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

Abstract

Urine represents a valuable biofluid for noninvasive measurement of Human Growth Hormone (HGH) secretion. Unfortunately, currently available commercial HGH immunoassays do not achieve the sensitivity needed for urinary HGH measurement in the low picogram per milliliter range, the expected normal concentration range of HGH in urine. A nanotechnology based sample preprocessing step was used to extract and concentrate HGH in urine so that urinary HGH could be measured with a clinical grade standard immunoassay designed for serum (Immulite 1000, Siemens). We applied the nanoparticle enhanced immunoassay to evaluate the baseline value of urinary HGH in a population of healthy young adults (age 18-30, N=33, median 21, M: F=39%:61%, with no reported medical therapies). Nanoparticle sample preprocessing effectively improved the lower limit of detection of the Immulite HGH assay by more than 50 fold, shifting the linear range of the assay to encompass the expected value of urinary HGH. The full process between run and within run CV% was 7.9 and 9.0%, respectively. On 33 healthy volunteers, the 95% reference values for hGH in spot urine normalized to specific gravity were 0.64 - 16.85 pg/mL (0.05-5.82 ng/g creatinine). Nanoparticle preprocessing constitutes a reliable means of measuring urinary HGH with a clinical grade immunoassay, now establishing a normal baseline value for HGH in urine. Nanoparticles can be used to study the kinetics of HGH excretion in urine, and the factors that influence urinary HGH secretion and HGH isoform proportions.

Keywords: Human growth hormone; Immunoassay; Nanoparticles; Reference values; Urine.

Figure 1

Capturing hydro gel nanoparticles pre-processing step. Well disperse and buoyant particles are introduced in the urine and all the solution phase Human Growth Hormone (HGH) is sequestered. Particles are separated from the urine and captured HGH is eluted and analyzed with a sandwich immunoassay.

Figure 2

Dissociation constant (K_D) values calculated for nine organic chemical baits assuming hGH-bait equimolar binding. AB22 = Acid blue 22, RBB = Remazol brilliant blue R, AB1 = Acid black 1, CB = Cibacron blue F3GA, DO3 = Disperse orange 3, PRR = Pararosaniline base, R250 = Brilliant blue R250, R123 = Rhoda mine 123, AAc = acrylic acid.

Figure 3

A) Atomic force microscope images demonstrate uniformity of the spherical poly (NIPAm/RBB) nanoparticles. B) Nanoparticle-Immulite GH assay has a within run CV = 9% (over ten replicates) and virtually 100% yield. Mean +/− CV. IS = initial solution, S = Supernatant, E = nanoparticle eluate, C100% = calculated GH concentration based on 100% yield. C) And D) Linearity of the immunoassay (9 calibrator values, range 0.750-0.05 ng/mL) with and without nanoparticle preprocessing, respectively. Nanoparticle preprocessing maintains the linearity of the immunoassay (R²=0.986, 0.982, with and without the nanoparticles, respectively).