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Assoc. Prof. Ma. Jennifer R.

Tiburcio, MSMT Department of Med Tech UST Faculty of Pharmacy

•Antibodies (Immunoglobulins)
bifunctional glycoproteins serum portion of the blood gamma band

humoral branch of the IR

•Structure of Immunoglobulins
basic structural units - 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 location – between CH1 and CH2 high proline content - flexibility hydrophobic residues

•Carbohydrate portions
localized between CH2 domains

functions:
increase solubility of the Igs provide protection against degradation

enhance functional activity of the Fc domains

Variable region
Differ between Igs Antigen binding fragment (Fab) Determines specificity Amino terminal end (NH2) Amino acid sequence is changing Binds to antigens

Constant region
Shared by many Igs Crystallizable fragment (Fc)

Binds to effector cells Carboxy terminal end (COOH) Amino acid sequence is fixed

Binds to host tissues

•Light Chains
kappa& lambda chains carboxy terminal ends differ – amino acid substitution both present in all classes but only

one type is present in a given mol.
kappa chains – predominant in humans allotype - Km

lambda chains – invariant in the pop’n

•Heavy chains
5 classes of Igs w/ different constant regions alpha, delta, gamma, mu, epsilon

allotypic forms of IgA & IgM – allotypes Am & Gm
constant regions – unique to each class

•Domains
globular regions disulphide intrachain bridges light chains – VL & CL heavy chains – VL & CH1, CH2 & CH3

Domain
VL & VH CH1 Antigen binding

Functions

Binds C4b fragment

CH2
CH3

Site for C’ system activation
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

•Amino Terminal Ends of Each Chain
hypervariable loops – binding site for an antigen
complementary determining regions (CDRs) monoclonal antibodies transplantation & cancer therapy

•Structure Function Relationship of Antibodies

2 fragments Fc (fragment crystalline) – no antigen binding capacity

Fab (fragment antigen binding)
w/ antigen binding capacity

•Papain cleavage
amino terminal side of the disulphide bridges 2 identical fragments w/ antigen binding activity

(2 arms of the Ab as separate Fab fragments)
1 Fc (carboxy terminal halves [heavy chain] linked)

•Pepsin cleavage
carboxy terminal side of the disulphide bridges F(ab)2 (2 arms remain linked)

 several Fc portions

•Fd
 papain digestion & reduction w/ mercaptoethylamine  amino terminal half (heavy chain)

including the variable portion
 immunologic function – enhanced by the light chain

•Properties of Ig Isotypes IgG blood & interstitial fluid transported across the placenta major role in elimination of micobes opsonization C’ activation ADCC neutralization

appear as major antibody in the secondary IR
participates better at precipitation reaction

IgM
•Macroglobulin – mol. wt. of 990,000 •5 monomeric units held by a J chain •Assumes a star-like shape •3 dimensional structure – crab-like mainly in intravascular pool can not cross the placenta first to appear in the primary response no memory cell for IgM IgM receptor – mature B cells

functions
most efficient in triggering the classical C’ pathway powerful agglutinator involves in opsonization useful indicator of intrauterine infection

IgA
10 to 15% of the Ig pool

serum – monomer, dimer & trimer
body secretions – as dimer serves to keep antigens from penetrating further secretory component (protect it from enzymatic digestion) “antiseptic paint” act as opsonin

IgD
less than 0.2% in the serum extended hinge region

surface of IC but unstimulated B cells
second type to appear & immunoregulator anti-idiotypic antibody no protective function in the serum an association between IgD & certain autoimmune diseases

(RA, LE, Hashimoto’s disease, scleroderma)

detectable by a highly sensitive assay
(radiolabeled antisera) 2 heavy chains are bridged by a single interchain

disulphide bonds

IgE
0.004% not capable of crossing the placenta heat-labile --- conformational changes no binding to target cells does not participate in typical Ig reactions

homocytotropic antibody (mast cells & basophils)
reaginic antibody (attach to human skin) immediate hypersensitivity reactions

immunity to helminthic infection

Feature shared by IgA, IgM and IgD carboxy terminal octa-deca peptide tail CH4 – IgM and IgE

•Amino Acid Variations in Ig Chains
Isotype –
between constant regions normal individuals

same in all individuals
unique to each Ig class IgG subclasses – 4 IgA subclasses - 2

Allotype

unique differences
within the constant regions gamma & alpha heavy chains kappa light chains present in different individuals IgG – Gm designation IgA – Am designation Kappa light chain – Km designation

Idiotype

variations in variable region specific

Ehrlich’s Side Chain Theory
• Based on the selection of the correctly programmed B lymphocytes • Certain cells had specific receptors for Ag • Selection of a cell with proper receptors • Combination will take place, receptors will break off • Represents Lock & key concept for the fit of Ab for an Ag Idea that Ag selected cells have built-in capacity to respond to the antigen

Haurowitz Instructional Theory
● Ab producing cells are capable of synthesizing a generalized type of Ab ● Ag serves as a template or pattern to which a standard unfolded gamma globulin is moulded ● Upon separation from the antigen, the Ab molecule would now have a shape complementary to the shape of the Ag template

•Clonal Selection Theory •Lymphocytes are generally preendowed to respond to one antigen or group of antigens. •That IgM &IgD act as surface receptors that interact w/ specific antigen to trigger proliferation of a clone of identical cells.

(gene coding for Igs) 1. Separate diversity exists for each chain since they are coded for on separate chromosomes Heavy Chain – Chromosome 14

Light Chain (Kappa) – Chromosome 2
Light Chain (Lambda) – Chromosome 22

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 3. Four separate chromosomes code for heavy chains, three separate chromosomes code for light chains Heavy Chain – VDJC Light Chain - VJC

a. During the process of B cell maturation, the pieces are sliced 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 resulting in rearranged VDJ complex. This results in pro B cell.

c. When RNA synthesis occurs, C gene is joined to the VDJ complex & all intervening DNA is lost. In general, the C gene which is immediately located next to the VDJ complex is expressed. Since C mu gene is the closest to the J segment, it is the Ab that is recognized d. W/ the exception of C delta gene which lies next to the C mu gene is often transcribed along with the C mu gene. Thus, B cell will have IgM & IgD at the same variable domain on the surface membrane at the same time The process of switching to other classes of Ig occurs later due to the looping out & deletion of other constant regions

A productive rearrangement of the kappa chains keeps other chromosome 2 from rearranging. In addition to this, it shuts off any recombination of lambda chain locus on chromosome 22. This process is known as allelic 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 IgM Ab which appears first in immature B cell. Once the IgD & IgM are present on the surface membrane of B cell, the B cell is fully mature & capable of responding to an antigen.

The large variety of VDJ & C combinations for each type of chain plus the different possibilities for light & heavy chain make more than enough configurations that allow us to respond to any antigen in the environment. Genetic preprogramming of lymphocytes can best be explained by the concept of gene recombination. More than 1 gene controls synthesis of a particular Ig and through a random selection process these individual segments are joined to commit that lymphocyte to making Ab of a single specificity.

•Production of Monoclonal Antibodies
a cancerous cell or myeloma is fused w/ an antibodyproducing cell forming a hybridoma

can produce antibody – normal B cell
can reproduce indefinitely – myeloma cells (deficient in HGPRT & thymidine kinase & presence of aminopterin)

HAT medium

Classification 1. Sedimentation constant IgM IgA IgE Serum IgA 19S 13S, 11S, 9S 8S 7S

IgG
IgD

7S
7S

2. Temperature
Cold – 4oC or RT Warm – 37oC

3. Occurrence
Natural Immune

4. Species which produce them
Isoantibodies Heterophile antibodies

5. Reaction with an antigen agglutinins hemaglutinins precipitins

lysin
opsonin blocking or inhibitory antibodies complement fixing antibodies

6. In vitro behaviour Complete thermolabile can’t cross the placenta early in occurrence saline acting Incomplete thermostable can cross the placenta late in occurrence albumin acting

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

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

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

The antibody isotype (when complexed with antigen) that serves as an opsonin for phagocytes is of the __________isotype. IgG

Natural isohemagglutinins are of the ______ isotype

IgM