Combinational Use of Antibody Affinities in an Immunoassay for Extension of Dynamic Range and Detection of Multiple Analytes

Abstract

Here, we describe the coordinated use of two antibodies with different affinities in a single immunoassay to extend the dynamic range and to enable detection of multiple analytes. The combination of dual antibodies was permitted with a flow-based assay at the antibody concentration below the dissociation constant, enabling affinity to govern the antibody-antigen binding. Both high and low affinity antibodies to estriol were used in combination to extend the range. The binding of each antibody was mutually independent and individually occurred over concentration ranges of 10 pM(-1) nM and 100 pM(-1) microM. The wide dynamic range of 10 pM(-1) microM was thus achieved as summation of the proportional signals to the total binding. When a combination of antibodies toward different antigens was used, it effectively detected multiple analytes within a mixture. In simultaneous analysis of a mixture of estradiol and estriol, the total signal was the sum of the binding signals from anti-estradiol and anti-estriol antibodies. In a further refinement, the individual antibodies were flowed through the flow cell sequentially, allowing the quantification of each binding signal within the combination. With this sequential format, measurement of the individual hormones in the range of 1.6 pM(-1) nM was shown. Furthermore, the same flow format was successfully applied to assay estriol and estradiol hormones in mixtures of six related compounds.

Extending the Working Range of Immunoanalysis by Exploitation of Two Monoclonal Antibodies

“…In addition to the usage of the antibodies separately, a mixture of both antibodies was used. A similar approach was described earlier by Ohmura et al (8) utilizing two mAbs with different affinities for estriol and demonstrating this technique in a flow immunoassay system.…”

Pretreatment Method for Immunoassay of Polychlorinated Biphenyls in Transformer Oil Using Multilayer Capillary Column and Microfluidic Liquid-Liquid Partitioning

“…The immunoassay system was based on theory and operation that were previously described by us [15][16][17] and others. [18][19][20] The antigen-immobilized solid-phase, which was prepared as described previously, 21 was captured in a small flow cell held at the focus of a fluorometer.…”

Sensitive detection of ochratoxin A in wine and cereals using fluorescence-based immunosensing

“…Details of the KinExA instrument performance have been described elsewhere (Blake, Pavlov, & Blake, 1999;Blake et al, 2001;Khosraviani et al, 2000;Kim, Shelver, Hwang, Xu, & Li, 2006;Ohmura, Tsukidate, Shinozaki, Lackie, & Saiki, 2003;PrietoSimón, Miyachi, Karube, & Saiki, 2010). Briefly, the capillary flow cell of the automated flow immunoassay system is fitted with a microporous screen where beads are packed acting as the solid phase.…”