n ANTIGEN
n ANTICORP
Antigene - Anticorpi
Dr. Minodora Dobreanu
Antigens
Antigen (Ag) the molecule an antibody (Ab) binds
to
usually a foreign substance
each antigen has different sites that antibodies
can bind to, so that one antigen can be bound by
several different antibodies
Allergen
examples in the case of allergy could be pollen,
cat dander, or a chemical in soap
Antigens, immunogens, epitopes:
nantigen: a substance that interacts with either
antibodies (immunoglobulins) or antigen-specific T-
cell receptors (TCRs) found on T lymphocytes
nimmunogen: substance that induces an immune
response (able to trigger) - Not every antigen can
trigger an immune response.
molecular size (MW of 10,000+), molecule
complexity, molecular form important in
determining immunogenicity
nonrepeating polymers (e.g., proteins,
carbohydrates) = good immunogens
repeating polymers (e.g., lipids, nucleic acids) =
poor immunogens
n REACTIA ANTIGEN-ANTICORP
n Aplicatii ale reactiei Ag-Ac
Haptens
n Small organic molecules can become antigens
if they bind to proteins.
n Become antigenic determinant sites on the
proteins.
By binding haptens to proteins in the
laboratory, new antigens are created for
research or diagnostic purposes.
n hapten: low molecule weight substance that combines
with specific antibodies but is incapable of eliciting an
immune response by itself
(e.g., sugars, amino acids)
Antigene
Factorii care determin / influeneaz potenialul imunogen al unui
antigen sunt:
n Dimensiunea moleculei (>10.000 D)
n gradul de diferen structural fa de componentele endogene
n distana filogenetic ntre antigen i gazd
accesibilitatea epitopului pentru recunoatere imun
n
n calea de administrare a antigenului (intravenos pentru Ag
corpusculare virusuri, bacterii; subcutan pentru Ag solubile
proteine, fosfolipide, acizi nucleici)
n doza de antigen: ptrunderea ntr-un organism a unui antigen n
cantiti excesive, poate s produc paralizia sitemului imun al
gazdei, ducnd la manifestarea unei imunotolerane; tolerana imun
apare i dac un antigen este administrat n doze similare pe ci
diferite: enteral, subcutanat sau intravenos.
n frecvena de administrare a antigenului (administrri multiple l fac
mai imunogen)
imunogenicitatea Ag solubile este crescut prin adugarea
n
adjuvanilor (de exemplu la vaccinuri, adugarea srurilor de
aluminiu sau de calciu); nu se cunoate cu precizie mecanismul, dar
se pare c sunt angrenate i macrofagele
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 Antigene Antigene
Criterii de clasificare Criterii de clasificare

Sursa
Capacitatea de a induce un raspuns imun -externa
-antigene complete -interna (normale, modificate de diferiti factori)
-antigene incomplete (haptena)
Modalitate de obtinere Grad de inrudire genetica
-antigene naturale -antigene autologe
-antigene artificiale (antigene naturale modificate) -antigene alogenice
-sintetice (homopolimeri, heteropolimeri liniari sau -antigene xenogenice
ramificati)
Origine Tip de celula implicata in raspunsul imun
-antigene animale -antigene timodependente (antigene T-dependente)
-antigene vegetale limfocit T - limfocit B - sintetiza anticorpi :
-antigene bacteriene majoritatea antigenelor
-antigene virale -antigene timoindependente (antigene T-independente)
-heteroantigene (origini multiple) direct limfocitului B):
antigene cu epitopi repetitivi

How Antibody Binds to Antigen

n ANTIGEN
n ANTICORP
n REACTIA ANTIGEN-ANTICORP
n Aplicatii ale reactiei Ag-Ac
The top part of this figure shows how different shaped antigens
can fit into the binding site of antibodies: left, pocket; center,
groove; right, extended surface. The panels below show space-
filling or computer-generated models indicating where contact
between the peptide antigen and antibody occurs.

Recunoasterea Ag scopul raspunsului imun Imunoglobulinele

o imunoglobulinele
o receptorii pentru Ag de pe celulele sistemului imun Diversitate si heterogenitate

Definitie - Glicoproteine prezente in ser, fluide

tisulare i ca receptori membranari pe limfocitul B
(rol n contactul cu Ag pentru diferenierea Ly B n
plasmocit).

Clase i subclase (Izotopuri si Allotipuri)

(secventa de Aa, mrime, ncrcarea cu carbohidrai)
IgG : IgG1, IgG2, IgG3, IgG4
IgM
IgA : IgA1, IgA2
IgD
Abdul K. Abbas, Celular and Molecular Immunology, 6th Ed, 2007 IgE

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 Bifunctionalitatea moleculei de imunoglobulina Imunoglobuline - structura

Situsuri de legare specifica pentru antigen

Regiunea Fab

C1q
Regiunea Fc

Imunoglobuline - structura Antibody Structure

n 100 million trillion
antibody molecules that
contain 4 polypeptide
chains (Idiotypes).
n 2 long H chains are joined
to 2 shorter L chains.
Fab regions are
variable.
Provide specificity
for a binding to an
antigen.
Fc region of different
antibodies are
constant.
n B cells have antibodies
that serve as receptors
for antigens.

Several Flavours of Abs: Antibody Structure

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 Major antibody classes

Properties of antibody classes

Properties IgM IgD IgG IgE IgA

Heavy m
chain
mean 1 0.03 12 0.0003 2
human
adult
serum level
(mg/ml)
Half-life in 5 3 25 2 6
serum
(days)
Number of 5 1 1 1 1-3
four-chain
monomers
Special Early Found Activates Stimulate Found
properties appearance; mainly on complement; s mast mostly in
& function fixes B-cell crosses cells to secretions
complement; surfaces; placenta; secrete
activates traces in bind to histamine
macrophage serum macrophage s
s s and
granulocytes

Distributia Ig in diverse lichide biologice The Number Dilemma

Concentratiile imunoglobulinelor in secretiile umane externe (mg/l)

Secreie IgA IgG IgM n You have about a trillion different antibodies able to react
with millions of different types of Ag
Ser 2.500 10.047 1.500
Lacrimi 80-400 0-16 0-18 n but you only have about 30,000-60,000 genes which code for
all the proteins you need in your entire body, most of which
are not Ab
Fluid nasal 70-846 8-304 0
Saliv 194-206 42 64 n so there cannot be one gene for one antibody to code for
Fluid Intestinal 166 4 8 these we wouldnt have enough antibodies!
Urin 0,1-1,0 0,06-0,56 -
So how can your body produce Ab to so many antigens,
Fluid Cervical 3-133 1-285 5-118
even those its never seen?
Fluid Vaginal 35 52 -
Tonegawa Theory
Fundamental Immunology (5th Edition)
Lippincott, Williams & Wilkins Chapter 31 (p.971)

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 Since there are multiple types of each gene segment, there
are many thousands of possible V-D-J combinations so that
Antibody Genes each B cell gets a unique combination of segments! Additional
diversity occurs because there are two types of light chains.

Genes for antibodies arent like most other genes -

they come in pieces (gene-lets):
k - crz 2
l - crz 22
, , m, , - crz 14

variable segments (V) many different versions

diversity segments (D) several different versions
joining segments (J) a few different versions
constant segments (C) a few different versions that are
nearly identical

A unique recombination A unique recombination occurs in each B cell

occurs in each B cell

n each B cell combines these

gene segments to make an
Ab chain like shuffling a Unique combination of segments becomes joined by
deck of cards somatic gene rearrangement

- V, D, and J for the

heavy chain, V and J for
the light chain

n since there are multiple

types of each gene each B cell combines these gene segments to make an
segment, there are many Ab chain like shuffling a deck of cards
thousands of possible V-D-
J combinations so that - V, D, and J are joined to C for the heavy chain, V
each B cell gets a unique and J are joined to C for the light chain
combination of segments!

Mecanisme care contribuie la generarea

diversitii anticorpilor
H Lk Ll
Gene din linia
germinativ n ANTIGEN
Gene V 250-1000 250 2
Gene J 4 4 3 n ANTICORP
Gene D 12 0 0

Diversitate
n REACTIA ANTIGEN-ANTICORP
combinatorie
V x J (x D) 10.000-40.000 1000 6
n Aplicatii ale reactiei Ag-Ac
Asocierea lanului H si L
H x Lk 1 - 4 x107
H x Ll 5 - 10 x104

Total potenial
cu diversitate joncional 109 - 1011

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 Question:

What types of immune

reactions are IgG, IgA, IgM,
IgE and IgD involved in?

Functii efectoare Funcii efectoare (1) - Opsonizare

Imunoglobuline de membrana - receptori pentru antigen de pe

limfocitul B
ontogenia B - IgM, IgM+IgD, orice fel de Ig de suprafata
activarea B - legarea incrucisata a Ig de membrana de catre
Ag multivalente

Neutralizarea Ag de catre Ac circulanti

toxine, medicamente, virusuri, bacterii, paraziti se
complexeaza cu Ac si astfel nu se leaga de tinte celulare

Activarea complementului (IgG, IgM)

complexele imune (Ag-Ac) se leaga de C1q - activarea caii
clasice a complementului (IgG-C2, IgM - Cm3)
Opsonizarea (IgG) - creste capacitatea fagocitant
fagocitele au FcR si leaga particule opsonizate cu Ac
Ac se leaga la bacterii si maresc capacitatea de fagocitoza
Cresterea fagocitozei implica intensificarea legarii de suprafata a
bacteriilor opsonizate si activarea fagocitului (procese mediate de FcR)
IgM creste indirect fagocitoza C3b

Funcii efectoare (2) How an Antibody Works

When an Ab finds its Ag on an invader, it will bind there and
Citotoxicitatea celular mediat de Ac (IgG, IgE, IgA) act as a trash tag, marking it for destruction by killer
Celule care lizeaz celule: NK, neutrofile, eozinofile cells, macrophages or complement
Ac mbrac intele celulare care urmeaz a fi lizate
Receptor for
agregarea FcR de pe NK activare, TNF, IFN constant region The target cell dies
After binding, the
Antibody binds to
eozinofilele au FcR si leaga Ac (IgE, IgA) care au complexat Ag target antigen
of antibody on NK NK cell is signaled by apoptosis and/or
cell recognizes a to kill the target membrane damage
parazitare secretie de enzime bound antibody cell

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 Funcii efectoare (3) Functii efectoare (4)
Hipersensibilitatea imediata (IgE)
Imunitatea de la nivelul mucoaselor
mastocitele si bazofilele au FcR pentru IgE monomeric, agregarea FcR (IgA)
in prezenta alergenului duce la eliberarea de mediatori ai inflamatiei celulele epiteliale au FcR,
capteaz IgA din snge i l trec n
(amine vasoactive, leukotriene, prostaglandine, citokine)
secreiile mucoaselor ca IgAs,
hipersensibilitatea imediata unde neutralizeaz Ag

Imunitatea neonatala la mamifere (IgG

matern)
IgG matern trece prin bariera
placentar n circuitul sanguin
fetal (om)
IgG din lapte e captat de FcR din
intestinul nou-nscutului i intr n
snge (porc, oarece) invers ca
IgA

Inhibarea rspunsului imun (IgG)

LyB au FcR i leag Ac din
complexele imune se inhib
activarea B

Antibody functions Antibody functions

Monoclonal Antibodies n ANTIGEN

n Commercially prepared antibodies are used: n ANTICORP
To provide passive immunity
In research, clinical testing, and treatment of certain n REACIA ANTIGEN-ANTICORP
cancers
n Monoclonal antibodies are pure antibody preparations
n Aplicaii ale reaciei Ag-Ac
Specific for a single antigenic determinant
Produced from descendents of a single cell
n Hybridomas cell hybrids made from a fusion of a tumor cell
and a B cell
Have desirable properties of both parent cells
indefinite proliferation as well as the ability to produce a
single type of antibody

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Aplicaiile reaciei Antigen-Anticorp
Tehnici de imunodetecie

Western Blotting
Imunoprecipitarea
Imunohistochimia / Imunocitochimia
Citometria in flux
Radioimunoanaliza (RIA)
ELISA - Elispot
Mutiplexare