Joseph Cannon, PhDClinical Laboratory Sciences ProgramAugusta University

Antibody synthesis(B cells)diversity

specificity

Lecture overview

Antibody structurecharacteristics of monomers

isotypesbinding sites

Functioninteraction with antigen

microbial evasioninteractions with host cells

(and other host defense molecules)

Distributionbody compartmentsepithelial transportfetal environment

Laboratory reagentdiagnostic in vitro assays

In vivo imagingtherapy

Antibody synthesis• B cells• diversity• specificity

The source of antibodies

B cells express CD194-8% of circulating leukocytesResponsible for humoral immunity ≡ antibody-mediateddifferentiate into memory cells or plasma cells

the B cell receptor is a monomeric form of IgM

plasma cells secrete antibodies

B

CD20

CD19

CD24

• CD19 is expressed early (Pro-B cell)

• B cell receptors (monomeric IgM) develop in bone marrow phase and are present on the surface of immature cells

• IgD is expressed on B cellsin later stage of development -“mature” B cells

• “virgin” means that the cell has not yet encountered the antigen that will stimulate it

• class switching and somatic mutation occur after stimulation with antigen

• remember that memory cells are also generated after antigen stimulation

B cell developmentCD19

CD19

CD19

CD19

CD19

Figure: Rittenhouse-Olsen & De Nardin

Antigen-independent Diversity

V1 V2 V3 Vn D1 D2 D3 Dn J1 J2 J3 Jn Cm Cd Cg3 Cg1 Cg2 Cg4 Ce Ca

ǁ ǁǁ

excised

excised

DNA

V2D2J3Cm

mRNA

Transcription and splicing

V2D2J3Cm

IgM heavy chain

Translation

V1 V2D2J3 Jn Cm Cd Cg3 Cg1 Cg2 Cg4 Ce Ca

ǁ DNA

V/D/J joining

V1 V2 V3 Vn D1 D2J3 Jn Cm Cd Cg3 Cg1 Cg2 Cg4 Ce Ca

ǁǁ DNA

D/J joining

(random recombination of gene segments to create heavy chains)

A similar process (on different chromosomes) creates the light chains (but no D segments). These chromosomes have a segment that codes for k or l constant region.

Cytokines from TH cells signal for excision of Cμ gene and recombination of VDJ region with a different constant gene(e.g., Cg, resulting in IgG)

Diversity: Millions of lymphocytes exist, each one recognizes only a single epitope on a single antigen.

Memory and effector T cells are generated the same way.

MemoryB cells

clonal selection

proliferationand differentiation

of clonesPlasma cells(effectors)

secreteantibodies

bind antigen

All clones share same antigen specificity

Clonal expansionFigure: Germann & Stanfield

Polyclonal antibodies

• Most antigens have many epitopes• Therefore not one, but many B cells are

activated• When an animal or human is vaccinated,

antibodies against each of the epitopes are produced and circulate

• Therefore, serum contains polyclonal antibodies

Antigen

Differentepitopes

B cell

Immunological Memory

Secondexposure

to the sameantigen

Antibody structure• characteristics of monomers• isotypes• binding sites

Isotype

Isotype refers to major classes of antibodies (IgA, IgG, IgM, IgD, IgE), based on heavy chain constant regions (a, g, m, d, e). Isotype is relatively constant within each species.

Idiotype

Idiotype refers to the specific binding behavior conferred by hypervariable region (antigen binding site).

Two types of light chains: kappa (k) and lambda (λ) exist in 2:1 ratio (no known functional differences)

Allotype refers to small differences among individuals in the constant regions of light and heavy chains (inherited).

Allotype

Immunoglobulin classification

IgG IgEIgD

U

IgM IgA

U

Isotypes

# of HC domains: 4 5 4 4 5

‘meric structure: mono- penta- di- mono- mono-

# antigen binding 2 10 4 2 2 sites

Monomer characteristics

Heavy chain (Fc) is constant for each Immunoglobulin class (isotype) within each species.

Papain breaks heavy chains above the disulfide bonds that connect them.

Antigen binding sites are separate

Pepsin breaks heavy chains below the disulfide bonds that connect them.

The two antigen binding sites remain connected

F(ab’)2

Fc’

FabFab

Fc

Enzymatically-produced fragments

F(ab’)2: minimum structure capable of agglutination

Fab: minimum steric hindrance

Fc is the key domain for:

• Distribution in body (neonatal FcR)• Binding to specific cells (Fcg -vs- Fce)• Classical complement activation (C1q)• Anchor points for J chain• Recognizing/measuring patient antibody• Microbial evasion

• ~80% of total Ig in serum• Passes into interstitial fluid• Biological functions:

o agglutination o neutralization o activates (“fixes”) complemento opsonization (Fcg receptors on phagocytic cells)o promotes antibody-dependent cell-mediated cytotoxicity (ADCC)

• Passes through placenta, confers infant protection for ~6 months

IgG

• ~10% of total serum Ig (monomer: has little apparent function)• Dimer is secreted onto mucosal surfaces, preventing pathogen entry• 2 Fc’s joined by J chain• Secretory piece added in passage through epithelial cell• Agglutinates, neutralizes, opsonizes• Transferred to infants through breast milk and colostrum, protects

against enteric pathogens

IgA

U

• Pentamer

• Largest mass: 900 kDa (macroglobulin)

• ~10% of total serum Ig

• 10 antigen binding sites (each with low affinity, but high total avidity)

• First Ig produced, no somatic mutation

• Can be secreted (J chain binds to poly-Ig receptor)

• Surface receptor on B cells (as monomer)

• Best preciptator, agglutinator and complement fixer. Also opsonizes and neutralizes.

IgM

U

Efficient at agglutination

Flexibility of IgMFigure: Kuby et al.

• Very little in serum (0.02% of Ig)

• Has extra domain in the heavy chains(compared to IgG)

• Made by plasma cells near interface with external the environment

• Mast cells, basophils, eosinophils, and Langerhans cells* have Fce receptors

• Induces degranulation by mast cells (release of histamine, heparin & chemoattractants)

• Mediates allergic responses

• Mediates protective responses against pathogens that penetrate mucosal barriers (especially parasites)

• Does not agglutinate, opsonize, or fix complement

IgE

*specialized dendritic cells in the skin

• 0.2% of serum Ig

• antigen receptor on B cellsappearing in late stage of development (“mature”)

• Responds to T cell signals for class switching (eg. IgM to IgG)

• does not agglutinate, opsonize, or fix complement

IgD

Antibody distribution• body compartments• epithelial transport• fetal environment

from: Janeway’s Immunobiology 8, Garland Science, 2011

• Heart represents bloodstream in this figure.

• In addition to bloodstream, IgM also concentrated in pleural & peritoneal spaces (also some secretion)

Distribution of Immunoglobulins

Epithelial cellIgA poly-Igreceptor

U

U

Plasma cell

endocytosis

U

transcytosis

U

IgA + secretorycomponent

exocytosis

U

Transepithelial passage of IgA

Transplancental passage of IgG

neonatal Fc receptor

same mechanismfor transendothelial

IgG passage

plasma

endothelialcell

extracellularfluid

Antibody function• interaction with antigen• microbial evasion• interactions with host cells

and molecules

Antibody function #1: Opsonization

Coating antigen with antibody enhances phagocytosis

Phagocyte

Pathogens coated with antibodies, CRP, or C3b

Phagocytosis of Opsonized Pathogen

Fc receptor

“Zipper effect”

Phagocytosis of Opsonized Pathogen

Phagocytosis of Opsonized Pathogen

Protein A binds Fc region of antibodies, therefore Fc receptors on phagocytic cells cannot bind

Pathogen evasion mechanism

protein A

Staphylococcusaureus

Antibody function #2: Agglutination

The basis for many non-labeled serological assays

Unlabeled Immunoassays• Can actually see the formation of immune complexes, either by their

ability to scatter light (precipitation) or the actual complexes themselves (agglutination).

• Unamplified reaction, so not very sensitive (~20 μg/ml for precipitation).

• Optical equipment can improve sensitivity (turbidimetry and nephelometry) to ~1 μg/ml (for nephelometry)

• Each antigen and antibody must have at least 2 binding sites in order for complexes to form.

Precipitation Agglutination

Agglutination of red blood cells

U

U

U

U

U

Zeta potential (surface charge): RBCs typically separated by 25 nm (IgM diameter = 35 nm)

IgM agglutinates best at 4-27°CIgG is best at 37°C

Prozone Zone of equivalence Postzone

Antigen concentration

Antig

en/a

ntibo

dy c

ompl

exes

Ag binding sites must equal Ab binding sites

false

nega

tive

false

nega

tive

Antibody function #3: Neutralization

Antistreptolysin O test

Anti-strep antibodies are detected in vitro by the ability of a patient’s serum sample to neutralize the bacterial exotoxin

Antibody function #4: Complement activation(by the classical pathway)

(C1)

Antibody function #5: ADCC

Fce

Appropriate, defense response

Antibody-dependent cell-mediated cytotoxicity

epitopesEosinophil

perforin & lytic enzymes

IgE

Antibody as a laboratory reagent

• production• application• problems

Polyclonal antibody production

• Immunize animal• Take blood• Whole serum X• (NH4)2SO4 precipitate X

• Affinity purification (protein A) X• Antigen-affinity purification X

ProteinsLipidsCHO

Largeproteins

Single isotype

Ig

Single antigen

Ig

Antigen

Differentepitopes

manyidiotypes

Monoclonal AntibodiesMouse is injected with antigen

Many antibody-producing B cells in spleen

Spleen cells mixed with myeloma cells, some fuse together (hybridize)

In “HAT” media, only hybrid cells survive Single hybrid cells

in separate wells

Single hybrid cellsproliferate, antibody secreted is screened Hybridomas

Desired hybridoma is cultured, large amounts of monoclonal antibody produced

Labeled Assays

Competitive Immunoassay

• Measure signal generated by label(radioactivity, color, light, or polarity).

• Unlabeled and labeled analytes compete for binding sites.

• Incubate samples with immobilized antibody (or antigen)

sample A sample B

• Signal measured is inversely related to analyte concentration.

AnalyteConcentration

Sign

al

A

B

• Add labeled analyte to sample.

• Wash away unbound material(then add substrate).

Noncompetitive Immunoassay

• Measure signal generated by bound, labeled antibody

• Incubate samples with immobilized capture antibody (or antigen)

sample A sample B Antibodycapture

• Add labeled detection antibody

• Wash away unbound material

• Wash away unbound material(then add substrate)

• Signal measured is directly proportional to analyte conc.

• Indirect: label not involved in first antigen-antibody reaction

• “Sandwich” assay AnalyteConcentration

Sign

alA

B

Interfering Substances

polystyrene surface

Intended reactionBinds antigen

Rheumatoid factorBinds Fc

U

Heterophilic antibodyBinds animal antibody in

a constant region

Human antimouseantibody (HAMA)

false

Positiv

e

false

Positiv

e

Flow Cytometry

Cell suspension

charging collardeflection plates

collection tubes

waste

Fluorochromes:FITC: fluorescein isothiocyanatePE: phycoerythrinPerCP: peridinin chlorophyllAPC: allophycocyanin

sheathfluid

FITC

PE

APC or PerCP

Dichroicmirrors

Argonlaser

To Computer

Forward- and Side-Scatter Analysis

• Lymphocytes: smaller size, no granules, spherical nucleus. Low complexity.

• Monocytes: larger size, some granules, indented nucleus. Some complexity.

• Granulocytes: medium size, many granules, segmented nucleus. Most complexity.

• Data for individual populations can be selected and analyzed separately by computer: “gating”

Single/Dual Parameter Plots

• Incubate cells with FITC-labeled anti-CD3 and PE-labeled anti-CD4 antibodies.

• Collect flow cytometry data.

• Gate on lymphocytes.

• Top figure: histogram for CD3 only - differentiates between T cells and other lymphocytes

• Bottom figure: dot plot for CD3 and CD4 - differentiates between helper T cells and other T cells.

• % of lymphocyte gate

• (% of total leukocytes)

52% (15.6%)

28% (8.4%)

1% (0.3%)

19% (5.7%)

Pathologies caused by antibodies

Waldenstrom’s Macroglobulinemia

• Proliferation of IgM-secreting cells.

• Tumors localize in lymphoid organs (enlarged lymph nodes and spleen)

• Increased blood viscosity impedes blood flow through vessels.

• The IgM paraproteins can behave as cryoglobulins that precipitate (or agglutinate with RBC) and occlude small vessels in extremities during cold weather (Raynoud phenomenon).

• Neuropathies can develop if IgM attacks peripheral nerves.

• Vasculitis develops if IgM is directed against IgG.

“lumpy-bumpy” immune complexes trapped by filtration

“linear” antibody deposition on glomerular basement membranes

Impaired renal function

Systemic Lupus Erythematosis Goodpasture’s

Hypothalmus

Pituitary

Thyroid

TRH

TSHstimulatory inhibitoryGraves’

disease

Na+

AChR

ACh

motor neuron

skeletal muscle fiber

A

B

Autoantibodies attack receptors

Myasthenia gravis

Autoantibody detection

Fluorescence Antinuclear Antibody (ANA) Test

Fix HEp-2 (human epithelial cell line) on microscope slide

Permeabilize

• >95% of SLE patients have a positive ANA, but other conditions (e.g., RA), and even some healthy individuals, can be positive

• Titers >80 are usually reliable indicators of SLE

Wash, add fluorescent anti-Ig, wash

Add patient serum (with autoantibodies)

Plasma membraneNuclear membrane

(for systemic autoimmune diseases)

interphase

metaphase

homogeneous rim speckled nucleolar centromere

ANA staining patterns

ANAPattern Homogeneous Speckled

Coarse Fine Coarse Atypical

Nucle-olar

Centro-mere

Confirmatory

testing for

specific autoantibodies

Histone

dsDNA*PCNA

SSA/RoRNP high titer

SCL-70 none noneSm

DNP SSB/LaRNP

Diagnosis DIL Systemic lupus

erythematosus SS MCTD Scleroderma

Diagnostic decisions using ANA

*Most specific for SLE, can also cause a rim (peripheral) patternDIL: drug-induced lupus, SS: Sjogren’s syndrome, MCTD: Mixed connective tissue diseaseDNP: deoxyribonucleoprotein, PCNA: proliferating cell nuclear antigen, RNP: ribonucleoprotein

Antibodies in viral diagnosis

Typical Time Course for Viral Infection

• Earliest indicators: cultured virus or viral antigen

• IgM: only present during or soon after infection

• IgG: measure soon after onset of symptoms, then 10-30 days later - 4x increase will indicate recent infection

• Newborns: Must measure IgM, IgG could be maternal

Incubationdays-weeks

Acute Infectionweeks-months

Recoveryweeks-months-years

viralantigen

symptoms

anti-viralIgM

Time

Relativemagnitude of response

anti-viral IgG

symptoms

Incubation8-13 weeks

Acute Infection2 weeks-3 months

Early Recovery3-6 months

HBsAg

anti-HBcIgM

Time

Relativemagnitude of response

total anti-HBc

HBeAg

Full Recovery>6 mo-years

anti-HBe

anti-HBs

Hepatitis B time course (acute infection)

• Anti-HBs is the only serological marker in a vaccinated individual.• Patients can be treated by passive transfer of immune globulin.

symptoms

Hepatitis B (chronic infection)

Incubation8-13 weeks

Acute Infection2 weeks-3 months

Symptoms subside2-4 months

HBsAg

anti-HBcIgM

Time

Relativemagnitude of response

total anti-HBc

HBeAg

Chronic carrier

anti-HBe

• HBsAg remains in serum

• Anti-HBs not produced in patients who become chronically infected.

• HBeAg may or may not be present, depending on stage of disease (it indicates active viral replication).

Epstein-Barr virus diagnosis

• Heterophile antibodies are produced that react with horse, sheep and cow (bovine) red blood cells.◦ Monospot test: serum agglutination to horse RBC.◦ Paul-Bunnell test: serum agglutination to sheep RBC.◦ Replaced by latex agglutination or point-of-care

immunochromatographic tests using purified antigens.

• For symptomatic patients with negative heterophile antibody results (10-15%), retest for anti-EBV antibodies:◦ ELISA using several recombinant EBV antigen for capture (easier),

- or -◦ Indirect immunofluorescence assay using EBV-infected cells

(“gold standard”)

Primary infection Convalescence Reactivation

Symptoms Symptoms

Antig

en ti

ter

EA IgG EA IgG

Early Antigen

VCA IgM VCA IgG

VCA IgM

Viral Capsid Antigen

EBNA IgG

EBV Nuclear Antigens

Antib

ody

titer Heterophile

IgM

Time~2 months months, years later

EBV Time Course

Laboratory Testing: Antibody to HIV

• Standard screening test: hybrid ELISA, solid phase coated with several HIV-1 and HIV-2 proteins, and antibody against p24. Bound antibodies detected with labeled HIV antigens, bound p24 detected with labeled antibody.◦ detects antibodies of several isotypes◦ ability to detect p24 allows earlier diagnosis

• If positive → retest twice by same ELISA

• ELISA has low positive predictive value in low-risk populations (only 13% of positives are actually infected)

• If one/both of retests are positive → confirm, usually by western blot

Western blot for detecting antibody to HIV

• Western blot has several antigens applied to membrane (separate tests for HIV-1 and HIV-2).

• Profile of antibodies indicates stage of infection:◦ p24 and p55: appear early, then fade◦ gp41, gp120, gp160: appear later, sustained

throughout disease

• Western considered positive if two of the follow-ing bands are present: p24, gp41 and gp120/160.

• Can be read visually or quantified with a densitometer.

• High positive predictive value > 99%.

Administering antibodiesto patients

Immunolocalization

• Intravenous injection of low-intensity radiolabeled monoclonal antibody to image metastasis.

• Example: Prostascint, which is an antibody against prostate-specific membrane antigen (PSMA) labeled with 111indium.

• Prostate must be removed first.

• Arrows indicate tumor metastasis to lymph nodes.

• Large mass is the liver, which is clearing the compound.

Liver

Radioimmunotherapy

• Intravenous injection of high-intensity radiolabeled monoclonal antibody to kill tumor.

• Example: Bexxar (antibody against CD20 conjugated with 131iodine) is a treatment for B-cell non-Hodgkin’s lymphoma.• Unlabeled antibody given first to reduce

binding to normal B cells in spleen.• Bexxar administered, tumor cells destroyed,

but normal B cell count is reduced.

BCD20

Immunotherapy

Passive immunotherapy• Infusion of monoclonal antibody against tumor antigen.• Example: Herceptin interferes with the human epidermal growth

factor receptor-2 (HER-2), inhibiting tumor growth.

Active immunotherapy• Vaccine (Gardasil) produced from purified, inactive human

papillomavirus is injected i.m. → primary immune response → generate memory cells to combat subsequent infection of HPV, which can cause cervical and vaginal cancers

Reducing Immunogenicity of Therapeutic Antibodies

mouse humanchimeric humanized

Murine variable domains grafted

onto human constant domains

Murine hyper-variable regions

grafted into human variable

domains