How to Set Up an ELISA
The enzyme-linked immunosorbent assay (ELISA) represents a simple and sensitive technique for specific quantitative detection of molecules to which an antibody is available (1 ,2 ). Although there are a huge number of variations based on the original ELIS
- PDF / 111,757 Bytes
- 7 Pages / 418.32 x 664.56 pts Page_size
- 80 Downloads / 306 Views
373
30 How to Set Up an ELISA Bill Jordan 1. Introduction The enzyme-linked immunosorbent assay (ELISA) represents a simple and sensitive technique for specific quantitative detection of molecules to which an antibody is available (1,2). Although there are a huge number of variations based on the original ELISA principle, this chapter will focus on the two perhaps most useful and routinely performed: the indirect sandwich ELISA, providing high sensitivity and specificity; and the basic direct ELISA, useful when only one antibody to the sample antigen is available. During the indirect sandwich ELISA (Fig. 1A), an antibody specific for the substance to be measured is first coated onto a high capacity protein-binding microtiter plate. Any vacant binding sites on the plate are then blocked with the use of an irrelevant protein, such as fetal calf serum (FCS) or bovine serum albumin (BSA). The samples, standards, and controls are then incubated on the plate, binding to the capture antibody. The bound sample can be detected using a secondary antibody recognizing a different epitope on the sample molecule, thus creating the “sandwich.” The detection antibody is commonly directly conjugated to biotin, allowing an amplification procedure to be carried out with the use of streptavidin bound to the enzyme horse-radish peroxidase (HRP). Because streptavidin is a tetrameric protein, binding four biotin molecules, the threshold of detection is greatly enhanced. The addition of a suitable substrate, such as 3,3',5,5;-tetramethylbenzidine (TMB), allows a colormetric reaction to occur in the presence of the HRP that can be read on a spectrophotometer, with the resulting optical density (OD) relating directly to the amount of antigen present within the sample. In some cases, however, only one specific antibody may be available, and in such a small quantity that directly conjugating it to biotin would be impractiFrom: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
373
374
Jordan
Fig. 1. Diagramatic representation of (A) the indirect sandwich ELISA, and (B) [facing page] the basic direct ELISA.
cal. In this situation a direct ELISA should be used. During the direct ELISA the sample itself is coated directly onto the microtiter plate and is then detected using the specific antibody. If this antibody is biotinylated, the procedure can proceed as in the indirect ELISA; if not, then a secondary biotinylated antibody directed against the species of the detecting antibody itself can be used. Figure 1B demonstrates this technique. To set up a reliable and durable ELISA it is essential to first optimize a number of the parameters mentioned above. The level of optimization will, of course, depend on exactly what is required from the assay. In some cases a simple “yes or no” answer is desired and a simple standard procedure may be sufficient. If, however, high sensitivity is the aim with accurate quantatation of the molecule in questi
Data Loading...
