There are some important points to follow for choosing the right secondary antibody. The first important point concerns species reactivity. As a rule, a species is chosen from which the primary antibody originates. Especially in the case of indirect multiple labelling, attention must be paid to cross-reactivity. The next important aspect is the choice of host species of the secondary antibody. In order to avoid cross-reactions between secondary antibodies, all secondary antibody conjugates should be from the same host species in the case of indirect multiple labelling with unconjugated primary antibodies. The third step is to choose the structure of the secondary antibody: total molecule (H+L), F (ab') 2 or Fab fragment. Another important criterion is specificity, which defines which part of the primary antibody is recognized by the secondary antibody. Since polyclonal antibodies of the IgG type (e.g. from rabbit, goat, chicken, etc.) or monoclonal antibodies from mouse, rat or hamster are mainly used for immunological assays, secondary antibodies should have the highest possible sensitivity for this primary antibody. An important selection criterion is the choice of conjugates, which depends on the method used and the desired type of detection (enzymatic, amplified, direct, etc.). The last important criterion is the degree of adsorption, since Ig-specific antibodies of a given species may cross-react to a greater or lesser extent with immunoglobulins and serum proteins of other species. For this reason, to eliminate cross-reactivity with molecules of that species, antibodies are partially preadsorbed to immunoglobulins and serum proteins of one or more foreign species.
