Biomedical subjects

F T Chen

Publications and source records attributed to F T Chen.

30 records · Page 2Linked to original sources

PubMed1994

Characterization of digoxigenin-labeled B-phycoerythrin by capillary electrophoresis with laser-induced fluorescence. Application to homogeneous digoxin immunoassay.

A laser-induced fluorescence (LIF) immunoassay technique based on capillary electrophoretic (CE) separation is demonstrated. The analysis of digoxin in serum at clinically useful concentration levels of 10(-9) to 10(-10) M is achieved using this technique. The chemistry presented here using digoxigenin-labeled B-phycoerythrin was selected as a convenient model for the exploration of CE-LIF-based immunoassays. The LIF system described here exhibits detection limits in the low 10(-11) M range for several common fluorophores. The data presented in this report are one of the first examples of nanomolar quantitative analysis in a human serum matrix by CE.

Binding, Competitive↗

PubMed1994

Analysis of structural specificity in antibody-antigen reactions by capillary electrophoresis with laser-induced fluorescence detection.

A rapid and simple procedure for screening antibodies for binding to an antigen is proposed. A fluorescent hapten-dye conjugate was prepared by labeling the amino moiety of the hapten with a commercially available reactive cyanine dye, Cy5 (excitation maximum: 650 nm, emission maximum: 670 nm). A fixed amount of the Cy5-hapten was titrated with serial dilution of the antibody. Each of the titration mixture was analyzed by capillary electrophoresis (25 cm x 20 microns column) monitored by laser-induced fluorescence (laser: 10 mW helium-neon, 632.8 nm). Free and antibody-bound Cy5-hapten were analyzed simultaneously on the electropherogram. Competitive immunoassay of hapten was demonstrated with low-end sensitivity of 5.10(-8) M, about 10x more sensitive than the present drug screening methods. Using morphine as an example, the screening of various antibodies (from different vendors) and cross-reactivity of morphine analogues using the present procedure will be discussed.

Antibodies↗

PubMed1994

Characterization of proteins by capillary electrophoresis in fused-silica columns: review on serum protein analysis and application to immunoassays.

Protein mixtures can be characterized in terms of their separations by capillary electrophoresis (CE). The separation of proteins by CE is performed in untreated fused-silica columns. Model proteins and complex protein mixtures with pI values ranging from 4.0 to 11.0 are separated in such columns in less than 10 min in the presence of phosphate buffer with a pH between 4.0 and 9.0. The application of CE separation procedures for routine analysis of protein in serum, urine, and cerebrospinal fluid in borate-based buffer is also demonstrated. The detection of protein in CE is usually based on the intrinsic ultraviolet (UV) absorbance of the peptide bond at or near 200 nm, which provides a detection limit of about 10(-5) M. The same protein separation procedures can also be applied to immunochemical reaction systems in which one component is labeled. Thus, an antigen analyte, or the antibody to the analyte, may be labeled with a fluor and detected by laser-induced fluorescence (LIF). With a fluorescent-labeled reactant, the use of LIF detection further extends the detection limit to 10(-11) M. The CE separation technique for proteins provides a means to separate the bound and free species of the labeled antigen or antibody without the use of a solid support. The application of these separation techniques in conjunction with laser-induced fluorescence detection to make possible the homogeneous immunochemical measurement of species at concentrations in the range of 10(-9) to 10(-10) M is shown.

Blood Proteins↗

PubMed1994

Feasibility studies for simultaneous immunochemical multianalyte drug assay by capillary electrophoresis with laser-induced fluorescence.

We present a method for the simultaneous quantification of multiple drug analytes in urine, based on combining immunochemical binding with capillary electrophoretic separation. Two fluorescent drug-cyanine (Cy) dye conjugates were prepared as competing species for the immunoassay. Morphine was derivatized with Cy5 (lambda max = 652 nm, epsilon = 215,000 mol-1cm-1 L), phencyclidine (PCP) with Cy5.5 (lambda max = 675 nm, epsilon = 200,000 mol-1cm-1L). The high-efficiency resolving power of the capillary electrophoresis system (20 microns x 27 cm column) separated the individual labeled drugs, and the antigen-antibody complexes were detected by laser-induced fluorescence (laser: 10 mW He-Ne at 632.8 nm) with Cy5 diacid as internal standard. Simultaneous competitive immunoassay of morphine and PCP in urine showed that the free labeled-drug peak areas were proportional to the concentrations of the drug species present in the urine sample. This immunoassay can be performed routinely and reproducibly in < 5 min with analytical detection limits of 4 nmol/L for PCP and 40 nmol/L for morphine.

Antigen-Antibody Reactions↗

PubMed1993

Semiconductor laser-induced fluorescence detection in capillary electrophoresis using a cyanine dye.

Cy5, an activated carboxyl cyanine fluorophore, was characterized by capillary electrophoresis (CE) using a semiconductor laser at 652 nm to induce fluorescence. Hydrolysis of the activated Cy5 in the presence of ammonia results in the formation of a mono- and diamide and a dicarboxylic acid. A Cy5-labeled oligonucleotide M13 primer for DNA sequencing (M13mp18 template) was synthesized with a purity of better than 95%. The labeled primer was analyzed by liquid chromatography, using UV-visible detection, and by CE, monitored by laser-induced fluorescence (LIF) detection. Analysis of the Cy5-labeled oligonucleotide primer by CE-LIF in a 9% polyacrylamide gel-filled capillary indicated the purity of the major Cy5-oligonucleotide primer was greater than 90%. The detection sensitivity for Cy5-based CE-LIF detection system with a 2.5-mW red semiconductor laser is about 10(-10) M.

Base Sequence↗

PubMed1993

[Immunohistochemical localization of CEA,EMA and keratin in salivary mucoepidermal carcinoma]

Localizations of CEA,EMA and keration in 19 cases of mucoepidermal carcinomas were investigated using the indirect immunoperoxidase technique.The results showed CEA was negative in normal salivary glands and showed faint reaction in glands near carcinoma tissue.Keratin and EMA were localized in some myoepithelial cells.The positive rates in carcinoma tissue were 78.9%,89.5% and 84.2%,respectively.The positive rates and staining intensity of CEA and EMA in carcinoma tissue gradually decreased with the decline of tumor differentitation,but that of keratin showed no variation.the author consider that CEA and EMA could become good indices in clinically diagnosing mucoepithelial carcinoma and determining tumor differentiation type cell in mucoepidermal carcinoma and have a potential to multiply express the tumor elements of epithelium and/or mesenchyma.

Journal Article↗

PubMed1991

Capillary electrophoresis--a new clinical tool.

The potential clinical diagnostic utility of capillary electrophoresis in an open-tubular column is established. Separation patterns for serum proteins by conventional agarose gel electrophoresis can be faithfully reproduced by a capillary electrophoresis procedure that provides the complete run data in 8 min. Hemoglobin variants can be separated within 10 min. The capillary electrophoresis separations are performed reliably and reproducibly in an untreated 75 micron (i.d.) x 25 cm fused-silica column. Diluted serum or hemoglobin samples can be loaded on an automated instrument with on-line injection, detection, and quantification, providing a truly "walkaway" electrophoresis system.

Blood Proteins↗

PubMed1986