Concentration and preservation of very low abundance biomarkers in urine, such as human growth hormone (hGH), by Cibacron Blue F3G-A loaded hydrogel particles

Abstract

Urine is a potential source of diagnostic biomarkers for detection of diseases, and is a very attractive means of non-invasive biospecimen collection. Nonetheless, proteomic measurement in urine is very challenging because diagnostic biomarkers exist in very low concentration (usually below the sensitivity of common immunoassays) and may be subject to rapid degradation. Hydrogel nanoparticles functionalized with Cibacron Blue F3G-A (CB) have been applied to address these challenges for urine biomarker measurement. We chose one of the most difficult low abundance, but medically relevant, hormones in the urine: human growth hormone (hGH). The normal range of hGH in serum is 1 to 10 ng/mL but the urine concentration is suspected to be a thousand times less, well below the detection limit (50 pg/mL) of sensitive clinical hGH immunoassays. We demonstrate that CB particles can capture, preserve and concentrate hGH in urine at physiological salt and urea concentrations, so that hGH can be measured in the linear range of a clinical immunometric assay. Recombinant and cadaveric hGH were captured from synthetic and human urine, concentrated and measured with an Immulite chemiluminescent immunoassay. Values of hGH less than 0.05 ng/mL (the Immulite detection limit) were concentrated to 2 ng/mL, with a urine volume of 1 mL. Dose response studies using 10 mL of urine demonstrated that the concentration of hGH in the particle eluate was linearly dependent on the concentration of hGH in the starting solution, and that all hGH was removed from solution. Thus if the starting urine volume is 100 mL, the detection limit will be 0.1 pg/mL. Urine from a healthy donor whose serum hGH concentration was 1.34 ng/mL was studied in order detect endogenous hGH. Starting from a volume of 33 mL, the particle eluate had an hGH concentration of 58 pg/mL, giving an estimated initial concentration of hGH in urine of 0.175 pg/mL. The nanotechnology described here appears to have the desired precision, accuracy and sensitivity to support large scale clinical studies of urine hGH levels. Nano Research503 Nano Res (2008) 1:502 518

Application of Analyte Harvesting Nanoparticle Technology to the Measurement of Urinary HGH in Healthy Individuals

“…The affinity ranges from K D = 10 −9 to 10 −12 M. We selected the bait that had the highest affinity, which was Remazol Brilliant Blue R (RBB, Figure 1). This previously unpublished bait for HGH had an affinity ten times greater than Cibacron Blue F3GA [12,13] (Figure 2). …”

“…Nanoparticles are added to the urine samples to concentrate the analyte into a small volume, which can then be eluted and measured by a clinical grade standard immunoassay designed for serum ( Figure 1). The nanoparticles can rapidly harvest, protect from degradation, and concentrate, low abundance analytes from biologic fluids while actively excluding high abundance proteins such as albumin [10][11][12][13].…”

Investigation of the Ovarian and Prostate Cancer Peptidome for Candidate Early Detection Markers Using a Novel Nanoparticle Biomarker Capture Technology

“…Particles were synthesized as previously described (26)(27)(28) using NIPAm (Sigma-Aldrich) and BIS (Sigma-Aldrich) by precipitation polymerization. Acrylic acid (AAc; SigmaAldrich) was incorporated into NIPAm particle to provide a charge-based affinity moiety bait for affinity capture of peptides and small molecules…”

“…The dried-up samples were stored at −20°C until use. Eluted proteins from the nanoparticles were reduced by dithiothreitol, alkylated by iodoacetamide, and digested by trypsin overnight at 37°C as described (27).…”

Microspheres Containing Cibacron Blue F3G-A and Incorporated Iron Oxide Nanoparticles as Biomarker Harvesting Platforms

“…The incorporation of Cibacron Blue F3G-A in pNIPAm-co-AA core particles has been previously described [14]. The protocol described here addresses the incorporation of Cibacron Blue F3G-A in the amine functionalized magnetic hydrogel particles.…”