countercurrent&rocket electrophoresis - أطلس مختبرات العرب

Countercurrent electrophoresis is performed in agar gels where the pH is chosen so that the antibody is positively charged and the antigen being tested is negatively charged. By applying a voltage across the gel the antigen and antibody move towards each other and precipitate. The principle is the same as for immuno-double-diffusion but the sensitivity is increased 10–20-fold. Antigens may be quantitated by electrophoresing them into an antibody-containing gel in the technique termed rocket electrophoresis. The pH of the gel is chosen so that the antibodies are immobile and the antigen is negatively charged. Precipitin rockets form; the height of the rocket is proportional to antigen concentration, and unknowns are determined by interpolation from standards. The appearance of stained rockets is shown on the right. Both techniques rely on the antigen and antibody having different charges at the selected pH; this is true for most antigens since antibodies have a relatively high isoelectric point (i.e. they are neutrally charged at a more alkaline pH than most antigens). If the charges on the antigen and antibody do not differ sufficiently, the antibody or antigen can be chemically modified to alter its isoelectric point. Rocket electrophoresis can be reversed to estimate antibody concentration if a suitable pH gel can be found to immobilize the antigen, without damaging it or preventing the antigen–antibody reaction.