FRET Assay Histag

FRET immunoassay

AntiHis antibody: ‘‘8-4-4’’, is  labeled with Black hole quencher 10 (BHQ-10) as an acceptor dye for EuLH

His-peptide that is labeled with the phosphorescent donor dye EuLH10 - a phenanthroline-based europium chelate

His6 samples (TEV-His6, 50 mL, dissolved in E. coli cell
lysate or buffer) were premixed with donor peptide (10 mL, 40 nmol/L)
and then mixed with BHQ-10-labeled anti-His-tag mAb ‘‘8-4-4’’ (40 mL,
80 nmol/L). Phosphorescence intensity was measured after a 90 s shaking
step.

One of the fastest sensors
described so far, is a label free His-tag sensor chip based on the
interaction with Ni-NTA and surface-plasmon resonance with a
total assay time of approx. 12 min3. A similar approach to this
FRET-based homogenous assay are the sensitive HTRFH assays from
Cisbio, France, using an europium cryptate as TRF donor coupled to
an anti-His-tag antibody and the phycobiliprotein XL665 coupled to
a His6 peptide.

However, the product
description13 suggests a similar sensitivity, but the HTRF assay needs
a rather long incubation time of 2 h at reduced temperature and is
therefore not suitable as a mix-and-measure assay. The detection
limit of the developed assay in the micro- to nanomolar range compares
well with other His-tag sensors. Darain et al. introduced an
approach which uses a label-free impedance-based sensor with a
detection limit of 1.5 mmol/L14. In addition, there are also His-tagassays
with sensitivities in the subpicomolar range as described by
Wasowicz et al. using gold nanorods and surface plasmon resonance
or impedance spectroscopy as detection system15,16. Another
example is an antibody-free approach using a chemiresistive sensor17.
While these detection systems are more sensitive than the assay
described here, the assay times are in the range of 10–30 min. This is
indeed faster than SDS-PAGE or ELISA techniques, but in our opinion
still not fast enough to be able to screen protein fractions during
the process of purification. Furthermore, a detection limit in the
lower mg/L-range as obtained for this assay compares well with
typical concentrations of expressed proteins in lysed cell medium14.

The unique properties of the used lanthanide dye EuLH, i.e.
that it can be excited in the near-Vis region at 360 nm, enables the
use of simple and cheap glass optics instead of quartz glass for the
measurement10. The use of a His10- instead of a His6- tag could
potentially improve the detection limit of the assay described here
due to a better sterical accessibility of the epitope.

Peptide Synthesis. The peptides used in the homogeneous assay and for His6 protein
labeling were synthesized as C-amides on MBHA resin using standard solid-phase
synthesis. EuLH10 and carboxyfluorescein as donor in the homogeneous assay were
incorporated to the free N-terminus via a free carboxyl group using the same reaction
conditions as for each amino acid coupling before. All peptides were cleaved from the
synthesis resin with TFA and finally purified via RP-HPLC and characterized using
MALDI-MS.
Protein Labeling. For labeling of proteins (BSA, phosphorylase B and casein) with
His6, the protein was firstly maleoylated with SMPH and secondly labeled with an
excess of 9 eq His6-peptide with an N-terminal cysteine side chain (CHHHHHHNH2).
Unreacted linker and peptide were removed by dialysis (MWCO 10,000,
Sigma-Aldrich, Taufkirchen, Germany). Protein concentration was determined using
absorption at 280 nm. For the homogeneous assay, the ‘‘8-4-4’’ antibody was labeled
with BHQ-10 succinimidyl ester. Unreacted dye was removed via SEC on a HiTrap
desalting column (5 mL, GE Healthcare, Munich, Germany).
Antibody generation. The monoclonal mouse anti-His-tag antibody ‘‘8-4-4’’ was
generated in collaboration with Biogenes (Berlin, Germany). Murine hybridoma cells
(SP2/0) producing k-IgG1 were cultured in Dulbecco’s Modified Eagle Medium
(DMEM) containing 15% fetal bovine serum, 1% non-essential amino acids, 1%
penicillin-streptomycin, and 50 mmol/L beta-mercaptoethanol. The antibody from
cell culture supernatant was purified by affinity chromatography with protein Gsepharose
(GE Healthcare).
 

Homogeneous assay. For the homogeneous assay, analyte solutions (50 mL, dissolved
in assay buffer: PBS pH 7.4 with 2 mg/mL BSA, or E. coli cell lysate) were premixed
with donor peptide (EuLH-HHRHH-NH2, 10 mL, 40 nmol/L in assay buffer), then
BHQ-10-labeled ‘‘8-4-4’’ anti-His-tag antibody was added (40 mL, 80 nmol/L in assay
buffer). Phosphorescence intensity was recorded using the TRF-EuSa-Cartridge (lEx
5 370 nm, 80 nm bandwidth, lEm 5 616 nm, 10 nm bandwidth) in a SpectraMax
Paradigm plate reader (Molecular Devices) after a 90 s shaking step. The assay was
performed in white, non-binding 384-well plates with flat bottom (Greiner Bio-One).
All experiments were carried out as triplicates. The detection limit (LOD) was
determined to be the lowest tested concentration with a mean signal higher than the
background control with c 5 0 plus 3x its standard deviation.