8. antibody (reuploaded)
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Assoc. Prof. Ma. Jennifer R. Tiburcio MSMTDepartment of Med TechUST Faculty of Pharmacy
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Antibodies (Immunoglobulins)
bifunctional glycoproteins
serum portion of the bloodgamma band
humoral branch of the IR
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Structure of Immunoglobulins
basic structural uni ts - monomer
four chain of polypeptide unit
Light or L chains (200 amino acids & 25,000 daltons)
Heavy or H chains (2x the light chain)
noncovalent forces
disulphide interchain bridges
Hinge region
locationbetween CH1 and CH2
high prol ine content - f lexibil ity
hydrophobic residues
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Carbohydrate portions
local ized between CH2 domains
functions:
increase solubility of the Igs
provide protection against degradation
enhance functional activity of the Fc domains
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Variable region Constant region
Differ between Igs Shared by many Igs
Antigen binding fragment
(Fab)
Crystallizable fragment (Fc)
Determines specificity Binds to effector cells
Amino terminal end (NH2) Carboxy terminal end (COOH)
Amino acid sequence ischanging Amino acid sequence is fixed
Binds to antigens Binds to host tissues
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Light Chains
kappa& lambda chains
carboxy terminal ends
differamino acid substitution
both present in all classes but only
one type is present in a given mol.
kappa chainspredominant in humans
allotype - Km
lambda chainsinvariant in the popn
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Heavy chains
5 classes of Igs w/ different constant regions
alpha, delta, gamma, mu, epsilon
allotypic forms ofIgA & IgMallotypes Am & Gm
constant regionsunique to each class
Domains
globular regions
disulphide intr achain bri dges
light chainsVL & CL
heavy chainsVL & CH1, CH2 & CH3
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Domain FunctionsVL & VH Antigen binding
CH1 Binds C4b fragment
CH2 Site for C system activation
CH3 Binds to Fc receptor on macrophages & monocytes
Attachment site for phagocytes, killer/cytotoxic cells,
platelets & heterologous mast cells
CH2 & CH3 Binds to staphylococcal protein A on placental
syncitiotrophoblast, neutrophils & killer cells
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Amino Terminal Ends of Each Chain
hypervar iable loopsbinding site for an antigencomplementary determining regions (CDRs)
monoclonal antibodies
transplantation & cancer therapy
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Structure Function Relationship of Antibodies
2 fragments
Fc (fragment crystalline)no antigen binding capacity
Fab (fragment antigen binding)
w/ antigen binding capacity
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Papain cleavage
amino terminal side of the disulphide bridges
2 identi cal fragments w/ antigen binding activity
(2 arms of the Ab as separate Fab fragments)
1 Fc (carboxy terminal halves [heavy chain] l inked)
Pepsin cleavage
carboxy terminal side of the disulphide bridges
F(ab)2(2 arms remain linked)
several Fc portions
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Fdpapain digestion & reduction w/ mercaptoethylamine
amino terminal half (heavy chain)
including the var iable portion
immunologic functionenhanced by the light chain
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Properties of Ig Isotypes
IgG
blood & interstitial f luid
transported across the placenta
major role in elimination of micobes
opsonization ADCC
C activation neutralization
appear as major antibody in the secondary IR
participates better at precipitation reaction
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Macroglobulinmol. wt. of 990,000
5 monomeric uni ts held by a J chain
Assumes a star -l ike shape
3 dimensional structurecrab-like
IgM
mainly in intravascular pool
can not cross the placenta
f irst to appear in the primary response
no memory cell for I gM
IgM receptor mature B cells
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functions
most efficient in triggering the classical C pathway
powerful agglutinator
involves in opsonization
useful indicator of intrauter ine infection
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IgA
10 to 15% of the Ig poolserummonomer, dimer & tr imerbody secretionsas dimer
serves to keep antigens from penetrating fur ther
secretory component
(protect it f rom enzymatic digestion)
antiseptic paint
act as opsonin
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IgD
less than 0.2% in the serum
extended hinge region
sur face of I C but unstimulated B cells
second type to appear & immunoregulator
anti -idiotypic antibody
no protective function in the serum
an association between I gD & certain autoimmune diseases
(RA, LE, Hashimotos disease, scleroderma)
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detectable by a highly sensiti ve assay
(radiolabeled antisera)
2 heavy chains are bridged by a single interchain
disulphide bonds
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IgE0.004%
not capable of crossing the placenta
heat-labi le --- conformational changes
no binding to target cells
does not participate in typical I g reactions
homocytotropic antibody (mast cells & basophils)
reaginic antibody (attach to human skin)
immediate hypersensitivity reactions
immunity to helminthic infection
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Feature shared by IgA, IgM and IgD
carboxy terminal octa-deca peptide tail
CH4IgM and IgE
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Amino Acid Variations in Ig Chains
Isotype
between constant regions
normal individuals
same in all individuals
unique to each I g class
IgG subclasses4
IgA subclasses - 2
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Allotype
unique differences
within the constant regions
gamma & alpha heavy chains
kappa light chains
present in different individuals
IgGGm designation
IgAAm designation
Kappa light chainKm designation
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Idiotype
var iations in var iable region
specific
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Ehrlichs Side Chain Theory
Based on the selection of the correctly programmed
B lymphocytes
Certain cells had specif ic receptors for Ag
Selection of a cell with proper receptors
Combination wil l take place, receptors wil l break off
Represents
Lock & key concept for the fi t of Ab for an Ag
I dea that Ag selected cells have bui l t-in capacityto respond to the antigen
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Haurowitz Instructional Theory
Ab producing cel ls are capable of synthesizing ageneralized type of Ab
Ag serves as a template or pattern to which a
standard unfolded gamma globulin is mouldedUpon separation from the antigen, the Ab moleculewould now have a shape complementary to the shape
of the Ag template
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Clonal Selection Theory
Lymphocytes are generally pre-
endowed to respond to one antigen
or group of antigens.
That I gM & I gD act as sur face
receptors that interact w/ specif ic
antigen to trigger proli feration ofa clone of identical cel ls.
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Heavy Chain Chromosome 14
Light Chain (Kappa) Chromosome 2
Light Chain (Lambda) Chromosome 22
(gene coding for Igs)
1. Separate diversity exists for each chain since
they are coded for on separate chromosomes
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Gene organization (gene coding for Igs)
2. Analysis of Ig genes has revealed that the variable &constant regions are separately encoded & located on
different fragments of DNA
Heavy ChainVDJC
Light Chain - VJC
3. Four separate chromosomes code for heavy chains,three separate chromosomes code for light chains
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a.Dur ing the process of B cell maturation, the pieces are
sl iced together to commit the B cell to make Ab of single
specificity
b. Joining of the segments occurs in two steps: 1) at the
DNA level, 1D gene is joined to the J gene, with deletion of
intervening DNA 2) the V gene is joined to the DJ complex
resul ting in rearranged VDJ complex. This resul ts in pro B
cell.
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c. When RNA synthesis occurs, C gene is joined to the VDJ
complex & all intervening DNA is lost. I n general, the C
gene which is immediately located next to the VDJ complexis expressed. Since C mu gene is the closest to the J
segment, i t is the Ab that is recognized
d. W/ the exception of C delta gene which l ies next to the Cmu gene is often transcribed along with the C mu gene.
Thus, B cell wil l have I gM & I gD at the same var iable
domain on the surface membrane at the same time
The process of switching to other classes of I g occur s laterdue to the looping out & deletion of other constant regions
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A productive rearrangement of the kappa chains keeps
other chromosome 2 from rearranging. In addition to this,
i t shuts off any recombination of lambda chain locus onchromosome 22. This process is known as allel ic exclusion.
Lambda chain synthesis occurs only if nonfunctional gene
product arises from kappa chains
The light chain is joined to the mu chain to form a
complete I gM Ab which appears f irst in immature B cell .
Once the I gD & I gM are present on the sur face membraneof B cell , the B cell is ful ly mature & capable of responding
to an antigen.
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The large var iety of VDJ & C combinations for each type of
chain plus the dif ferent possibil i ties for l ight & heavy chain
make more than enough conf igurations that allow us torespond to any antigen in the environment.
Genetic preprogramming of lymphocytes can best beexplained by the concept of gene recombination. More than
1 gene controls synthesis of a particular I g and through a
random selection process these individual segments are
joined to commit that lymphocyte to making Ab of a single
specificity.
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Production of Monoclonal Antibodies
a cancerous cell or myeloma is fused w/ an antibody-producing cell forming a hybridoma
can produce antibodynormal B cell
can reproduce indef ini telymyeloma cells (deficient in
HGPRT & thymidine kinase & presence of aminopter in)
HAT medium
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Classification
1. Sedimentation constant
IgM 19S
IgA 13S, 11S, 9S
IgE 8S
Serum IgA 7S
IgG 7S
IgD 7S
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2. Temperature
Cold4oC or RT
Warm37oC
3. Occurrence
Natural
Immune
4. Species which produce them
Isoantibodies
Heterophile antibodies
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5. Reaction with an antigen
agglutinins
hemaglutinins
precipitins
lysin
opsonin
blocking or inhibitory antibodies
complement fixing antibodies
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6. In vitro behaviour
Complete Incomplete
thermolabile thermostable
cant cross the placenta can cross the placenta
early in occurrence late in occurrence
saline acting albumin acting
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The half life of most IgG subclasses is
approximately 23 days.
True or False
Some of the antibodies in the serum from a blood
group type A individual will bind to RBCs from an
individual who is type O.
True or False
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Treatment of antibody molecule w/ papain generates
two Fab fragments and one Fc fragment.
True or False
There are four IgM isotype subclasses.
True or False
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Plasma cells derived from one B cell clone, secrete
antibodies that all recognize the same epitope.True or False
The predominant antibody isotype found in
mucosal secretion is _________.IgA
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