A novel bead-based fluorescence immunoassay for aldosterone

Abstract

Aldosterone quantification helps evaluate the rennin-angiotensin-aldosterone system. The new bead-based multiplex platform has not been applied in aldosterone detection to achieve simultaneous measurements of multiple hormones. A new sensitive competitive bead immunoassay based on Luminex technology for detecting aldosterone in small sample volumes was developed using two-antibody coupled beads and biotinylated aldosterone as tracer in combination with an extraction step. The assay was validated in human and mouse samples and exhibited a linear working range from 10 to 1,000 pg/mL. The assay was reproducible and precise with intra-assay coefficient of variations (CVs) from 6.0% to 11.2%, inter-assay CVs from 8.0% to 13.0% and good recovery [(90-110)%] and linearity [(89-107)%]. Excellent correlation was found between this new assay and the reference method (r = 0.96, P < 0.000,1). The successful establishment of this assay provides high possibility for carrying out bead-based multiplex assay measuring aldosterone and other parameters simultaneously in one 50 µL sample so that the efficiency can be improved and precious samples can be saved.

Keywords: aldosterone; bead; extraction; immunoassay; multiplex.

Fig. 1. Titration of rabbit anti-mouse IgG coupled to beads.

The median fuorescence intensity (MFI) generated by the biotinylated anti-rabbit IgG antibody was used to demonstrate the relative amount of the aimed antibody coupled to the beads. MFI increases with the amount of rabbit anti-mouse IgG added to each scale of beads and reaches the plateau when 12 µg is added.

Fig. 2. Titration of anti-aldosterone antibody.

Standard curves for the displacement of aldosterone obtained by different titrations of anti-aldosterone antibody used for the second step of bead coupling. The curves show median fuorescence intensity (MFI) at maximal binding when no unlabeled aldosterone is present in the calibrators and decreasing counts with reduced binding due to increasing concentrations of unlabeled aldosterone competing for binding.

Fig. 3. Titration of biotinylated aldosterone (tracer).

Displacement of calibration curves obtained by different titrations of biotinylated aldosterone added into the assay. Y axis is median fuorescence intensity (MFI) (A) and B/B0 (B), respectively. B/B0 is the percentage of MFI at each point compared with that of calibrators zero. The arrows show the ED50 of each calibration curve.

Fig. 4. Impact of antigen and antibody incubation temperature and time on standard curves.

Fig. 5. Correlation between bead-based assay and a TR-FIA.

A: Comparison of aldosterone in 82 extracted human plasma samples measured by bead-based assay and the reference in-house TR-FIA. B: Bland-Altman plots of the same data.