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