Anti phospho-(tyrosine)-peptide binding immunoassay

Fig. 1: Schematic of FRET immunoassay competition assay.

The desire to measure specific antibody-antigen interactions has resulted in a search for appropriate technologies to satisfy the demands of the immunodiagnostic sector. In particular, there has been interest in homogenous fluorescence immunoassays(1). To reflect this requirement a Fluorescence Resonance Energy Transfer (FRET) immunoassay has been developed. The assay comprises a Cy™5 N-terminally labeled phosphopeptide (a 13-mer and a substrate for src kinase) which is recognised by an anti-phosphotyrosine primary mouse antibody, followed by a Cy3 labeled secondary antibody.

Energy transfer occurs when the components are sequentially bound together and excitation at Cy3 wavelengths produces emission at Cy5 wavelengths. Disruption of the interaction between the phosphopeptide and primary antibody using an excess of unlabeled specific phophopeptide will result in a reduction of the FRET signal observed (Figure 1).

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Methods

Assays performed in black opaque 384-well (maximum working volume 80 ml) microtitre plates (Greiner Labortechnik GmbH, Frickenhausen, Germany) contained 25 ml of 3.9 to 62.5 ng/ml 13-mer Cy5-labeled phosphopeptide (final assay concentration of 0.098 to 1.56 ng/well ) with 12.5 ml of a 20mg/ml preparation of the 4G10-anti-phosphotyrosine (Upstate Biotechnology, Lake Placid, NY, USA. catalog number; 05-321) antibody (final assay concentration 0.25 µg/well), all dilutions carried out in assay buffer, 50 mM Hepes, pH 8.5 containing 10 mM MgCl₂, 0.1% BSA and 0.005% Triton-X 100. Following incubation for 2 hours at room temperature, 12.5 ml of a 25mg/ml preparation of Cy3-labeled anti-mouse secondary antibody (final assay concentration of 0.25mg/well) was added to give a final assay volume of 50 ml. Reactions were left to proceed for a further 2 hours prior to imaging in LEADseeker™ Homogeneous Imaging System for 40 seconds at 540 nm excitation and 690 nm emission wavelengths. A well-by-well correction method was used with 5 standard concentrations of between 0 and 100 nM. The standard used for the corrections was the Cy3-Cy5 calibrator.

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Results

Figure 2: Binding of Cy5 phosphopeptide in homogeneous FRET immunoassay

Binding of the Cy5-labeled phosphopeptide to the Cy3-labeled secondary antibody, mediated by the anti-phosphotyrosine antibody, is shown in Figure 2. Values are means (n=2) and are expressed as nominal signal units obtained either for the Cy5 phosphopeptide alone (—), for the Cy5 phosphopeptide in the presence of the Cy3 labeled secondary antibody (—) and for the binding assay between these two fluor labeled components mediated by the anti phosphotyrosine antibody (—). For reference maximum signal:noise levels achieved were in the order 2:1 (expressed as specific FRET signal above the Cy3 and Cy5 background signals alone).

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References

1. WU, P.G. and BRAND, L.
Resonance energy transfer – methods and applications.
Anal. Biochem., 218, 1-13, (1994).

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