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 becau...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.展开更多
A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed. Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the ...A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed. Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the central compartments. The presence of an electric field accelerated the migration of proteins inside the gel matrix during adsorption and desorption processes. This led to the increase of the overall speed of separation. The present study was focused on the effect of the strength of the electric field on adsorption and desorption processes.展开更多
基金This work was partly supported by the Italian Istituto Superiore di Sanita’in the framework of the Italy/USA cooperation agreement between the U.S.Department of Health and Human Services,George Mason University and the Italian Ministry of Public HealthThis work was partially supported by the U.S.Department of Energy grant number DE-FC52-04NA25455。
文摘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.
基金Supported by the State Key Projects(No.96c-03-04-05).
文摘A new kind of electrophoretic affinity chromatography (EAC) for bioseparation was proposed. Separation by EAC was conducted in a multicompartment electrolyzer in which the affinity gel media were packed in one of the central compartments. The presence of an electric field accelerated the migration of proteins inside the gel matrix during adsorption and desorption processes. This led to the increase of the overall speed of separation. The present study was focused on the effect of the strength of the electric field on adsorption and desorption processes.
