Introduction

The dual-antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA; Fig. 1) is a sensitive and specific technique for quantifying molecules in solution. The technique is dependent upon the availability of two antibodies recognizing separate epitopes upon the antigen to be measured such that they are able to bind to the molecule simultaneously (1,2). The "capture" antibody specific to the substance to be measured is first coated onto a high-capacity

From: Methods in Molecular Biology, vol. 295: Immunochemical Protocols, Third Edition. Edited by: R. Burns © Humana Press Inc., Totowa, NJ

Monoclonal Antibody
Fig. 1. The dual-antibody sandwich enzyme-linked immunosorbent assay.

protein-binding microtiter plate. Any vacant binding sites upon the plate are then blocked with the use of an irrelevant protein such as bovine serum albumin (BSA). Samples, standards, and controls are then incubated on the plate, allowing the antigen to bind to the capture antibody. The bound sample can be detected using a secondary antibody (recognizing a different epitope on the antigen), thus creating the "sandwich." The detection antibody is sometimes directly conjugated to an enzyme such as horseradish peroxidase (HRP), or, more commonly, biotin-conjugated, allowing an amplification procedure to be conducted with the use of streptavidin-HRP. As 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) results in a colorimetric reaction to occur in the presence of the HRP. The color can then be measured using a spectrophotometer with the resulting optical density (OD) relating directly to the amount of antigen present within the sample. Comparison of OD within a sample to a standard curve of known concentrations allows the concentration within that sample to be quantified.

0 0

Post a comment