8. Antibody (Reuploaded)

8/13/2019 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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8. Antibody (Reuploaded)

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