Indirect ELISA is illustrated in the figure below and Figure 2. Phases I and II are similar to the direct system.Phase III involves adding unlabeled detection antibodies that are diluted in the buffer to prevent the proteins in the antiserum from nonspecifically attaching to the solid phase (the blocking buffer). It is then incubated and washed away the excess (unbound) antibodies to achieve specific binding (stage IV).Stage V is the addition of the conjugate (enzyme-labeled), anti-species antibody, dilution in a blocking buffer, and then incubating and washing again to achieve binding of the conjugate (Stage VI).Vii) develop colour, then stop it (stage VIII) and read it in a spectrophotometer (stage IX).
The indirect system is similar to the direct system in that the antigen is directly attached to the solid phase and is targeted by the added antibody (detection antibody).However, these added antibodies are not labeled by the enzyme, but are targeted by antibodies that are bound to the enzyme.These antibodies, which produce immunoglobulins for the species in which they are produced, are called antispecies conjugates.Therefore, if the detection antibody is produced in rabbits, the enzyme-labeled antibody must be anti-rabbit Ig in nature.This allows for great flexibility in the use of anti-species binding, as the different specificities of the binding can be used to detect specific immunoglobulin binding in the assay, and there are literally thousands of commercially available binding substances.For example, anti-species conjugates may be anti-igm, anti-igg 1, IgG 2, etc.
Indirect ELISA. Antibodies from a particular species react with antigen attached to the solid phase. Any bound antibodies are detected by the addition of an antispecies antiserum labeled with enzyme. This is widely used in diagnosis.
The indirect system offers the advantage that a single anti-species conjugate can be used to check the binding of any number of antisera to a given antigen. Such systems have been used extensively in diagnostic applications, especially when examining (screening) large numbers of samples. One problem with this system is the varying degrees of non-specific binding in a single serum. This tends to amplify the degree of dispersion (variability) in the analysis results, thus increasing the need to process many sera to assess confidence.