Lecture No. 3 - Date 13/2/2010
Immunoglobulins (antibodies)
*Immunoglobulins are part of the immune system
*Function of Immune system is to protect the body from the invasion of
foreign molecules, bacteria and microorganism (Any foreign substance
or organism that gets into the body should be inactivated)
*There are two components for the immune system:
1) B Lymphocytes
-Responsible for humoral immune response
-Humoral means liquid
-B Lymphocytes secrete proteins (Soluble antibodies or Immunoglobulin)
-B Lymphocytes are converted into Plasma cells that secrete the
immunoglobulin
-Function of immunoglobulin: recognize and bind to foreign molecules
2) T Lymphocytes
-Responsible for cellular immune response that contain killer cells
-Function of killer cells : Kill cells that display foreign substances on their
surface (they recognize cells by specific receptors on their surface )
*Binding of Immunoglobulin to foreign molecules is specific and w/
high affinity:
Every Immunoglobulin is responsible for binding to specific foreign
molecule and the binding occurs with high affinity and even though its
non-covalent binding but it's a strong Binding
*What happens after binding of Immunoglobulin to foreign molecules is:
Effector functions (Could include inactivation, degredation, lysis etc)
*Immunoglobulins can recognize and interact with foreign molecules
even if not encountered before (Even if these molecules enter the body
for the first time the antibodies can recognize them)
-Immunoglobulins can recognize any substance if it is a foreign
substance
-Recognition by specific binding (there should be a sort of
complementary between immunoglobulin and foreign substance)
-This kind of recognition reminds us of key and lock model (Substrate
and enzyme should fit each other) and immunoglobulin and foreign
body should fit in the active site.
*huge # of immunoglobulins because of the huge number of foreign
bodies (10^8)
*Synthesis is stimulated by invasion of the body (the body is not ready to
have immunoglobulins for all kinds of foreign bodies at adequate
concentration)
-The body response by stimulating the synthesis of suitable
immunoglobulins that are specific for these foreign bodies
>>Formation of immunoglobulins
**Antigen:
- Foreign molecule to which immunoglobulin binds (substance that allow
the body to produce antibodies)
-Can elicit antibodies formation (Antigen is also known as immunogen)
-Antigen can be macromolecule (protein, polysaccharide and nucleic
acid)
>> Antigen must be large molecule … small molecules even if they are
foreign substances, they are not antigens
**Epitope:
A particular site on the antigen to which the antibody binds is known as
epitope or antigenic determinant
-Large molecule (antigen) can be recognized by having areas on the
surface and more than one area can be recognized >>Large molecule has
more than one epitope
-Antigen can stimulate the synthesis of many immunoglobulins and each
immunoglobulins can recognize one certain site on the large molecule
**Hapten
-Small foreign molecule … by itself is not an antigen
-To make it antigenic (Stimulate synthesis of antibodies) must be
attached strongly to macromolecule
>>Example: Formation of antibodies against small molecules
-If we inject a small molecule like dinitrophenol (DNP) it will not
produce antibodies, so we attached it to a large molecule like Bovine
Serum Albumin (BSA), Covalently (Covalent bond between DNP and
lysine side chain)
* Bovine means from cows ( albumin from cows)
-After we inject this DNP-BSA complex to a rabbit and take the serum
from the rabbit after several days … we will notice that the serum will
contain Classes of antibodies against DNP-BSA (IgM and IgG) ..
>initially there is a production of IgM
>IgM will decrease while the production of IgG will increase (production
of IgM takes time to occur)
If we inject the same complex the level of immunoglobins will increase
immediately (Sort of memory)
Both IgG & IgM can bind to DNP-BSA complex
*How to recognize the antibodies
By isolation of antibodies against DNP from the rabbit
-Take the blood from the animal >Allow it to clot >then take the serum we called it Antiserum (plural: antisera) because the serum (from the
rabbit) after several days contain antibodies
-Antibodies obtained can bind to free and attached hapten (after
formation of antibodies , there is no need for the DNP to be part of
macromolecule )
Being part of macromolecule is just for synthesis of antibodies, After we
get the antibodies it will bind to the DNP as well as to the DNP attached
to the macromolecules (Free and Attached hapten)
-Antibodies can be purified by affinity chromatography (Depends on the
ability of the protein to bind specifically to small molecules).
-Antibodies obtained are not one kind of molecules (maybe there is
more than one epitope on the surface of Bovine Serum Albumin) as a
result there are several antibodies all of them are able to bind to DNP 
mixture of antibodies with the same specificity <--.
*Look at the Handout of lecture (slide 8 and 9)
-This is a column of antigen bound to inert matrix (polymer)
-The inert matrix must be solid substance and covalently attached to the
antigen
-If we pass the antisera through the column the proteins will bind to the
matrix (Any protein that can bind specifically to the antigen will be
retarded and other proteins will just pass through)
-We wash the column with buffer and the result will be only inert matrix
that bound to the antibodies
> to get the purified antibodies
1-we add free antigen, it will compete with bound antigen (the binding
reversible though it's strong)
2-Change pH or salt concentration
*Polyclonal antibodies: mixture of antibodies (come from number of
cells)
*Monoclonal antibodies : one kind of antibodies produced from one
clone of one single cell
*Principles of preparation of monoclonal antibodies
-depending on Multiple Myeloma which is a sort of cancer, a malignant
growth of a single type of B Lymphocytes that are producing single type
of antibodies
-The result of Multiple Myeloma is large amount of monoclonal
antibodies with unknown or unwanted specificity
-They found that mice strains with multiple myeloma are available and it
can be transplanted from one mouse to another (taking the serum and
inject it to another animal or mouse ), the mouse will have multiple
myeloma and it will produce same kind of antibody and cells can be
cultured (Cell line)
*Steps to produce monoclonal antibodies of given specificity (Slide 12)
1-Take specific antigen and inject it into the mouse
2-The mouse will produce antibodies after few days by proliferation of
spleen cells that will produce plasma cells
3-There are large number of spleen cells will produce antibodies against
the specific antigen (but we can't culture these cells, its not a cancer
cells)
4-so they make a mixture of Multiple Myeloma cells and spleen cells
with addition polyethylene glycol under specific conditions, they will
fuse
5-Fused cells (Also called hybrid cells because they come from spleen
cells that produce antibody and from myeloma cells that are cancer
cells)
6-They select the cells that make antibody or desired specificity
7-They take it and grow it in culture
***The result is (Antibodies are produced, monoclonal, large amount,
desired specificity and can be transplanted from one animal to another)
<<SO>>the purpose of mixing the normal cells with the cancer cells is to
obtain an immortal cells that can be cultured (the cancer ones) with the
ability to produce the required antibody (from the normal cells)
> Structure of Antibodies
*Immunoglobulin G is the major class of antibodies in the plasma
*150 kDa
*Tetramer of:
-Two Heavy chains (every one 50 kDa)
-Two light chains (every one 25 kDa)
-The chains are connected by disulfide bond between the heavy chains
and between the light and heavy chain
*The antigen binding site is a combination of light (N terminal part) and
heavy chain (N terminal part)
-Each Ig molecule can bind two antigens
-Binding of immunoglobulin to the antigen can form cross links (Every
antibody has two arms to bind and the antigen has several epitopes)
*IgG is not a rigid molecule (it shows hinge when the two antigens they
are not at suitable distance)
*Classes of immunoglobulins
IgG, IgA, IgM, IgD and IgE
* Deciliter (dl) = 100 ml
*Properties of immunoglobulins classes
-IgG >the largest in concentration( about 1/3 the total Ig)
-Concentration in mg/dl : IgG>IgA>IgM>IgD>IgE
-Mass in kDa :
**IgG, IgD, IgE is around 150
**IgA is around 3*150
**IgM is around 5*150
-Type of Heavy Chain
IgG = γ (Gamma) , IgA = α (alpha) , IgM = μ (Mu) , IgD = δ(delta) , IgE = ξ
(epsilon)
-Chain structure
** IgG, IgD, IgE is Monomer (2 heavy chain and 2 light chain)
**IgA is dimer or trimer ( 4 heavy chains and 4 light chains)
**IgM is pentamer (10 light chain and 10 heavy chain)
Done by : Ahmad Kharabsheh
Good luck all 