Homogeneous Fluorescence-Based Immunoassay Detects Antigens Within 90 Seconds

Abstract

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.

Novel peptide–protein assay for identification of antimicrobial peptides by fluorescence quenching

“…2c). The corresponding quenching curves of the modified oncocin peptides showed a binding to the protein in the same range as oncocin (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11) and oncocin (12)(13)(14)(15)(16)(17)(18)(19) (Fig. 2c, d).…”

“…Fig. 3 Correlation between the determined K d values of the investigated peptide-DnaK interactions using the quenching assay and the FP assay; error bars show a confidence interval of 95 % (n03) Table 2 Analysis of significant differences in the quenching effects of analyte peptides compared to the negative control apidaecin 1b (9)(10)(11)(12)(13)(14)(15)(16)(17)(18) in the prescreening test using a one-way ANOVA test (significance level, 0.01 %) and determined K d values (quenching assay) …”

“…As reported previously, the Black Hole Quencher 10™ (BHQ10) is able to efficiently quench the fluorescence of fluorescein due to a nearly complete overlap of the fluorescein emission spectrum with the absorption spectrum of BHQ10 [15]. As a consequence, the peptides used in the quenching assay developed here were modified with 5(6)-carboxyfluorescein at the N-terminus.…”

“…2b). Furthermore, the native oncocin showed a strong binding towards DnaK whereas the N-and C-terminal fragments oncocin (1-11) and oncocin (12)(13)(14)(15)(16)(17)(18)(19) displayed weaker binding, although these truncated analogues still afforded significant differences compared to the control peptide apidaecin 1b (9-18) (Fig. 2c).…”

“…After dialysis of DnaK against the modification buffer and determination of the protein concentration with a Micro BCA Protein Assay (Thermo Scientific, Bonn, Germany), the DnaK was diluted to a concentration of 2 g/L and then labeled with a 10-fold molar excess of BHQ10-NHS-ester [15]. After further dialysis to remove excess BHQ10, the protein concentration was determined as above.…”