Thomas Kreisig

Homogeneous Fluorescence-Based Immunoassay Detects Antigens Within 90 Seconds

Homogeneous immunoassays are prevalent tools for the detection of antigens. The major advantage over heterogeneous immunoassays is the absence of numerous incubation and washing steps, reducing the assay time and allowing rapid on-site detection of antigens (e.g., toxins and pollutants). The simple experimental setup of a homogeneous immunoassay also allows a robust analysis even when performed by non-laboratory-trained personnel. Here we present a homogeneous immunoassay for the rapid determination of antigens. As a proof of concept, a phosphorylation-specific anti-human tau monoclonal antibody was labeled with an acceptor and the corresponding peptide probe with a donor fluorophore. The analyte sample is spiked with a fixed amount of donor peptide before acceptor-labeled antibody is added leading to a donor fluorescence quenching. Thus the intensity of the fluorescence signal of the donor peptide probe depends on the concentration of the target antigen. The sequence of the donor peptide was optimized to lower its affinity to the antibody giving a higher response for the analyte antigen compared to the native epitope. This allowed a semiquantitative analysis of the antigen within only 90 s.

A luminescence-based probe for sensitive detection of hydrogen peroxide in seconds.

Here, we present a fast and simple hydrogen peroxide assay that is based on time-resolved fluorescence. The emission intensity of a complex consisting of terbium ions (Tb(3+)) and phthalic acid (PA) in HEPES buffer is quenched in the presence of H2O2 and this quenching is concentration-dependent. The novel PATb assay detects hydrogen peroxide at a pH range from 7.5 to 8.5 and with a detection limit of 150 nmol L(-1) at pH 8.5. The total assay time is less than 1 min. The linear range of the assay can be adapted by a pH adjustment of the aqueous buffer and covers a concentration range from 310 nmol L(-1) to 2.56 mmol L(-1) in total which encompasses four orders of magnitude. The assay is compatible with high concentrations of all 47 tested inorganic and organic compounds. The PATb assay was applied to quantify H2O2 in polluted river water samples. In conclusion, this fast and easy-to-use assay detects H2O2 with high sensitivity and precision.

His-tag protein monitoring by a fast mix-and-measure immunoassay

Here, we present a fast mix-and-measure immunoassay for the specific semiquantitative detection of His-tagged proteins, for example in E. coli cell lysate. The assay is based on Förster resonance energy transfer (FRET) between a lanthanide dye-labeled low-affinity His-peptide and an acceptor-labeled anti-His-tag antibody. The targeted His-tag protein in the sample displaces the donor-labeled peptide and leads to a concentration-dependent time-resolved fluorescence signal. The assay has a total assay time of less than two minutes including sample preparation. The assay recognizes both, N- and C-terminally tagged proteins. The detection limit is comparable to those obtained in SDS-PAGE or Western Blot, which are used as standard methods for the characterization of His-tag protein expression. Additionally, we demonstrate a full compatibility of the developed assay to cell lysate, and a correlation to detectable bands in a western blot application. In conclusion, this fast, sensitive, specific and affordable mix-and-measure assay provides a timesaving and user-friendly way to quantify recombinant protein expression. It substantially reduces the workload for recombinant protein detection, especially when His-tag-protein-containing fractions in manual chromatographic purifications have to be identified.

Highly Efficient Förster Resonance Energy Transfer in a Fast, Serum‐Compatible Immunoassay

Highly efficient FRET leads to important enhancements for homogeneous immunoassays. By using the novel phosphorescent dye EuLH and BHQ-10 as a donor-acceptor pair, the FRET efficiency increases to >99.5 %, leading to significantly improved signal-to-background ratio, precision and linear range. The phosphorescence detection enabled full compatibility to serum samples for this fast-responding immunoassay.

A homogeneous assay principle for universal substrate quantification via hydrogen peroxide producing enzymes.

H2O2 is a widely occurring molecule which is also a byproduct of a number of enzymatic reactions. It can therefore be used to quantify the corresponding enzymatic substrates. In this study, the time-resolved fluorescence emission of a previously described complex consisting of phthalic acid and terbium (III) ions (PATb) is used for H2O2 detection. In detail, glucose oxidase and choline oxidase convert glucose and choline, respectively, to generate H2O2 which acts as a quencher for the PATb complex. The response time of the PATb complex toward H2O2 is immediate and the assay time only depends on the conversion rate of the enzymes involved. The PATb assay quantifies glucose in a linear range of 0.02-10 mmol L(-1), and choline from 1.56 to 100 μmol L(-1) with a detection limit of 20 μmol L(-1) for glucose and 1.56 μmol L(-1) for choline. Both biomolecules glucose and choline could be detected without pretreatment with good precision and reproducibility in human serum samples and infant formula, respectively. Furthermore, it is shown that the detected glucose concentrations by the PATb system agree with the results of a commercially available assay. In principle, the PATb system is a universal and versatile tool for the quantification of any substrate and enzyme reaction where H2O2 is involved.

Protein surface charge of trypsinogen changes its activation pattern

BackgroundTrypsinogen is the inactive precursor of trypsin, a serine protease that cleaves proteins and peptides after arginine and lysine residues. In this study, human trypsinogen was used as a model protein to study the influence of electrostatic forces on protein–protein interactions. Trypsinogen is active only after its eight-amino-acid-long activation peptide has been cleaved off by another protease, enteropeptidase. Trypsinogen can also be autoactivated without the involvement of enteropeptidase. This autoactivation process can occur if a trypsinogen molecule is activated by another trypsin molecule and therefore is based on a protein–protein interaction.ResultsBased on a rational protein design based on autoactivation-defective guinea pig trypsinogen, several amino acid residues, all located far away from the active site, were changed to modify the surface charge of human trypsinogen. The influence of the surface charge on the activation pattern of trypsinogen was investigated. The autoactivation properties of mutant trypsinogen were characterized in comparison to the recombinant wild-type enzyme. Surface-charged trypsinogen showed practically no autoactivation compared to the wild-type but could still be activated by enteropeptidase to the fully active trypsin. The kinetic parameters of surface-charged trypsinogen were comparable to the recombinant wild-type enzyme.ConclusionThe variant with a modified surface charge compared to the wild-type enzyme showed a complete different activation pattern. Our study provides an example how directed modification of the protein surface charge can be utilized for the regulation of functional protein–protein interactions, as shown here for human trypsinogen.