immunological techniques

What is immunological techniques what types of antigen- antibody reactions occurs in immunological techniques

Immunological techniques

What is immunological techniques? what types of antigen- antibody reactions occurs in  immunological techniques?


Immunological techniques:-

Types of antigen- antibody reactions:-

Precipitation: 

It refers to a antigen antibody reaction between is soluble antigen and its corresponding antibody resulting in the formation of insoluble precipitate. Antibody causing precipitation is precipitinin.

Mechanism:


Precipitation is due to antigen antibody complex. An antigen is multi-valent and antibody is bivalent. Each antibody can bridge to multivalent antigen molecule. This bridging leads to the formation of a lattice which forms the precipitate.

precipitation mechanism
precipitation mechanism

Agglutination:-


Is an antigen antibody reaction is the antibody of the serum binds to the cellular antigen to form clumps. The antibody that causes agglutination are called agglutinins. And the particular antigen are called agglutinogens. When red blood cells [rbc} are agglutinated this type of agglutination is called haemagglutination.

Mechanism:


Agglutination is brought by the linking of antigen and antibody. Most of the antibodies are bivalent they can link to two adjacent antigen. Igm antibody is multivalent and it capacity to make clamps more effectively with lesser number of molecules.

Complement fixation:-


In this antibody is combined with surface antigen of the bacteria. The antigen antibody complex the compliment system. The activated complement is attached to antigen antibody complex. The phagocytic cells has a receptor for the binding of complement already linked to antigen antibody complex. The phagocytic cell undergo phagocytosis to destroy the antigen.
 

Complement fixation
Complement fixation

Hybridoma technology monoclonal antibody:-


A hybridoma is a hybrid cell obtained by fusing a b lymphocyte with as usually a famous cell of the antibody forming system { these are maylomas}. The hybrid cells thus produced possesses the ability to produce antibodies. Due to b lymphocytes genome and the capacity for indefinite growth in vitro due to the tumor cell involved in the fusion. Therefore, hybridoma cultured in vitro or passage through mouse peritonial activity to obtain clonal antibodies. This is called hybridoma technology. This technology was developed by g choler and c.mistine in 1975 for which they were nobel prize in 1984.

Antibodies are produced by b lymphocytes. Each b lymphocytes cell being pre programmed to respond to a single antigenic determinant. When an antigen reacts to the cell surface receptors of a b lymphocyte. It proliferates rapidly field relation of b cells all of which produce antibodies of the same specificity. This is called clonal selection. Thus a b lymphocytes cell produces antibodies of only one specificity.

The myloma cells are selected for mainly two futures:

  1. These cells not produce antibodies themselves.
  2. They must contain a genetic marker i.e., hgprt trait, which permits an easy selection of the result in hybrid cells. When hgprt fused with b lymphocytes the result in the cell population will consists of


  • Hybrid cells
  • Myloma cells
  • B lymphocytes



This cell population is now cultured in hat medium containing the drug aminopurin. The hgprt myloma cells unable to divide in the hat medium due to aminoptruin. Similarly the b lymphocytes do not grow for long periods of time in tissue culture and eventually die. In contrast only the hybridoma cell proliferate on the head medium. Cincinnati lymphocytes genome markers then hgprt and they have the capability for indefinite growth from the myloma cells. Thus hybridomas are selected by using a suitable selective medium hat which allows for to proliferate.
The hybridoma cells are suspended suitably diluted and distributed into microwells. One cell in each microwell are allowed to grow. The hybridoma cells grow and secrete antibodies into the medium. The supernatant from which microwell is sampled and assayed for the presence of antibodies by Elisa.
Hybridoma technology monoclonal antibody
Hybridoma technology monoclonal antibody

Applications:-

  1. Mab’s can administrate to provide passive immunity against diseases.
  2. Mab’s very useful in purification of antigens specific to pathogens, these purified antigens are used as vaccines.
  3. Mab’s have used to isolate m rna encoding the protein to which mab’s are specific.
  4. Mab’s are used to isolate the cells displaying a specific antigen on their surface.
.

Immunodiffusion:-

Immunodiffusion also indicates identity, cross reaction and non identify between different antigens.
  • Single diffusion in one dimension { oudin method}- syllable antigen is directly layered body containing gel. The antigen diffuses into the gel and forms a line of precipitation.
  • Double diffusion in one dimension { oakely ful trophy method} – this method separate antigen and antibody solution with a layer of clear agar.
  • Single radial immunodiffusion {mancini method} – it is used to quantitate serum proteins, immunoglobulins, complement factors.
  • Double diffusion agar assay { ouch teritony method} – it is used for the comparison of various antigens and antisera or for comparison of several antigens at a time on one agar plate.
  • It is also used to detect entirotoxin in cases of suspected food poisoning. It is used for the diagnosis of smallpox.

Elisa:-

Enzyme linked immuno sorbent assay {Elisa} is highly sensitive, high specific and less expensive technique used in serology to detect antigens or antibodies. This test involves the linking of various “label” enzymes to either antigens{or} antibodies. Elisa is based on two principles.
  • Antibodies and some antigens can absorb to a solid support such as polystyrene, polyvinyl {or} polycarbonate wells.
  • Antigens and antibodies can be linked to enzymes with the resulting complexes still fully functional, both immunologically and enzymatically.
Enzyme activity is used to measure the quantity of antigen {or} antibody present in the test sample. Enzymes used in elisa include b galactosidase, glucose oxidase, peroxidase, and alkaline phosphatase.

  • There are two basic elisa techniques.
  1. The double antibody sandwich technique
  2. Indirect immuno solvent technique

  • Double antibody sandwich assay:-

  1. It is used for the direction of antigens.
  2. In this technique specific antibody is placed in wells of a micro titer plate.
  3. The test antigen is added to each well. If the antigen reacts with the antibody, the antigen is retained when the well is washed to remove unbound antigen.
  4. An antibody- enzyme conjugate specific for the antigen is then added, it will bind to the antigen already fixed by the first antibody, forming and antibody { with enzyme} – antigen- antibody. Sandwich.
  5. A substrate that the enzyme will convert to a coloured product is then added, and any resulting product is quantitatively measured by optical density scanning an elisa reader.
  6. If the antigen has reacted with the adsorbed antibodies the first step, the elisa test is positive. If the antigen is not reacted with the adsorbed antibody the elisa test is negative.

  • Indirect immuno sorbent assay:-

  1. It is used for the detection of antibodies { example:- antibodies to hiv and tubella virus in serum sample}.
  2. In this technique the antigen is observed on the inner surface of well wall.
  3. Test antiserum is added and incubated
  4. If any antibodies in the test antiserum have bound to the antigen, the presence is detected by adding an enzyme linked anti immunoglobulin { anti antibody}.
  5. Enzyme substrate is then added and the colour change can be detected by elisa reader in terms of optical density.
 

The double antibody sandwich technique Indirect immuno solvent technique
The double antibody sandwich technique and Indirect immuno solvent technique

Leave a Comment