Development and evaluation of an immuno-MALDI (iMALDI) assay for angiotensin I and the diagnosis of secondary hypertension

Abstract

Plasma renin activity (PRA) is an essential analytical tool for screening and diagnosis of secondary forms of hypertension. Typically, PRA is measured by competitive radioimmunoassay, but there are significant drawbacks to this technique including non-specificity, long analysis times, narrow calibration range, and the requirement for radionucleotides. In this paper, we report a method for plasma renin activity determination by immuno-MALDI mass spectrometry detection. This method overcomes the issues of non-specificity and long analytical times present with RIA, and does not require the use of radionucleotides. As an initial methodological evaluation, plasma renin activity results obtained by radioimmunoassay, LC/ESI-MS/MS, and immuno-MALDI on 64 samples from an outpatient primary aldosteronism screening program have been compared. A strong correlation was found between immuno-MALDI and radioimmunoassay (R2 = 0.9412, 62/64 within the 95% CI of the Bland-Altman plot), and iMALDI and LC/ESI-MS/MS (R2 = 0.9471, 62/64 within the 95% CI of the Bland-Altman plot). Technical replicates showed a 4.8% CV, while inter- and intra-day replicates showed CVs of 17.3% and 17.2% respectively. We have developed an assay capable of measuring PRA without the use of radionucleotides. This immuno-MALDI approach affords the specificity of MS while avoiding the long analytical run times and technical problems associated with HPLC. With the use of robotic sample preparation to optimize precision, this assay should be adaptable to clinical environments.

Figure 1

iMALDI workflow. Stable-isotope labelled internal standard (SIS)-Ang-I (green) is spiked into plasma and the sample is incubated with anti-Ang-I-antibody conjugated beads. After immunoprecipitation of endogenous (blue) and SIS-Ang-I the beads can be placed directly on a MALDI target. To elute the analytes and permit MALDI-MS analysis, CHCA matrix is applied. The relative abundances of endogenous to SIS-Ang-I are used for quantitation.

Figure 2

Correlation of RIA with LC/ESI-MS/MS. PRA was determined by RIA and LC/ESI-MS/MS for 64 patients. The correlation is shown by Passing and Bablok regression as well as a Bland Altman difference plot. Shaded regions represent a 95% confidence interval.

Figure 3

Correlation of iMALDI with RIA and LC/MS/MS. A) Difference plot comparisons for RIA and iMALDI. B) Difference plot comparison for LC/ESI-MS/MS and iMALDI.

Figure 4

The determination of PRA by iMALDI. A) A standard curve is created from antibody captures of natural and SIS peptides. B) MALDI spectra showing capture of natural and SIS Ang-I in a 4°C sample. C) MALDI spectra showing capture of natural and SIS Ang-I in a 37°C sample.