Purification of antibodies on protein blots

Affinity purification of antibodies on protein blots has been described by Olmsted (1981). In this study DPT filters were used, but CNBr-activated paper or nitrocellulose works equally well (Gershoni and Palade, 1983). 

The blotted proteins are reacted with their corresponding antibodies (Section 16.3.2). The bands are excised and eluted with 3 ml 200 mM glycine-HCl buffer, pH 2.8, during 2 min at 0 C. The solution is then neutralized with NaOH and concentrated to 0.1-0.2 ml. The monospecific antibodies obtained can then be used for other EIA (blots, titration, EIH). Though the blot can be used several times to extract monospecific antibodies from the serum, the amounts obtained are not excessive. It can be advantageous to link the antigen to the blot by the method given in Table 16.11C. 

The ideal enzyme label for EIH should convert a soluble substrate to an insoluble product so that an identifiable precipitate forms immediately at the site of enzyme action, whereas the substrate should not contribute to background staining. The precipitate in the cell can then be seen with a light microscope and may be rendered, in some systems, electron opaque for studies by electron microscopy. In particular POase (Avrameas and Uriel, 1966 Nakane and Pierce, 1966) and GOase are useful for this purpose. In EIH an amplification of the signal is possible to a degree seldom attained in enzyme cytochemistry and often much less tedious. Recent developments in EIH made routine applications of these techniques possible (reviews Bullock and Petrusz, 1982 Van Noorden and Polak, 1983). 

The low penetration rate of antibodies through cell membranes 

Advantages and disadvantages of methods using enzyme-labeled antibodies compared to fluorescent antibodies 

---