Immunoglobulin

Lecture 6

Wesam Mohammed Emharib

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 Introduction
* In 1900 immunization of patient with life threating diphtherial toxin using serum horses
containing antibodies against diphtherial toxin was the first experiment demonstrate presence
of adaptive immunity .

*Reorganization of antigenic molecules by adaptive immunity occur through; antibodies, MHC

and T cell receptor

* Daily production of immunoglobulin in healthy adult human is about 3g

* immunoglobulin ( referred to immunity related protein )
* All immunoglobulin have the same structure but differ in antigen binding site, in human
there are about 109 different immunoglobulin

 Definitions
* Serum; it’s the remaining fluid which persist after blood or plasma form clot and contain
antibodies

*Antiserum; its serum contain detectable amount of antibodies against particular antigen
* Serology; mean study of antibodies –antigens – recognition
* Titer; concentration of antibodies molecules in given serum sample and determined by how
many sequential dilutions of serum can be prepared before antibody antigen binding on longer
be witnessed . Highest concentration of antibodies called high titer.

*polyclonal antibodies; different antibodies against different epitope of antigen produced by

different B cell clones.

*monoclonal antibodies; it are identical immunoglobulin molecules specific for particular

epitope.

 Functions

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* There are two form of immunoglobulin secretory and membrane bounded form and each of
them provide different function

 Membrane bound form

It’s bound to cell membrane of naïve B cell , so act as B cell receptor to recognize antigen, thus
play important role in recognition phase of adaptive immunity
 Secretory form
In adaptive immune response and after antigen recognition, B initiate immune response by
production secretory form of anti-bodies which represent the effector mechanism of humoral
immunity that include the following ;

*Neutralization of microbial product e.g. ( toxins)

*complement activation
*Enhancing phagocytosis by opsonization of microorganism
* Mast cell activation and initiate immediate hypersensitivity
 Structure
*Each immunoglobulin molecules has basic structure of two identical heavy chains and two
identical light chains

*Molecular weight of light chain is about 24Kd and heavy chain from 55-75KD
*Disulfide bounds link light chains to heavy chains and heavy chains to each other.
*Both heavy and light chains formed of units of amino acid each unit about 110 amino acid in
length and form globular part named domain.

*Antigen binding site formed of amino end variable region (V) of both heavy and light chains
and the constant region formed from carboxy end of heavy chain which mediates the biological
function of immunoglobulin.

*Each heavy chain consist of variable region (V) and constant regions (C), variable region
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formed of one domain and constant regions formed of three domain. Light chain consist of two
domains one in variable region and one in constant region.

* Variability of amino acid sequence in variable region responsible on diversity of

immunoglobulin which produced by different B clones.

*There are two antigen binding site in each immunoglobulin molecule.

* The constant region of light chain not contributes in biological function of immunoglobulin
and not attached to cell membrane of B cell.

*Most of differences in immunoglobulins are limited in three short parts in variable region of
both heavy and light chain named hyper-variable segments and formed of 10 amino acid.
Because the surface of hyper-variable region form complementary site for three dimensional
antigens, the hyper-variable region is also called complementary determining region.

*Molecular movement between constant heavy domain 1 (CH1) and constant heavy domain 2
(CH2) responsible on flexibility of Immunoglobulin this flexible region called hinge region

*There are two type of light chain Kappa and lambda (K and λ )which can differentiate
between them by amino acid sequence in constant region.

* Both light chain never persist together in antibody, but each immunoglobulin has either two
K or two λ.

*immunologist estimate that 60% of immunoglobulin has K light chain and the remaining
contain λ light chain

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 Antigen
Antigen binding site
binding site +
NH 3

Disulphide
bound Disulphide
bound

Hinge
region

CH2 CH2

Disulphide Disulphide
bound bound

CH3 CH3

COO- COO-

Biological activity

Immunoglobulin

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 Types
*Immunoglobulin molecules divided into five main type with subtypes depending on type of
heavy chain

*The isotypes of immunoglobulin are (IgA,IgD,IgE,IgG and IgM). IgA and IgG subdivided into
IgA1 and IgA2 and IgG1, IgG2, IgG3 and IgG4

*Heavy chains are nominated by letter of Greek symbols crossponding to isotype of antibody
IgA α (Alpha)
IgD δ(Delta)
IgE ε (Epsilon)
IgG γ (Gamma)
IgM μ (Mu)

*Different types and subtypes of antibodies perform different function

IgA Mucosal immunity
IgD Naïve B cell receptor
IgE Defense against helminthic parasite and immediate hypersensitivity
IgG Oposinisation, complement activation , antibody dependent cell cytotoxicity and
neonatal immunity
IgM Naïve B cell receptor and complement activation

*There are different secreted form of antibodies

IgA present as monomer, Dimer and trimer
IgD there is no secretory form
IgE present as monomer
IgG present as monomer
IgM present as pentamer

*IgG has longest half-life (23 day ) and IgE has shortest half-life (2 days )
*IgG has highest concentration in serum while IgD has the lowest concentration in serum. IgA
produced in large amount in gastrointestinal and respiratory tract.

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 Enzymatic action on association between
heavy and light chain.
*The Immunoglobulin can be cleaved into structural and functional fragments by proteolytic
enzyme

* Papain enzyme cleaves the immunoglobulin at hinge region into three fragments, two of
them are identical to each other and consist of complete light chain associated with part of
heavy chain ( VL +CL) +(VH1+CH1). These parts maintain the capacity to bind antigen and
called Fab (Fragment – Antigen- Binding). Third fragment is consisting of two identical, disulfide
linked heavy chains (CH2+CH3). This part of immunoglobulin has tendency to self-association
and crystallization so called FC (fragment crystallizable)

*Pepsin enzyme cleavage action on the immunoglobulin is limited to carboxy end of hinge
region generating antigen binding fragment with hinge region and intact inter-chain-disulfide
bound this fragment called F(ab)2

*Fab and F(ab)2 are suitable experimental tool because they can bind antigen without FC
dependent biological function

Enzymatic cleavage of Immunoglobulin

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 Valency and avidity of antibody –antigen –interaction
* Large space or area between epitopes of monovalent antigen will lead to low avidity
interaction between antibody and antigen despite high affinity interaction between single
antigenic binding site of antibody and single epitope of monovalent antigen, when antigenic
epitopes are close to each other and both antigen binding site of antibody interact with it, this
called higher avidity or bivalent interaction.

* IgM has 10 antigen bonding site, when bind with 10 epitopes on cell surface resulting
polyvalent – very high- avidity- interaction

 Monoclonal antibodies production

*Hybridoma Technology
There are two essential steps first induction of antibody producing cell (In vivo) the second
selection of antibody producing hypridoma (In vitro ). These two step done as the following :

 Antigen of interest injected into the mice

 After proper period (5-21)days, the mice killed and lymphoid tissue containing plasma
cells producing antibody against antigen of interest are isolated from spleen.
 Lymphoid cells ( plasma cells )and myeloma cells fused together to form Hybridoma in
vitro
 Two original cell type and hybridoma cultured together in selective media (HAT) which
allow only hybridoma cell to grow
 Supernatant from many micro-cultures show pattern growth of hybridoma collected
and examined by immunoassay for desired antibody.
 The selected cells sub-cultured in vitro (hybridoma of specific antibody production)

* HAT “Selective media of hybridoma”

 Animal cells normally use purine and thymidylate as precursor of DNA. In de novo
pathway this required tetrahydrofolate.
 Aminopterin (anti folate drug ) prevent DNA production by de novo pathway by
inhibiting the tetrahydrofolate, thus stop production purine and thimdylate , therefor
prevent DNA production.
 In presence (HAT) “aminopeterin, hypoxanthine, and thymidine” normal animal cells can
grow normaly. Because aminopeterin- treated- cells use another pathway (Salvage
pathway ) in which thymidylate is synthesized from thymidine in presence thymidine

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 kinase (TK) and purine synthesized from hypoxanthine in presence (HGPRT) enzyme
(Hypoxanthin-guanine-phsopho-ribosyl-transferase).
 Myeloma cell lines made by mutagenesis (Defect ) in HGPRT or TK ..
 Deficit myeloma cell cannot use salvage pathway will die in HAT media unless fusing
with normal B cell deliver the genes which transcript essential enzymes for the
hybridoma.
 Concluding mark ; myeloma cells can grow in normal culture media but not in HAT
media because it lack necessary gene responsible in production of enzymes which
essential for DNA synthesis. Thus fusing myeloma cells with normal B cell providing
myeloma with necessary gene for DNA synthesis. Thus only somatic hybridoma can
grow in in selective media. Also uncontrolled growth property of myeloma makes
hybridoma immortal. Deficit myeloma cells cannot use the salvage pathway therefor
will die and B cell cannot survive for more than two weeks because they are not
immortalized. Thus only hybridoma will grow in HAT media

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*Uses of monoclonal antibodies
 Identification of particular cell line by interaction with specific marker e.g. (CD)
 Diagnosis of many infectious diseases
 Diagnosis and therapy of cancer
 Immunological research
*Production of monoclonal antibodies by genetic engineering:
This approach done through generation cDNA encoding antigen binding site of antibody
these cDNA are produced from RNA sample isolated from spleen of mice immunized by
antigen of interest. Then put the cDNA in bacteriophage to form phage display libraries .

References
Marx et al (1997). Monoclonal antibody production. ATLA 25,121-137
Abbas AK, Lichtman. AH, eds.2005. Cellular and molecular immunology .Elsevier Saunders