The novel FluoroChrome ImmunoAssay -- (FCIA). The role of molecular environment upon molecular structure exemplified by constriction of a flourescence-photochrome flanked by two proteins.

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

A rapid, sensitive, and quantitative novel immunoassay [FluoroChrome ImmunoAssay, FCIA] technique was developed which auspiciously combines both the high sensitivity of fluorescence measurements with the high specificity of an antibody. As opposed to existing immunoassays, FCIA is performed without separation of antibody-bound haptens from those that are free, and utilizes fluorescence measurements from widely available standard commercial fluorimeters. FCIA is based on the hypothesis that an appropriately designed stilbene-antigen analogue probe will suffer considerable steric hindrance to trans-cis photoisomerization when bound within the combined constraints of both an antibody binding site and a second globular protein. Specifically, an appropriately designed 2,4-dinitrophenyl-hapten derivative of fluorescent trans-4,4'-diaminostilbene (DAS), was squeezed between two large globular proteins: lysozyme (Lys) from one side, and anti-2,4,6-trinitrophenyl antibody (antiTNP) from the other side, in order to provide the desired constricted environment to restrict trans/cis-stibene isomerization within the antiTNP-DNP-DAS-Lys adduct. As was theoretically predicted and then experimentally verified, the trans-cis photoisomerization rate for the bound probe was found to be markedly inhibited, compared to that expected for the free probe in solution. The fluorescence-photochrome labeled probe was competitively displaced from the antiTNP binding site in the presence of the picric acid hapten, and photoisomerization then commenced to produce the fluorescence-silent cis-stilbene diastereomer. The process of association and dissociation of a hapten-antibody complex was readily monitored by the fluorescence technique in the presence of both antibody-bound and free haptens.

Original languageEnglish
Pages (from-to)1073-1079
Number of pages7
JournalJournal of Biochemical and Biophysical Methods
Volume70
Issue number6
DOIs
StatePublished - 1 Jan 2008

Fingerprint

Dive into the research topics of 'The novel FluoroChrome ImmunoAssay -- (FCIA). The role of molecular environment upon molecular structure exemplified by constriction of a flourescence-photochrome flanked by two proteins.'. Together they form a unique fingerprint.

Cite this