Antibodies, A Laboratory Manual, Second Edition, Edited by Edward A. Greenfield

Table of Contents

Expand All | Contract All

Immunometric Antibody Sandwich ELISA

(Protocol summary only for purposes of this preview site)

The antibody sandwich ELISA is the most commonly used assay format for rapid and accurate detection of antigens. The assay displays greater sensitivity compared with the indirect ELISA. This assay can be used to determine absolute antigen concentrations in unknown samples provided purified antigen standards are available. This assay, however, requires the use of two different antibodies. These can be polyclonal/monoclonal, monoclonal/monoclonal, or monoclonal/polyclonal antibody combinations (Rabin et al. 1992; Hornbeck et al. 2001). The sensitivity of the sandwich ELISA is dependent on four factors: (1) the number of molecules of capture antibody immobilized on the well's surface; (2) the avidity of the capture antibody for the antigen; (3) the avidity of the detection antibody for the antigen; and (4) the specific activity of the detection antibody in the ELISA. When using either monoclonal/monoclonal or polyclonal/polyclonal combinations, a subset of the antibody must be biotinylated or directly labeled with a reporter of choice in order to detect the antigen. Furthermore, when using two monoclonal antibodies, they must recognize discrete epitopes on the antigen under investigation.

Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition
Antibodies: A Laboratory Manual, Second edition

Search for information about other protocols included in the book: