RIA commonly used separation methods

RIA commonly used separation methods

1. Second antibody precipitation method The principle is to immunize another animal (such as sheep) with reagent antibody (primary antibody) in the RIA reaction, such as guinea pig IgG, to prepare goat anti-guinea pig IgG serum (primary antibody). At the end of the RIA reaction, add a secondary antibody to form a double antibody complex of antigen-primary antibody-secondary antibody; but because the content of primary antibody is very small, this complex is also small, and it is not easy to centrifuge. Generally, a certain amount of Serum or IgG of the same animal as the primary antibody, so that it forms a large amount of visible precipitate with the secondary antibody and co-precipitates with the dual antibody complex; after centrifugation, B and F can be effectively separated. The secondary antibody can also be connected to certain particle solid phases to make a solid phase secondary antibody, and the effect of separating B and F is also good.

2. Polyethanol (PEG) precipitation method PEG can non-specifically precipitate large molecule proteins such as antigen-antibody complexes without precipitating small molecule antigens. Therefore, PEG is widely used as a precipitant in RIA experiments. The advantage is that the precipitation is complete, economical and convenient, but there are more non-specific precipitations, and when the temperature is higher than 30 ~ C, the precipitates are easily reconstituted.

3. PR reagent method First, the secondary antibody and PEG are mixed into a suspension in a certain ratio and then tested. It is a method that combines the principle of secondary antibody and PEG precipitation. This method retains the advantages of the two, saves the amount of both, and the separation is quick and simple.

4. Activated carbon adsorption method Activated carbon can adsorb small molecules of free antigens or haptens, while large molecular proteins (such as antibodies and immune complexes) remain in solution. After the antigen-antibody reaction, activated carbon particles are added to adsorb the free labeled antigen (F) onto the particles, and then the particles are precipitated by centrifugation. The labeled antigen-antibody complex (B) contained in the supernatant is measured. This method is mainly used to determine the RIA of small molecule antigens or drugs.