Enhanced detection sensitivity of prostate-specific antigen via PSA-conjugated gold nanoparticles based on localized surface plasmon resonance: GNP-coated anti-PSA/LSPR as a novel approach for the identification of prostate anomalies.

Prostate-specific antigen (PSA) is used to screen for prostate disease, although it has several limitations in its application as an organ-specific or cancer-specific marker. Furthermore, a highly specific/sensitive and/or label-free identification of PSA still remains a challenge in the diagnosis of prostate anomalies. We aimed to develop a gold nanoparticle (GNP)-conjugated anti-PSA antibody-based localized surface plasmon resonance (LSPR) as a novel approach to detect prostatic disease. A total of 25 nm colloidal gold particles were prepared followed by conjugation with anti-PSA pAb (GNPs-PSA pAb). LSPR was used to monitor the absorption changes of the aggregation of the particles. The size, shape and stability of the GNP-anti-PSA were evaluated by dynamic light scattering transmission electron microscopy (TEM) and zetasizer. The GNPs-conjugated PSA-pAb was successfully synthesized and subsequently characterized using ultraviolet absorption spectroscopy and TEM to determine the size distribution, crystallinity and stability of the particles (for example, stability of GNP: 443 mV). To increase the stability of the particles, we pegylated GNPs using an N-(3-dimethylaminopropyl)-N*-ethylcarbodiimide hydrochloride (EDC)/N-hydroxylsuccinimide (NHS) linker (for example, stability of GNP after pegylation: 272 mV). We found a significant increase in the absorbance and intensity of the particles with extinction peak at 545/2 nm, which was shifted by ~1 nm after conjugation. To illustrate the potential of the GNPs-PSA pAb to bind specifically to PSA, LSPR was used. We found that the extinction peak shifted 3 nm for a solution of 100 nM unlabeled antigen. In summary, we have established a novel approach for improving the efficacy/sensitivity of PSA in the assessment of prostate disease, supporting further investigation on the diagnostic value of GNP-conjugated anti-PSA/LSPR for the detection of prostate cancer.

Preparation, purification and virus inactivation of intravenous immunoglobulin from human plasma.

IVIG can be prepared from fractionation of normal human plasma and it is used as a therapeutic drug for treatment of various diseases. IVIG has been for some time the high-growth product within the plasma derived products, at both a global and a national country level. Fractionation was performed according to Cohn method with some modifications. Fraction II was first produced and then it was used for purification and virus inactivation steps. Two methods of virus inactivation (pasteurization at 60 degrees C for 10 hours and solvent/detergent treatment with TnBP and Tween 80) were used and validated. A chromatography method (cation exchange chromatography on CM Sepharose FF) was also added to obtain high purity. The final product (in liquid and freeze dried formulation) meets European Pharmacopeias requirements. The amount of PKA and aggregates was beyond the acceptance limit. The intactness of the IVIG was also examined by circular dichroism (secondary and tertiary structure). It was stable after 6 months of storage. Since Iran market is completely dependant on importation of plasma derived products, it is important to develop such methods for production of IVIG to obtain regional demands.

Inactivation of virus in intravenous immunoglobulin G using solvent/detergent treatment and pasteurization.

The safety of plasma derived medicinal products, such as immunoglobulin, depends on viral inactivation steps that are incorporated into the production process. Several attempts have been made to validate the effectiveness of these inactivation methods against a range of physio-chemically diverse viruses. Treatment with solvent/detergent (S/D) and pasteurization (P) has been continuously used in our IgG production and these methods were analysed in this study as models of viral inactivation. Bovine Viral Diarrhoea Virus (BVDV), Herpes Simplex Virus (HSV) and Vesicular Stomatitis Virus (VSV) were employed as models of HCV, HBV and HIV respectively. Polio and Reo viruses also were used as stable viruses to chemical substances. The infectivity of a range of viruses before and after treatment with two methods of viral inactivation was measured by end point titration and their effectiveness expressed as Logarithmic Reduction Factors (LRF). Solvent/detergent treatment reduced the amount of enveloped viruses by 5-6 logs. The reduction factor was between 5-6 logs for all viruses used in the pasteurization process. A final log reduction factor was obtained as the sum of the two individual methods. Both inactivation methods have advantages and disadvantages with respect to their ability to inactivate viruses. Thus,combination of two robust virus inactivation steps, solvent/detergent and pasteurization, increases the safety margin of immunoglobulin preparations.

Evaluation of the homing of human CD34+ cells in mouse bone marrow using clinical MR imaging.

There have been several reports using various superparamagnetic iron oxide (SPIO) nanoparticles to label mammalian cells for monitoring their temporal and spatial migration in vivo by Magnetic Resonance Imaging (MRI). The purpose of this study was to evaluate the efficiency of labeling cells using two commercially available FDA-approved agents: ferumoxide, a suspension of dextran-coated SPIO used as an MRI contrast agent and protamine sulfate used ex vivo as a cationic transfection agent to evaluate the use of clinical 1.5 Tesla magnetic resonance imaging equipment in showing the in vivo homing of iron oxide-labeled human CD34+ cells in irradiated mouse. After labeling human Hematopoietic (CD34+) stem cells with ferumoxide-protamine sulfate complex (FE-Pro), cellular toxicity, functional capacity and quantitative cellular iron incorporation were determined. FE-Pro labeled cells demonstrated neither short or long-term toxicity nor changes in colonogenic assay of the stem cells and their phenotype when compared with unlabeled cells. Efficient labeling with FE-Pro was observed with iron content per cell varying between 1.91 +/- 0.1 pg for CD34+ cells with 100% of cells labeled. After irradiation, Female Balb/c mice underwent MR imaging before and 12, 24, 48 and 72 h after intravenous injection of 0.5-1.5 x 10(7) labeled CD34+ cells. A significant decrease in MR signal intensity was observed in bone marrow at 24 and 48 h after injection. Our observations confirm that efficient labeling of cells with appropriate contrast agents should facilitate the translation of this method to clinical trials for evaluating the trafficking of infused or transplanted cells by MR Imaging.