Immunology

Fluorescence is the property of absorbing light ray of particular wavelength and emitting rays in different wavelength.

Antigens that are bound to cells or tissue sections can be visualized by tugging the antibody molecule with a fluorescent dye or fluorochrome.

This technique is applied to detect the antibody against hepatitis-B and to detect antibodies against SLE (systemic leupus erythromotosis) and used to detect specific antigen foe- meningo coccus in cerebrospinal fluid.

This technique involves the simultaneous electrophoresis of antigen and antibody in the gel in the opposite direction resulting in precipitation of point where there is optimum concentration of antigen-antibody.

This method produces visible precipitin with in 30 minutes and is 10 times more sensitive than the standard double diffusion technique.

In paper electrophoresis, serum proteins can be separated into 5 different bands but the same protein using immuno electrophoresis can be separated into 30 different proteins.

The resolving power of immuno diffusion was greatly enhanced bye immuno electrophoresis. This involves the electrophoretic separation of antigen into its constituent proteins followed by immuno diffusion.

This technique is performed on 1% agarose gel. Antigen mixture is first electrophori zed and separated based on charge, troughs are then cut in the agarose gel, and antiserum is added to the troughs.

The agarose gel is then incubated 18-24hrs during which the antigen and antibody diffuse towards each other. The formation of precipitin bands can be observed for the individual antigen components.

1. This technique is useful for testing normal and abnormal proteins in serum and urine.

2. It is useful to determine whether a patient produces abnormally a low amount of one or more proteins.

3. It is also used if a patient over produces some serum proteins.

This method cannot the antigens present in concentration below 5-10 micro grams/ml.

It is used to qualitate the antigen. Suitable dilution of antiserum is incorporated in the agar gel. Antigen is added to the wells cut on the surface of the gel. As the antigen diffuses into the agar region, equivalence is established and ring of precipitation is formed. The area of precipitin ring is directly proportional to the concentration of antigen. By comparing the area of precipitin with a standard curve obtained by measuring the precipitin area of known concentration of antigen, the concentration of antigen in the given sample can be determined.

► Radial immuno diffusion method and

► Double immuno diffusion in two dimensions

These reactions can be used to determine relative concentrations of antigens and antibodies to compare antigens and to determine the relative purity of an antigen. They are mainly preformed in 1% agarose gels.

1. Precipitation reaction is the basic reaction for a number of techniques.

2. It is less sensitive for detecting antibodies.

3. Precipitation reactions in gels have several advantages rather than in liquid medium.

4. They have forensic application in identification of blood and seminal stains.

In this, ratio of antigen-antibody is seen optimal which results in large multimolecular lattice, hence maximum precipitation is observed.

The three distinct phases are

Ascending part called ‘zone of antibody excess’.

A peak called ‘zone of equivalence’.

A descending part called ‘zone of antigen excess’.

In this, the first available antigen is completely filled by antibody molecules. Hence, no antigenic determinant is left out free. Unreacted antibody is seen in large amount, hence poor lattice formation.

Marrak proposed the lattice hypothesis to explain the mechanism of precipitation.

The amount of precipitate formed is greatly influenced by relative proportions of antigens and antibodies.

The valency of antigens is multivalent.

When antigen-antibody is in optimal concentration, the precipitation is complete. So that, large lattice is formed.

When a soluble antigen combines with corresponding antibody in the presence of electrolyte at a suitable temperature and pH, the antigen-antibody complex forms an insoluble precipitate .Antibodies that form precipitate ate called precipitants.

It is broadly classified into five-

1. Precipitation

2. Agglutination

3. Complement fixation

4. Immunoassay using labeled reagents

5. Immunohistrochemistry (Immunoflourescence)

The glycolipid antigens are present in most tissues of guinea pigs but not in the RBC. They are found in gastrointestinal mucosa in some people. This horseman antigen will not induce antibody formation.

Heterophile antigens are polysaccharides, which are structurally similar because of their limited complexity. They are derived from members of widely separated taxonomic groups.