Actual Biological Samples Research Articles

Colorimetric heparinase assay for alternative anti-metastatic activity

Heparanase has been previously associated with the metastatic potential, inflammation, and angiogenesis of tumor cells. Heparanase activity has been detected by means of UV absorption, radiolabeled substrates, electrophoretic migration, and heparan sulfate affinity assays. However, those methods have proven to be somewhat problematic with regards to application to actual biological samples, the accessibility of the immobilized substrates, experimental sensitivity, and the separation of degraded products. Rather than focusing on heparanase activity, then, we have developed a rapid, alternative colorimetric heparinase assay, on the basis of the recent finding that sulfated disaccharides generated from heparin by bacterial heparinase exhibit biological properties comparable to those from heparan sulfate by mammalian heparanase. In this study, the concentrations of porcine heparin and bacterial heparinase I were determined using a Sigma Diagnostics Kit. Morus alba was selected as a candidate through this assay system, and an inhibitor, resveratrol, was purified from its methanol extract. Its anti-metastatic effects on the pulmonary metastasis of murine B16 melanoma cells were also evaluated. Our findings suggest that this assay may prove useful as a diagnostic tool for heparinase inhibition, as an alternative anti-metastatic target.

Determination of nucleic acids at nanogram level using resonance light scattering technique with Congo Red

Based on the enhancement of the resonance light scattering (RLS) of Congo Red (CR) by nucleic acid, a new quantitative method for nucleic acid is developed. In the Tris–HCl buffer (pH 10.5), the weak light scattering of CR is greatly enhanced by addition of nucleic acid and CTMAB, the maximum peak is at 560 nm and the enhanced intensity of RLS is in proportion to the concentration of nucleic acid. The linear range is 1.0×10 −9 to 1.0×10 −6 g ml −1, 7.5×10 −8 to 1.0×10 −6 g ml −1 and 7.5×10 −8 to 2.5×10 −6 g ml −1 for herring sperm DNA, calf thymus DNA and yeast RNA, and the detection limits are 0.019, 0.89 and 1.2 ng ml −1 ( S/ N = 3), respectively. Actual biological samples were satisfactorily determined.

Ultra-fast gradient vs. fast isocratic chromatography in bioanalytical quantification by liquid chromatography/tandem mass spectrometry.

Liquid chromatography/tandem mass spectrometry (LC/MS/MS) methods developed for quantification using rapid ('ballistic') gradients on narrow bore, short HPLC columns have been previously described by this laboratory. This paper compares the fast gradient approach with the more traditional high-organic isocratic LC/MS/MS methods. The comparison is based on an analysis of the effectiveness of the chromatographic separations when using the two approaches (i.e. k', N, and W). The data presented herein are derived from actual biological samples analyzed as part of the drug discovery process.

Ultrasensitive heterogeneous immunoassay using photothermal deflection spectroscopy

A novel ultrasensitive, highly selective immunoassay method that uses gold ultrafine particles as the labeling material and laser-induced photothermal beam deflection quantitation is proposed. Ultrasensitive laser spectrometric analysis of actual biological samples is always obstructed by the background and its fluctuation. However, we could adequately suppress this obstruction by substituting polystyrene microspheres in the antigen-antibody solid-phase reaction instead of the conventional nitrocellulose membrane because the nonspecific adsorption velocity of the admixture proteins was highly restrained and the area of the antigen-antibody reaction was increased