We recently described a novel homogeneous assay principle based on upconversion fluorescence resonance energy transfer (UC-FRET), where an upconverting phosphor (UCP) is utilized as a donor. The UC-FRET has now been applied to a competitive homogeneous immunoassay for 17β-estradiol (E2) in serum, using a small-molecular dye as an acceptor. The assay was constructed by employing an UCP coated with an E2-specific recombinant antibody Fab fragment as a donor and an E2-conjugated small-molecular dye, Oyster-556, as an acceptor. Standard curves for the assay were produced both in buffer and in male serum. Sensitized acceptor emission was measured at 600 nm under continuous laser diode excitation at 980 nm. In buffer, the IC50 value of the assay was 1 nM and in serum 3 nM. The lower limits of detection (mean of zero calibrators, 3 SD) were 0.4 and 0.9 nM, respectively. The measurable concentration range extended up to 3 nM in buffer and 9 nM in serum. Equilibrium in the assay was reached in 30 min. The novel pr...

Upconversion Fluorescence Resonance Energy Transfer in a Homogeneous Immunoassay for Estradiol

Development of a screen-printed carbon electrochemical immunosensor for picomolar concentrations of estradiol in human serum extracts.

A flow-based immunoassay that uses microspheres as the solid phase accomplished the theoretical limit of detectability achievable with the antibody. An equilibrated mixture of anti-estriol monoclonal antibody and estriol was briefly exposed to a bead pack containing immobilized estriol in a flow cell. A small portion of free antibody was separated rapidly from the mixture by binding it to immobilized hormone, but the antibody-hormone complex was kinetically excluded from binding. This rapid separation prevented shift in the equilibrium of the liquid phase binding. Signals were generated by labeling the separated antibodies on the beads with a Cy5-conjugated anti-species secondary antibody. By labeling after the separation step, perturbing the liquid-phase or solid-phase binding was prevented. This assay allowed the reduction of the concentration of primary antibody by continuously accumulating free antibody onto the beads prior to quantification and, thus, offered ideal conditions to achieve theoretical l...

An immunoassay for small analytes with theoretical detection limits.

Development and validation of a simple and direct ELISA method for the determination of conjugated (glucuronide) and non-conjugated testosterone excretion in urine.

EGFRvIII is the type III deletion mutant form of the epidermal growth factor receptor (EGFR) with transforming activity. This tumor-specific antigen is ligand independent, contains a constitutively ac

Production and characterization of a new antibody specific for the mutant EGF receptor, EGFRvIII, in Camelus bactrianus.

Enzyme immunosorbant assay of oestradiol in unextracted plasma using penicillinase as label

A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes

P.K. Grover

Papers

Enzyme immunosorbant assay of oestradiol in unextracted plasma using penicillinase as label

A highly specific heterologous enzyme-linked immunosorbent assay for measuring testosterone in plasma using antibody-coated immunoassay plates or polypropylene tubes