Tuesday, 20 July 2021

Concept of Antigenicity and Immunogenicity

Here we will understand about two more confusing terms in immunology. These are antigenicity and immunogenicity. To understand these terms we should have an idea about how adaptive immune response works.

How adaptive immune system works?

  Main cells of adaptive immune system are known as lymphocytes. The two important lymphocytes are B-cells and T-cells. B-cells deal with extracellular pathogens and the T-cells deal with intracellular pathogens.

Both of these cells are produced in the bone marrow. Although B lymphocytes complete their maturation in the bone marrow itself. T lymphocytes travel to the organ called thymus where they finally mature.

After the maturation stage, These cells are released into the blood they circulate in blood and have ability to enter tissues where they can recognise pathogens, if present. For recognition purpose these cells possess specific receptors. which we know as B-cell receptors and T-cell receptors.

When this happens these lymphocytes are stimulated to respond and mount a specific immune response.
   When B cells recognize an antigen they get activated and get differentiated into plasma cells. These plasma cells produce antibodies specific to that antigen.
  The antibodies bind to those antigens and activate defense mechanisms that lead to the destruction of the pathogen. Some of the activated B-cells remain in circulation as memory B-cells.
This immune response is known as humoral immune response.

Similarly when T cells recognise the antigen. They differentiate into T cell subsets namely T helper cells and T cytotoxic cells. These subsets of T cells further lead to the destruction of the invader and the infected cells.
  Some of these cells also differentiate into memory T cells. This immune response is known as cellular immune response.

Immunogenicity and antigenicity

When pathogen such as bacteria say invaded the body, here bacteria are the antigens. once inside the body B-cells recognise these antigens or more specifically antigenic determinants or epitopes of the bacteria. After recognition of the antigen B cells differentiate into plasma cells which are specific to this antigen.

  These plasma cells produce specific antibodies which bind to the antigens and further lead to the destruction of that pathogen. So Substance and antigen induced an immune response.

The ability of a substance to induce an immune response, that is either humoral or cellular immune response is known as immunogenicity. And such substances are called immunogenic.

Now suppose a related but harmless bacteria with similar epitopes invades the body. This time since antibodies specific to the epitopes of this bacteria are already present inside the body. These antibodies bind to invaded bacteria.

" here note that the bacteria does not induce an immune response it just binds to product of an immune response that is antibodies."

So the ability of a substance to bind specifically to the products of an immune response is known as antigenicity. And such substances are called antigenic.

Definition of immunogenicity and antigenicity

Immunogenicity is defined as the ability of a substance to induce an immune response. Whereas antigenicity is defined as the ability of a substance to bind specifically to the products of the immune response.

"All molecules that are immunogenic are antigenic too."

All molecules which are able to induce an immune response are also able to bind to the final products of the immune response.

Humoral immune response was induced by the antigen (bacteria) and the antibodies produced bind specifically to that antigen. So substances which are immunogenic are also antigenic.

"All antigenic molecules cannot be considered immunogenic."

There are some substances which are able to bind to the final products of an immune response. That means they are antigenic but they cannot induce an immune response by themselves.
  Well-known example of such substances or haptens. Haptens are able to react with the antibodies, but they are unable to stimulate their production.

Thursday, 1 July 2021

Northern Blotting Steps

 Northern Blotting is a blotting technique for the detection of RNA. the overall technique and the steps involved in northern blotting is almost similar to the southern blotting. Northern Blotting is a technique which will be suitable for a study  gene expression and analysis.

Steps of Northern Blotting

1) RNA gel Electrophoresis

• The first step in northern blotting is RNA gel electrophoresis. The RNA molecules isolated from cells are separated according to size by Gel electrophoresis.

• RNA molecules are negatively charged, so they move from negative to the positive electrode during gel electrophoresis.

• RNA is a single-stranded nucleic acid but, still this RNA gel electrophoresis also includes the denaturation step.
- This is because RNA molecules fold onto themselves. Because of intramolecular base pairing they form secondary structures. So if we want to separate them on the basis of their molecular weights we need them to bring in the linear shape.
- Otherwise the secondary structures of RNA molecules will affect their electro phoretic mobility during gel electrophoresis.

• To denature RNA formaldehyde is used as a denaturing agent. Thus denaturring gel electrophoresis is used in this step.

2) Blotting (RNA)

• The second step is blotting.
The separated RNA molecules are now transferred from the gel to the suitable solid support, such as the Nylon membrane.

• There is the traditional way to transfer the RNA to the Nylon membrane. The basis of this transfer is the capillary action.  

System of Northern Blotting

  The tray is filled with the suitable transfer buffer. A support for gel on membrane is kept in this buffer. This is usually a glass plate an absorbent paper or blotting papers are placed on this support. These are placed such that they imbibe the buffer. They act as a wick

-  Next the gel containing RNA is placed on the top of it.
-  An nylon membrane of the same size as that of the gel is placed over it.
- Again a thick stack of blotting papers or absorbent paper is placed over the membrane.
- This is followed by three to four inches of paper towels and this complete stack of gel membrane blotting papers and paper towels are pressed down by putting a weight on top the buffer or liquid.

  From the tray now Rises through the gel taking with it the RNA molecules. Once the RNA molecules reach the nylon membrane they become adsorbed tightly to the membrane.

  The remaining liquid passes through the paper and is absorbed by the paper towels placed at the top. During this transfer the RNA fragments retain the same pattern of separation they had on the gel.  

3) Hybridization and washing

• The third step involves hybridization with probe and washing. Suppose these bands are the RNA molecules on the nylon membrane. For the detection of these RNA molecules first we need a probe that will specifically bind to these target RNA molecules.

• The probe can be a complimentary labeled RNA sequence or labelled complementary DNA sequence.

• When nylon membrane is incubated with these probe molecules probes will bind specifically to their complimentary target RNA molecules.

• Unbound probes are removed by washing.

4). Detection

• In the fourth and final step detection is done. Detection and visualization method depends on the type of labelled molecule we used for hybridization step.

• The probe is detected by autoradiography, fluorescence or a color change depending on what label we have used in the probe.

These were the steps involved in the northern blotting.

Applications of Northern Blotting

The main applications of northern blotting include
• Gene expression studies such as to determine when and where a particular gene is expressed.
• To identify the presence of closely related species
• To determine the size and abundance of RNA.
• For the analysis of RNA processing.

Modern methods such as PCR have replaced the methods of southern and northern blotting. This is because PCR and PCR based techniques are more simple quick and of more precise nature.