Friday, September 28, 2007


Precipitation Reactions
Agglutination Reactions
Western Blotting
The antigen-antibody interaction is a bimolecular association similar to an enzyme-substrate interaction but with the important distinction that it does not lead to an irreversible chemical alteration in either the antibody or antigen and therefore is reversible.

1. Precipitation Reactions:
The interaction between an antibody and a soluble antigen in aqueous solution forms a lattice that eventually develops into a visible precipitate. Antibodies that thus aggregate soluble antigens are called precipitins. Although formation of the soluble Ag-Ab complex occurs within minutes, formation of the visible precipitate occurs more slowly and often takes a day or two to reach completion.
Precipitate reactions in fluids: A quantitative precipitation reaction can be performed by placing a constant amount of antibody in a series of tubes and adding increasing amounts of antigen to the tubes. After the precipitate forms, each tube is centrifuged to pellet the precipitate, the supernatant is poured of, and the amount of precipitate against increasing antigen concentrations yields a precipitin curve.
Precipitation reactions in Gels: immune precipitates can form not only in solution but also in agar matrix. When antigen and antibody diffuse toward one another in agar or when antibody is incorporated into the agar and antigen diffuse into the antibody-containing matrix, a visible line of precipitation will form.

2. Agglutination reactions:
The interaction between antibody and a particulate antigen results in visible clumping called agglutination. Antibodies that produce such reactions are called agglutinins. Agglutination reactions are similar in principle to precipitation reactions. Just as antibody excess inhibits precipitation reactions, an excess of antibody inhibits agglutination reactions; this inhibition is called the prozone effect.
Hemagglutination: Agglutination reactions are routinely performed to type red blood cells. In typing for the ABO blood groups, RBCs’ are mixed on a slide with antisera to the A and B blood-group antigens. If the antigen is present on the cells, they agglutinate, forming a visible clump on the slide.
Bacterial agglutination: A bacterial infection often elicits the production of serum antibodies specific for surface antigens on the bacterial cells. The presence of such antibodies can be detected by bacterial agglutination reactions. Serum from patients thought to be infected with a given bacterium is serially diluted in a series of tubes to which the bacteria is added. The last tune showing visible agglutination will reflect the serum antibody titer of the patient. For example, if serial dilutions of serum are prepared and if the dilution of 1/20 shows agglutination but the dilution of 1/40 does not, then the agglutination titer of the patient’s serum is 40. This is widely used in typing Salmonella typhi.

4. Western Blotting:
Identification of a specific protein in a complex mixture of proteins can be done by a technique called Western Blotting, named for its similarity to Southern Blotting, which detects DNA fragments and Northern blotting, which detects mRNAs’. In Western blotting a protein mixture is electrophoretically separated on a polyacrylamide gel in the presence of sodium dodecyl sulfate (SDS), a dissociating agent. The protein bands are transferred to a nitrocellulose membrane by electrophoresis and the individual protein bands are identified by flooding the nitrocellulose membrane with radiolabeled polyclonal or monoclonal antibody specific for the protein of interest. The Ag-AB complexes that form are visualized by autoradiography. If labeled specific antibody is not available, Ag-AB complexes can be detected by adding a secondary anti-isotype antibody that is either radiolabeled or enzyme labeled; in this case the band is visualized by autoradiography or substrate addition.Western blotting can also identify a specific antibody in a mixture. In this case, the separated antibody bands are visualized with a labeled antigen. For example, this technique has been used to identify the envelope and core proteins of HIV and the antibodies to these components in the serum of HIV-infected individuals