Elsevier

Analytica Chimica Acta

Volume 585, Issue 1, 28 February 2007, Pages 120-125
Analytica Chimica Acta

Homogeneous non-competitive bioaffinity assay based on fluorescence resonance energy transfer

https://doi.org/10.1016/j.aca.2006.12.021Get rights and content

Abstract

A homogeneous non-competitive assay principle for measurement of small analytes based on quenching of fluorescence is described. Fluorescence resonance energy transfer (FRET) occurs between the donor, intrinsically fluorescent europium(III)-chelate conjugated to streptavidin, and the acceptor, quencher dye conjugated to biotin derivative when the biotin–quencher is bound to Eu–streptavidin. Fluorescence can be measured only from those streptavidins that are bound to biotin of the sample, while the fluorescence of the streptavidins that are not occupied by biotin are quenched by quencher–biotin conjugates. The quenching efficiencies of the non-fluorescent quencher dyes were over 95% and one dye molecule was able to quench the fluorescence of more than one europium(III)-chelate. This, however, together with the quadrovalent nature of streptavidin limited the measurable range of the assay to 0.2–2 nmol L−1. In this study we demonstrated that FRET could be used to design a non-competitive homogeneous assay for a small analyte resulting in equal performance with competitive heterogeneous assay.

Introduction

Haptens are low molecular weight analytes, e.g. many drugs, steroids, metabolites and pollutants, which are too small to be recognized simultaneously by two binders (for example, antibodies). Many of the traditional non-competitve assay techniques, however, require the analyte to be bound to two different binders in order to be detected. First the analyte is bound to the capture antibody and then detected with another analytic spesific labeled antibody. Therefore, the measured signal is obtained from those labeled recognizing agents, which are bound to the analyte. Thus, the signal increases with the concentration of the analyte. Since haptens are not suitable for two-site assays, most of the assays designed to measure haptens are competitive, where the analyte to be measured is competing with a labeled analyte analogue for the binding sites of a single recognizing agent. Thus, in competitive assay the signal is measured from those labeled analyte analogues that are bound to the recognizing agent. Increasing the amount of analyte decreases the amount of bound labeled analyte analogue, which means that increasing the amount of analyte decreases the obtained signal.

Sensitivity can be defined as the smallest amount of analyte, which generates a distinguishable difference in the signal from the background (zero amount of analyte) [1], [2]. In competitive assays the signal at zero concentration is high whereas in non-competitive assays the signal at zero amount of analyte is small. When the concentration is slightly elevated from zero only a small change in the signal occurs. It is typically easier to detect a small change from small signals than in high signals. Hence, the non-competitive assays are capable of detecting smaller changes in analyte concentrations and are, therefore, considered to have better sensitivity than competitive assays [3].

Since non-competitive assays have better sensitivity than competitive assays, quite a few different non-competitive assay techniques for haptens have been designed. For example, in immunometric assays antibodies bound to analyte are separated from those antibodies that are not bound to analyte. After the separation the analyte-bound antibodies are measured. A non-competitive digoxin assay uses digitoxigenin-coupled polyacrylamide beads to separate the unbound antibodies [4]. These techniques rely on the laborious physical separation step, so they are time consuming to do.

Homogeneous assays are separation free, and, therefore, less tedious to perform than heterogeneous assay. Fluorescence resonance energy transfer (FRET) is a commonly used technique in homogeneous assays. When the donor and the acceptor are in close proximity with each other, the non-radiative energy transfer occurs from the excited donor molecule to the acceptor [5]. In addition to small distance, an overlapping of the emission spectrum of the donor with the excitation spectrum of the acceptor is also required [6]. During measurement either the emission of the acceptor or the fluorescence of the donor, in which case the acceptor acts as a quencher, is measured. For example, Arai et al. has demonstrated a FRET based non-competitive open sandwich assay where separate VH and VL fragments are labeled with donor and acceptor. FRET can occur only if the antigen has induced heterodimerization between VH and VL fragments thus bringing the donor and acceptor in close proximity. However, this assay requires a suitable antibody fragment that has a weak VHVL interaction when the antigene is not bound to it [7]. Another FRET method was described by Pulli et al. which utilizes anti-immuno complex (anti-IC) Fab fragments [8]. In that assay europium labeled antimorfine and Cy5-labeled anti-IC Fab are in close enough proximity for FRET only when the antimorfine is bound to the antigen. However, anti-IC antibodies are rather difficult to develop.

This article presents a homogeneous non-competitive assay for biotin. Moreover, the objective is to demonstrate a principle for non-competitive homogeneous assay based on fluorescence resonance energy transfer for a small analyte, which can only be recognized by one binder at a time. Thus, biotin–streptavidin complex was used as a model system. Since the interaction of biotin and streptavidin is strong (Kd  10−15 mol L−1), it is ideal for testing this type of protocol. The assay requires only one binder/recognizing agent, which is labeled with europium chelate, and an analyte analogue, which is conjugated to quencher dye. The assay utilizes FRET to quench the fluorescence of those europium(III) labeled streptavidins that are not occupied by biotin of the sample.

Section snippets

Measurement buffer and instrument

Buffer for all assays and dilutions contained 0.05 mol L−1 Tris–HCl, pH 7.75, 0.9% (w/v) NaCl, 0.05% (w/v) NaN3, 0.01% (v/v) Tween 40, 0.05% (w/v) bovine-γ-globulin, 20 μmol L−1 diethylenetriaminepentaacetate (DTPA) and 0.5% (w/v) bovine serum albumin (BSA) (purchased from Innotrac Diagnostics, Turku, Finland).

Fluorescence was measured using time-resolved fluorescence mode at Victor 1420 Multilabel counter from Wallac, Perkin-Elmer Life and Analytical Sciences (Turku, Finland). Excitation and

Results and discussion

The different situations depending on the amount of biotin during measurement are presented in Fig. 1 Streptavidin labeled with europium(III)-chelate (Eu–SA) has emission at 615 nm when excited with 340 nm. Maximum signal of the assay is obtained when four biotins are bound to Eu–SA, hence no quenching occurs. If biotin has bound to some of the binding sites of Eu–SA and the rest are occupied by the biotin–quencher conjugates the emission of europium is decreased. This is due to fluorescence

Conclusions

We have demonstrated a novel principle for non-competitive homogeneous assay based on fluorescence resonance energy transfer between europium(III)-chelates and quencher dyes. Since the principle requires strong interaction between the analyte and the recognizing agent, the method was tested using biotin–streptavidin complex. The practical range was 0.2–2 nmol L−1, when using the most suitable quencher, Cy7. Due to the high quenching efficiencies of the quenchers and the quadrovalent nature of

Acknowledgement

This study was supported by TEKES, the Finnish Funding Agency for Technology and Innovation.

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