Horseradish peroxidase-anti-horseradish peroxidase method

The characteristics of the PAP complex are related to the preparation method of the reagent. HRP is first used to immunize animals to produce high titer, highly specific anti-HRP antibodies, and then HRP is used to bind them, because the HRP anti-HRP complex prepared in vitro is different from other antigen-antibody reactions . When the antigen is slightly excess, all anti-HRP antibodies participate in the formation of soluble PAP complexes, leaving only a little free HRP. However, most antigen-antibody reactions require an absolute excess of antigen to form a soluble complex. The shape of the PAP complex is quite stable and is not affected by the amount of antigen. Regardless of the initial addition of antigens and antibodies, the HRP / anti-HRP ratio of the resulting PAP complex is mostly 3; 2. Centrifugal sedimentation is applied. The methods of liquid phase diffusion and electron microscope observation showed that the PAP complex has a pentagonal ring structure, three corners are HRP, and the other two corners are anti-HRP antibodies with a molecular mass of 400-430 kDa, and this structure is very stable.