Secondary antibodies can be classified based on their specificity, which can be broad or narrow, and their fragment type, which can be whole, F(ab), or F(ab')2. 

The principle of F(ab')2 is to generate divalent antibody fragments that retain the antigen-binding Fab portions of whole IgG antibodies while removing most of the Fc region.

F(ab')2 fragments are generated by pepsin digestion of whole IgG antibodies to cleave the hinge region and leave intact some of the Fc region. The resulting fragments have two antigen-binding Fab portions linked together by disulfide bonds, making them divalent with a molecular weight of about 110 kDa. The use of F(ab')2 fragments has several advantages over whole IgG antibodies, including better penetration into tissues, reduced nonspecific binding to Fc receptors on live cells or to Protein A/G, and the ability to precipitate antigens. However, F(ab')2 fragments are not recommended for blocking since they have two binding sites that are available to capture the primary antibody introduced subsequently. The preparation of F(ab')2 fragments involves pepsin digestion of whole IgG antibodies, followed by purification using techniques such as FPLC with DEAE-