Immunology sheet # 4 - Haneen Hasan

بسم الله الرحمن الرحيم

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بسم الله الرحمن الرحيم


Immunology
Sheet # 4
5/12/2010


As we said before each B cell or T cell express a unique antigen receptor on it's cell surface.
• Receptor on B cell is IG or BCR.
• Receptor on T cell is TCR or T cell receptor.
These are the receptors which are specific for an antigen.

Antigen
* is a molecule which is able to connect with a receptor ( TCR or BCR ) or an antibody
Note: an antibody is a receptor, so when we say that a receptor which is specific for an antigen X it means that antigen X is specific nothing else.

How do we produce antibodies against an antigen?
There must be ways of producing antibodies against antigens.
Indeed in the human body if you are exposed to an antigen, your immune response is to produce antibodies against antigen.
BUT the problem is, it is does not necessarily that every time you put an antigen with an antibody, you will get an immune response!!
If you put an antigen with antibody, you may get an immune response but sometimes if you have small molecules it does not necessarily to produce immune response.
An antigen which is able to produce immune response when injected into the body and give rise to specific antibody is immunogen.

On the other hand there are other molecules ( usually small molecules ),
they are antigen but if you inject them into the body, there will be no an immune response and such these molecules are haptens.
The hapten becomes really immunogenic if it coupled with another protein and this protein is a carrier .

So immunogens and haptens are antigens. However immunogens are able to produce immune response and antibodies, but haptens can produce an immune response and give rise to antibodies only when it is coupled to a protein molecule and this protein molecule usually is a carrier.


Characteristics of Immunogenicity
Many different substances can induce immune responses. The following characteristics have an important influence in the ability that a substance has to behave as an immunogen.

A. Foreigness.
As a rule, only substances recognized as “non-self” will trigger the immune response. Microbial antigens and heterologous proteins are obviously “non-self” and are strongly immunogenic.
B. Molecular Size.
The most potent immunogens are macromolecular proteins [molecular weight (M.W.) > 100,000].
Molecules smaller than 10,000 daltons are weakly immunogenic
A very small molecules e.g haptens , are non immunogenic.
C. Chemical Structure and Complexity.
Large heterologous proteins is the potent immunogens,
Polysaccharides can be very large molecules but they are made of repeated structures so they are not very immunogenic (polysaccharides can be immunogenic but not as well as proteins)

Besides the chemical nature of the immunogen, other factors strongly influence the development of an immune response.

* Genetic Constitution (immune response genes)
Different animal species or different strains of one given species show
different degrees of responsiveness to a given antigen. In humans, different individuals can behave as “high responders” or “low responders” to any given antigen. THE DR. SAID THAT HE WILL TALK ABOUT IT IN DETAILS LATER

* Method of Antigen Administration and the amount of an antigen.
A given dose of antigen may elicit no detectable response when injected intravenously or given orally, but may elicit a strong immune response if injected intramuscularly or subcutaneously .
Note: high dose specially intravenously or low dose , there will no immune response.

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Interaction between antigen and a receptor ( immunoglobulin or antibody) which will be specific for an antigen, it will unite to each other
how do they unite ?!
the point of contact at an antigen which is recognized by the receptor is known as epitope
• an antigen can have a several hundreds of epitopes.
• the size of epitope is 5-7 aminoacids .
• the epitopes can be different.
so any antigen can have many epitopes of the same kind or can have many epitopes of different shapes and sizes.


an antigen when injected inside the body it wil give rise to many many antibodies
for example an antigen has X epitope, Y epitope, Z epitope, when injected (antiX antiY antiZ) antibodies will be formed.
this what happens due to an immune response.
The antibodies that produced against the antigen is polyclonal antibody.
note: we can separate these antibodies from one another and we can select for example the antibody against X epitope this antibody is a monoclonal antibody.
so in the monoclonal serum there will be 1 antibody against 1 epitope but polyclonal serum contains diff. types of antibodies specific to (X,Y,Z) epitopes on one antigen.
note:* monoclonal are mainly produced in the lab due to down selection until we get one B cell then it will produce a single antibody against one antigen.
*in nature we will respond by producing a polyclonal antibodies

Cross reactivity: an antibody binds to more than one antigen. (From the book)


Antigen-Antibody Reactions
General Characteristics of the Antigen-Antibody Reaction(epitope – paratope rxn)
note: paratope is an antigen binding site (which is found on antibody) that will bind to epitope.
Antigens and antibodies bind through noncovalent bonds in a similar manner to that in which proteins bind to their cellular receptors or enzymes bind to their substrates. The binding is reversible and can be prevented or dissociated.

The following intermolecular forces are involved in antigen-antibody binding:

1. Electrostatic bonds.
Electrostatic bonds result from the attraction between oppositely charged ionic groups of two protein side chains

2. Hydrogen bonding.
When the antigen and antibody are in very close proximity, relatively weak hydrogen bonds can be formed between hydrophilic groups

3. Hydrophobicity interactions.
Hydrophobic groups and hydrophillic groups

4. Van der Waals bonds. These forces depend upon interactions between the “electron clouds” that surround the antigen and antibody molecules.

Note:
If these forces are strong they will stick together easily and it will determines specificity, if it is specific there will be very strong attraction.
If there is no specificity, there is no attraction.

A measure of the attraction between epitope and paratope due to the forces that we described is known as affinity of antibodies.

Note: if we have specificity, it will be strong (high affinity) or weak (low affinity)

In immune response you will get a high and a low affinity antibodies but the more useful is high affinity antibody cuz they stick to the antigen easily.


Avidity ( الشراهة ) due to the summation of the affinity between paratopes and epitopes.
In monoclonal antibody, IGM is pentameric which is made of 5 immunoglobulin molecules together linked by J molecule, and each molecule has 2 paratopes so IGM molecule has 10 binding sites not only 2.
IGA is dimeric , it has 2 immunoglobulin molecule so it has 4 binding sites.

So avidity of IGM is better than avidity of IGA.


Adjuvant (محفز)
• Is a chemical that make the antigen more immunogenic.
• It will pokes immune response.

Ex. Froend adjuvant *is irritant chemicals , *it will make more macrophages to go to the site of injection , *is used in vaccines in human being to make it more immunogenic.
AL hydroxide *is usually used in human.
*used in vaccines and usually release the antigen (immunogen) slowly thereby prolonging the stimulus , *it is a good irritant, *it attracts microphages.
Note: we know that we need microphages to phagocytosis of antigen (breaking down the antigen) and presentation for T cells.

Don't confuse adjuvant with the carrier!!
Adjuvant: makes an immunnogen more immunogenic( promotes immune response)
Carrier: is a protein which makes the haptens( which is not immunogenic) make it immunogenic

Antigens: * T- dependent.
* T- independent.
For production of antibodies or any response against T- dependent antigens, we need helper T cells.
Note: *most proteins are T- dependent antigens.
*Polysaccharides don't need the presence of cytokines and they don't need the presence of the T cells, they will come in contact with B cells and make them produce produce antibodies independently of T cells.



Specific Types of Antigen-Antibody Reactions

*Precipitation.
Antigen ( soluble) + antibody( which is a bivalent ) = cross linking (antibody will be
in contact with 2 antigens


precipitation


*Agglutination
Antigens (cellular with epitope on them like bacterial cells, RBC ) + antibody =
Cross linking agglutination(clumping)

Precipitation=agglutination
Due to cross linking of antigen by means of antibodies
BUT
precipitation (its antigen must be soluble and need to have special optical instrument to detect it).
Agglutination (its antigen is cellular and can be detected by the naked eye) .
Ex. Blood grouping.

Note: to see the precipitation in the naked eye, you can use a semisolid media (jelly) instead of liquid media this produce opaque lines.

*RadioImmunoAssay "RIA"
-Radio: radio active.
-Immuno: relies in immune reaction.
-Assay: حسبة
antigen+ antibody (linked with radioactive ID), we will do washing then we will put it in a gamma radiation scanner.
If there is no radiation, the antigen didn't have an epitope which is specific to antibody cuz the antibody which has radioactive ID is washed away.
But if antigen will unit with antibody, the antibody will be specific for the antigen and there will be a radiation (when we put it in the gamma radiation scanner).
By this test we can also detect the amount of the antigen.
When the amount of antigen increase the antibodies will increase and the radiation will increase.
SO we can use it as a qualitative test cuz we will know that a specific antigen is present or not, and we can use it as a quantitave test cuz we can measure the amount of an antigen by mean of radiation that has been produced is called
Note: instead of radiation we can use fluorescent compound (it emits a greenish light when exposed to ultraviolet light then we call it an ImmunoFluorescence.


*ELISA (Enzyme linked immunosorbent assay) .
Antibody(enzyme) +antigen
The enzyme chosenis one that is capable of catalyzing a rxn to generate a colored product from a colorless substrate.
Note: the presence or absence of the color indicate if there has been a rxn or not so it is a qualitative test and it is a quantitative test in measure the amount of the change in color .

The end

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Written by: Haneen Bahzad Hasan