Biochemical Techniques and Instrumentation

3.1.6.1. Separation of bound antigen from free antigen

There are several ways for the separation of bound antigen from free antigen.

1. The antigen-antibody complexes are precipitated by the addition of a "second" antibody directed against the first antibody. eg. If a rabbit IgG is used to bind the antigen, the complex can be precipitated by the addition of an antirabbit-IgG antiserum
2. The antigen-specific antibodies can be coupled to the inner walls of a test tube. After incubation, the contents containing "free" antigen are removed and then the tube with bound antigen is washed and the radioactivity of both can be measured.
3. The antigen-specific antibodies can be coupled to particles, like Sephadex. The bound antigen can be separated as pellets from the free antigen in the supernatant fluid by centrifugation of the reaction mixture.