

Fourth generation SandwichSandwich ELISAELISA

PP PP

AbAg

Enzyme-conjugated Ab

AgAg

Enzyme conjugated Ag

Ab

Substrate Color Product

for detecting HIV Abs and Ag simultaneously

Substrate of ELISASubstrate 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)Immunoblot (Western blot)

Immunochromatographic assayImmunochromatographic assay (Strip test)(Strip test)

Ag assay Ag assay (Double Antibody sandwich assay)(Double Antibody sandwich assay)

Test band

Control band

Dye labeled reagent

Sample flow

= Dye-labeled Ab1= Dye-labeled Ab1 (mobile) (mobile)

= Ab2 coated bandAb2 coated band

Ab against Ab1 =Ab against Ab1 =

• Positive = test and control bandsPositive = test and control bands

• Negative = control band onlyNegative = control band only

Ag Test StripAg Test Strip

Ab against Ab1 Ab against Ab1

Dye-labeled Ab1Dye-labeled Ab1 (mobile) (mobile)

Ab2 coated bandAb2 coated band

Ab AssayAb Assay: :

Double Antigen sandwich assayDouble Antigen sandwich assay

Control band Control band (Ab against Ag)(Ab against Ag)

Dye-labeled Ag Dye-labeled Ag (mobile)(mobile)

Test band (Ag Test band (Ag coated band)coated band)

Sample flowSample flow

• Positive Positive

= test and control bands= test and control bands

• Negative Negative

= control band only= control band only