Dual Antibody Sandwich ELISA

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... 

These assays resemble a hybrid of an immunohistochemistry assay and ELISA. Whole cells are fixed, for example with 3.7% formaldehyde, to MTPs permeabilized by repetitive washing with 0.1% Triton X-100, blocked with a protein solution, probed with primary antibodies (phospho-spe-cific, and non-phospho-specific), washed, and subsequently the secondary antibodies labeled with infrared fluorescent tags are added. After washing, these assays are read in a reader (such as the Odyssey or Aerius) designed for high sensitivity detection of two colors. The two colors are useful because one color can be used to accommodate a stain assigned as a total protein or cell number control or as an antibody to total protein, which allows for normalization. These assays may only require a single antibody versus the dual antibody sandwich required for ELISA. 

The reverse capture autoantibody microarray is based on the dual-antibody sandwich immunoassay of enzyme-linked immunosorbent assay (ELISA) (see Fig. 1). The basic platform consists of a glass microarray slide arrayed with 1000 highly specific well-characterized monoclonal antibodies against 500 unique antigens. These antibodies are used to immobilize native proteins. Because the reagents used in the procedure are non-denaturing, antigens are... 

Key Words ELISA antibodies capture antibody sandwich detection antibody substrate dual antibody. 

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