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Biomedical subjects

N B Afeyan

Publications and source records attributed to N B Afeyan.

7 recordsLinked to original sources
PubMed1995

Capillary electrophoresis-based immunoassay for cortisol in serum.

A competitive immunoassay for cortisol based on capillary electrophoresis (CE) and laser-induced fluorescence is described. The work involved the production of assay reagents and the development of separation conditions allowing for routine analysis of serum samples. Fluorescein-labeled cortisol was synthesized and purified. Fab fragments were produced from mouse monoclonal anticortisol antibody and purified using a POROS cation exchange chromatography column. After incubation of these reagents with serum, free and bound labeled antigen were separated by CE with high reproducibility. No prior sample cleanup of the serum samples was necessary. Serum calibration curves were established and used for the quantitation of cortisol in serum. The results demonstrate feasibility for a cortisol assay based on CE operating directly on serum samples.

Electrophoresis↗
PubMed1995

Sequence analysis of phosphorothioate oligonucleotides via matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Modification of the natural phosphodiester backbone of deoxyribooligonucleotides can impart increased biostability via nuclease resistance. Further, uniform incorporation of phosphorothioate linkages renders oligonucleotides highly resistant to reagents traditionally used in sequencing reactions. As a consequence, analytical tests crucial for establishing the identity of such oligonucleotide drugs are less informative. To circumvent this problem, chemical oxidation has been employed for converting the phosphorothioate to the uniform phosphodiester, thereby facilitating enzymatic degradation. Following oxidation, exonucleases which sequentially cleave individual bases from the 3' or 5' terminus of the oligonucleotide or base-specific cleavage chemicals were used to facilitate sequence identification of the oligonucleotide. Matrix-assisted laser desorption ionization-time-of-flight/mass spectrometry (MALDI-TOF/MS), previously used to sequence natural phosphodiester DNA, was then used to sequence the chemically oxidized phosphorothioate. Sequential enzymatic cleavage of desulphurized phosphorothioates in combination with MALDI analysis not only provides a viable alternative to radiolabeling as used in conventional sequencing approaches (e.g. Maxam-Gilbert), but also enables rapid sequencing of phosphorothioate oligonucleotides, for routine drug analysis.

Base Sequence↗
PubMed1992

Automated real-time immunoassay of biomolecules.

ImmunoDetection is a novel technique combining perfusion chromatography technology with antibodies to perform the steps of an immunoassay in a flow-through column format. Sensitive and precise measurements are performed in seconds to minutes using automated liquid chromatography instrumentation.

Antibodies↗
PubMed1992

Preparative peptide purification by cation-exchange and reversed-phase perfusion chromatography.

Cation exchange was compared to reversed-phase chromatography for the preparative purification of a 28-residue peptide (vasoactive intestinal polypeptide) on the 100-mg scale. Optimized high-speed, high-resolution methods were developed for both chromatographic modes on POROS Perfusion Chromatography flow-through particle chromatography columns. While both methods appeared to provide similar purity, the cation exchange column had approximately ten times the loading capacity per unit column volume as the reversed-phase column. Five-minute methods for desalting the cation exchange-purified peptide and analysis of fractions were developed using small reversed-phase columns. The cation-exchange method was scaled up to process 95 mg of crude peptide in a 12-min run.

Amino Acid Sequence↗
PubMed1991

Antibody quantitation in seconds using affinity Perfusion Chromatography.

An extremely rapid assay technique for antibodies has been developed utilizing protein A or protein G bound to Perfusion Chromatography support matrices. Either dilute or concentrated samples are directly injected on a column that selectively binds antibody, which is quantitated directly by elution and UV absorbance. Due to the unique mass transport characteristics of the supports, total assay cycle times are typically 1 minute or less, with assays as short as 15 seconds possible. The assay system can accurately quantitate a 100,000:1 or greater dynamic range in sample concentration without sample dilution, is extremely repeatable and is easy to automate with conventional HPLC systems. Assay of antibodies in a wide range of sample types has been demonstrated.

Animals↗
PubMed1990

Flow-through particles for the high-performance liquid chromatographic separation of biomolecules: perfusion chromatography.

This paper reports a new technique for reducing resistance to stagnant mobile phase mass transfer without sacrificing high adsorbent capacity or necessitating extremely high pressure operation. The technique involves the flow of liquid through a porous chromatographic particle, and has thus been termed "perfusion chromatography". This is accomplished with 6000-8000 A pores which transect the particle. Data from electron microscopy, column efficiency, frontal analysis and theoretical modelling all suggest that mobile phase will flow through these large pores. In this manner, solutes enter the interior of the particles through a combination of convective and diffusional transport, with convection dominating for Peclet numbers greater than one. The implications of flow through particles on bandspreading, resolution and dynamic loading capacity are examined. It is shown that the rate of solute transport is strongly coupled to mobile phase velocity such that bandspreading, resolution of proteins and dynamic loading capacity are unaffected by increases in mobile phase velocity up to several thousand centimeters per hour. The surface area of this very large-pore diameter material is enhanced by using a network of smaller, 500-1500 A interconnecting pores between the throughpores. Scanning electron micrographs show that the pore network is continuous and that no point in the matrix is more than 5000-10,000 A from a through-pore. As a consequence, diffusional path lengths are minimized and the large porous particles take on the transport characteristics of much smaller particles but with a fraction of the pressure drop. Capacity and resolution studies show that these materials bind and separate an amount of protein equivalent to that of conventional high-performance liquid chromatography as well as low performance agarose-based media at greater than 10-100 times higher mobile phase velocity with no loss in resolution.

Chromatography, High Pressure Liquid↗