Also, indirect ELISA assays take longer to run than direct ELISAs since an additional incubation step for the secondary antibody is required. Among its disadvantages is the possibility of cross-reactivity of secondary antibody to the adsorbed antigen, which could increase background noise. ![]() Indirect ELISA delivers greater flexibility since different primary antibodies can be used with a single labeled secondary antibody. The indirect ELISA method has high sensitivity since more than one labeled secondary antibody can bind the primary antibody it is more economical than the direct ELISA as fewer labeled antibodies are needed. Second, an enzyme conjugated secondary antibody that is directed against the host species of the primary antibody is applied. ![]() First, an unlabeled primary antibody binds to the specific antigen. Less prone to error – as less reagents and fewer steps are requiredīest for: when analyzing the immune response to an antigen.įigure 3 demonstrates how an indirect ELISA is set up antigen is adsorbed to a well in an ELISA plate. No signal amplification - reduces assay sensitivity Less flexible - each target protein needs a specific conjugated primary antibody Mainly because all proteins in the sample, including the target protein, will bind to the plate Finally, the direct ELISA technique is typically used when the immune response to an antigen needs to be analyzed.įaster than other ELISA – the technique has fewer stepsĪntigen immobilization is not specific - may cause higher background noise than indirect ELISA. As no secondary antibody is used there is no signal amplification, which reduces assay sensitivity. Direct ELISA is less flexible since a specific conjugated primary antibody is needed for each target protein. This is primarily because all proteins in the sample, including the target protein, will bind to the plate. As the antigen immobilization is not specific, higher background noise may be observed in comparison to indirect ELISA (see below). Although there are some disadvantages to this method. no potentially cross-reacting secondary antibody needed. The assay is also less prone to error since fewer reagents and steps are needed, i.e. The antigen is then detected by an antibody directly conjugated to an enzyme such as HRP.ĭirect ELISA detection is much faster than other ELISA techniques as fewer steps are required. In general, ELISAs can be grouped into the four main categories:įigure 2 illustrates the setup of direct ELISA an antigen is immobilized in the well of an ELISA plate. The detection antibody is either directly conjugated to an enzyme, such as horseradish peroxidase (HRP), or provides a binding site for a labeled secondary antibody. After immobilization, a detection antibody is added, which binds to the adsorbed antigen thereby leading to the formation of an antigen-antibody complex. The capture antibody has to be specific to the target antigen and is mainly used in a specific ELISA type called “ sandwich ELISA”. This can be achieved by direct adsorption to the plate’s surface or by using a “capture antibody”. An adjuvant, which is a chemical that provokes a generalized activation of the immune system that stimulates greater antibody production, is often mixed with the antigen prior to injection.The first step in an ELISA experiment is the immobilization of the antigen in a sample to the wall of the wells of a microtiter plate. On re-exposure to the antigen, those B cells capable of producing antibody with higher affinity antigen-binding sites will be stimulated to proliferate and produce more antibody than their lower-affinity peers. Affinity maturation occurs because of mutations in the immunoglobulin gene variable regions, resulting in B cells with slightly altered antigen-binding sites. The memory cells also undergo affinity maturation, resulting in a pool of antibodies with higher average affinity. The second injection will activate memory cells that make class IgG antibodies against the antigen. ![]() Lab animals are usually injected at least twice with antigen when being used to produce antiserum.
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