Secondary Antibodies for ELISA

Enzyme-linked immunosorbent assay (ELISA) allows researchers to detect and quantify an analyte in solution. It can be configured in one of four main ways: direct, indirect, sandwich, and competitive ELISA. Of these, both the indirect and sandwich formats incorporate secondary antibodies for detection. A major advantage of using secondary antibodies for ELISA is that they can provide signal amplification as a result of multiple secondary antibodies binding to each primary antibody. If necessary, the assay sensitivity can be further enhanced with tertiary reagents targeting the secondary antibodies. In an indirect ELISA, the analyte of interest is bound to the microplate wells along with any other proteins present in the sample. An analyte-specific primary antibody is then added, followed by a labeled secondary antibody. Critically, the secondary antibody must recognize the host species of the primary antibody. For example, if the primary antibody was raised in a mouse host, an anti-mouse secondary antibody is required for detection. A sandwich ELISA follows similar principles, but a plate-bound antibody is used for analyte capture. In this scenario, researchers can either perform direct detection with a labeled primary antibody or indirect detection with an unlabeled primary antibody and a labeled secondary antibody. If indirect detection is employed, it is important to ensure that the capture antibody and the primary antibody have different host species to prevent the secondary antibody from cross-reacting with the capture antibody and yielding false positive results. Enzymes such as horseradish peroxidase (HRP) and alkaline phosphatase (AP) are the most common antibody labels used for ELISA. These allow for either chromogenic or chemiluminescent detection depending on the chosen substrate. Chromogenic substrates for HRP include 3,3′,5,5′-tetramethylbenzidine (TMB), o-phenylenediamine dihydrochloride (OPD), and 2,2′-azino-bis [3-ethylbenzthiazoline-6-sulfonic acid] (ABTS), which respectively yield blue, yellow/orange, and green end products. AP typically catalyzes the hydrolysis of para-nitrophenyl phosphate (PNPP) to produce a yellow reaction product. Advantages of chromogenic detection are that the substrates are relatively inexpensive and the readout can be measured with a standard spectrophotometer. Most chemiluminescent substrates are HRP-dependent and work by oxidizing luminol to form an excited state product that emits light as it returns to the ground state. Chemiluminescent detection offers higher sensitivity than chromogenic detection, but the reagents are more costly and a dedicated plate reader is required. Secondary antibodies for ELISA can also be labeled with fluorophores, when the technique is sometimes referred to as a fluorescence-linked immunosorbent assay (FLISA), or with biotin. If a biotinylated secondary antibody is used, a streptavidin conjugate is required for detection. This is usually streptavidin-HRP or streptavidin that has been conjugated to a fluorophore such as fluorescein, R-phycoerythrin (RPE), or allophycocyanin (APC). These types of reagents exploit the high-affinity multivalent biotin-streptavidin interaction to increase the amount of label bound to the target and thus boost the assay sensitivity beyond that of indirect detection.