Editing FRET Assay Histag

FRET immunoassay

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

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



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



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



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

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



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



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