Immunoelectrophoresis, immunoblotting, Complement fixation test.

Immunoelectrophoresis, immunoblotting (Western blotting), and the complement fixation test are immunological techniques used to detect and analyze specific antigens or antibodies in biological samples. Each of these methods has unique principles and applications:

1. Immunoelectrophoresis:

• Principle: Immunoelectrophoresis combines two techniques: electrophoresis and immunodiffusion. Proteins are separated by electrophoresis based on their charge, and then antibodies are used to form immune complexes with specific antigens in the gel. These complexes create visible precipitation bands.
• Procedure:

• Proteins from a sample are separated by electrophoresis on a gel, creating wells or troughs in the gel.
• Antisera containing specific antibodies to target antigens are added to these wells.
• As the antigens and antibodies diffuse towards each other through the gel, they form immune complexes, resulting in visible precipitation lines.

• Applications: Immunoelectrophoresis is used for the qualitative analysis of proteins in biological samples. It can identify and quantify specific antigens, such as proteins or immunoglobulins, in serum or other biological fluids. It has applications in clinical diagnostics and research.

2. Immunoblotting (Western blotting):

• Principle: Immunoblotting is used to detect and analyze specific proteins within a complex mixture. It involves the separation of proteins by electrophoresis (usually SDS-PAGE) and their subsequent transfer to a membrane (blotting). Then, specific antibodies are used to detect the target protein on the membrane.
• Procedure:

• Proteins are separated by electrophoresis and transferred to a membrane.
• The membrane is blocked to prevent non-specific antibody binding.
• Primary antibodies that specifically recognize the target protein are added and allowed to bind.
• After washing, secondary antibodies conjugated to a reporter enzyme or fluorophore are applied to detect the bound primary antibodies.
• The signal is visualized, and the presence and quantity of the target protein can be determined.

• Applications: Western blotting is widely used in research and diagnostics to detect and quantify specific proteins in biological samples. It is particularly valuable for studying protein expression, post-translational modifications, and protein-protein interactions.

3. Complement Fixation Test:

• Principle: The complement fixation test is an immunological assay used to measure the level of specific antibodies in a patient's serum. It is based on the ability of antibodies to fix and consume complement proteins when they bind to their specific antigens.
• Procedure:

• The patient's serum is heat-inactivated to remove any existing complement.
• The serum is mixed with a known amount of antigen.
• Complement proteins and sensitized sheep red blood cells are added.
• If the patient's serum contains specific antibodies against the antigen, the antibodies will bind to the antigen, leading to complement fixation and hemolysis (lysis of red blood cells).

• Applications: The complement fixation test is used for serological diagnosis of infectious diseases, such as syphilis and viral infections. It measures the presence and titer of specific antibodies in a patient's serum and is particularly useful for detecting antibodies at low concentrations.

These immunological techniques play critical roles in various areas of biomedical research, clinical diagnostics, and disease monitoring by allowing the detection, quantification, and characterization of antigens and antibodies in biological samples.