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Biomedical subjects

F E Regnier

Publications and source records attributed to F E Regnier.

At least 37 records · Page 2Linked to original sources
PubMed1999

Chromatography and electrophoresis on chips: critical elements of future integrated, microfluidic analytical systems for life science.

Liquid chromatography and electrophoresis played a major role in the life-science revolution, most strikingly in protein purification, peptide fractionation and sequencing, amino acid analysis, and DNA sequencing. The objective of this article is to examine the potential role of separation systems in the continuing evolution of biochemistry, biotechnology and molecular biology. Very small chip-based systems may change how chemical analyses in biology, medical research and health care evolve over the next decade.

Biological Science Disciplines↗
PubMed1998

Protein-protein interactions on weak-cation-exchange sorbent surfaces during chromatographic separations.

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized protein coats 24% or less of the sorbent surface. It was found that columns on which proteins were immobilized still behaved as a cation-exchange chromatography sorbents, but their selectivity was different from the parent weak cation-exchange column. This was interpreted to mean that in addition to the normal electrostatic interactions expected in ion- exchange chromatography, protein analytes interact with immobilized protein on the sorbent surface. Anionic proteins were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It is concluded that these protein-protein interactions occur after proteins are captured by the primary interaction mechanism of the column, in this case, electrostatic interaction. Protein-protein interaction is a secondary, lateral process. These lateral interactions were observed between 4% and 24% surface saturation. The significance of this observation is that in preparative chromatography and the case of "fouled" columns, strongly adsorbed proteins could alter the elution characteristics of sample proteins being target for analysis or purification.

Cation Exchange Resins↗
PubMed1998

Rapid verification of disulfide linkages in recombinant human growth hormone by tandem column tryptic mapping.

An automated tryptic mapping method was developed for characterization of disulfide linkages in recombinant human growth hormone (rhGH). The hormone was trypsin digested and the peptide fragments concentrated by eluting rhGH through an immobilized trypsin column and transferring the peptides directly to a reversed-phase liquid chromatography (RP-LC) column where they were collected. Reaction time was controlled by the flow-rate. Following tryptic digestion of a sample, the immobilized enzyme column was uncoupled from the flow train by a switching valve and the RP-LC column eluted with a solvent gradient ranging from 0.1% trifluoroacetic acid (TFA) with 1% acetonitrile (ACN) to ACN with 0.1% TFA and 5% water. This two-step mapping process was achieved within 2 h on both native and reduced rhGH samples. The chromatographic elution position and mass spectra matrix-assisted laser desorption ionization time-of-flight mass spectrometry of native rhGH and sulfur-containing peptides were determined with standards. Standards of the individual sulfhydryl (-SH) containing peptides and all possible disulfide linked peptides that could result from coupling the -SH peptides in disulfide linkages were obtained by synthesis and chromatographic purification. This approach allowed the chromatographic elution position of all possible mismatched disulfide containing peptides to be established and samples of rhGH to be examined for improper folding.

Amino Acid Sequence↗
PubMed1996

Study of protein binding to a silica support with a polymeric cation-exchange coating.

A silica-based, polyacrylate ion-exchange stationary phase has been prepared using Ce(IV) as the initiator. Analysis of the physical properties of the polymeric layer separated from the silica surface indicates that the polymeric coating is cross linked to some extent. The polymerization carried out at different concentrations of Ce(IV) demonstrated that the effective surface area can be increased by lowering the Ce(IV) concentration at higher monomer concentrations of the reaction mixture. These materials are quite reproducible and of high electrostatic binding capacity; 1.485 mumol/m2. The electrostatic binding capacity of a non-polymeric stationary phase reached the theoretical limit for a monolayer (0.16 mumol/m2). However, the covalent binding capacity of the same stationary phase was only 50% of the electrostatic binding capacity. The same trend was observed in all the polymeric stationary phases tested. This shows that the mechanism of protein binding in polymeric and conventional stationary phases is similar, and multilayer electrostatic binding is highly unlikely in these sorbents examined. Z numbers revealed that the contact area of the protein is independent of the polymeric character of the stationary phase and therefore, the increased loading of these polymeric stationary phases is due to the increased surface area.

Acids↗
PubMed1996

Lateral interaction between electrostatically adsorbed and covalently immobilized proteins on the surface of cation-exchange sorbents.

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized proteins coats 50% or less of the sorbent surface. It was found that although these sorbents still function as cation exchangers, covalently immobilized proteins frequently contribute to the ion-exchange behavior of some protein analytes. Chromatographic retention of analytes was equal to or greater on immobilized protein derivatized columns than underivatized sorbents. Anionic proteins, in contrast, were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It was concluded that electrostatic adsorption is a prerequisite for analyte protein/immobilized protein interactions of sufficient magnitude to impact ion-exchange separations. Large differences in protein resolution were observed on columns that were identical in all respects except for the immobilized protein, further confirming that analyte/immobilized protein interactions were unique to the interacting pair. The extent of interaction was also influenced by concentration of the immobilized protein in the case of lysozyme. Interactions between the analyte and immobilized protein were found to occur between both the same two proteins and dissimilar species. It was concluded that these phenomena are due to lateral interactions between immobilized proteins and analyte proteins subsequent to electrostatic adsorption of the analyte on the underivatized surface of ion-exchange sorbents.

Acrylic Resins↗
PubMed1996

Enzyme-linked immunosorbent assays in a chromatographic format.

This paper describes the execution of enzyme-linked immunosorbent assays (ELISA), (i) in immunosorbent columns with antibodies immobilized on porous particles, (ii) where samples and reagents are metered by valves and syringe pumps, (iii) samples, reagents, substrate, and wash buffers are transported into or through the system by high pressure liquid chromatography pumps, and (iv) enzyme reaction product is detected by absorbance. The normal protocol used for ELISA was modified in that antigen was complexed with enzyme conjugated antibody in the autosampler and an aliquot introduced into the system where it was transported to the immunosorbent and captured. Substrate was subsequently pumped into the immunosorbent bed and the product swept to an absorbance detector for quantitation.

Animals↗
PubMed1996

Dynamic modeling of electrophoretically mediated microanalysis.

A dynamic model is presented for simulation of reaction-based chemical analysis of enzymes and substrates in capillary electrophoretic systems by the methodology of electrophoretically mediated microanalysis (EMMA). The mathematical model utilizes mass balance expressions describing the time-dependent effects of electromigration, chemical reaction, and diffusional band broadening upon the concentration profiles of the various reagent and product species. The model is implemented in an iterative computer program in which the capillary is segmented into arrays of bins storing the concentration profiles of each of the chemical species. During each time increment, the effects of electrophoresis, reaction kinetics, and diffusion are calculated, and the concentrations stored in the arrays are updated. The flexibility of the model to accommodate various initial capillary conditions, sample introduction methods, and voltage programming allows diverse EMMA analyses to be simulated. The simulated results are shown to be in good qualitative agreement with experimental data for zonal injection and moving boundary EMMA determinations of leucine aminopeptidase as well as an EMMA analysis of ethanol.

Computer Simulation↗
PubMed1996

Moving boundary electrophoretically mediated microanalysis.

Moving boundary sample introduction is described as an alternative to zonal injection methods for the electrophoretically mediated microanalysis (EMMA) of leucine aminopeptidase (LAP). The capillary was initially filled with the analyte solution while the faster-migrating substrate, L-leucine-p-nitroanilide, was maintained in the inlet reservoir. Upon application of an electric field, electrophoretic merging of the reagents proceeded, and the detectable reaction product, p-nitroaniline, was transported to the detector. The area, maximum height, inclining slope, and declining slope of the resulting triangular product profile were each directly proportional to the activity of LAP, and the observed migration times of the product profile features defined the volume and time of the incubation. The moving boundary technique offered more than an order of magnitude greater concentration sensitivity than the zonal injection EMMA method. This heightened sensitivity facilitated rapid analysis as the use of elevated electric field strenghts and short capillaries allowed for a 24-s kinetic determination of LAP.

Animals↗
PubMed1995

C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.

The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.

Adrenocorticotropic Hormone↗
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

Contribution of electrostatic and hydrophobic interactions to the adsorption of proteins by aluminium-containing adjuvants.

The effect of ionic strength and ethylene glycol on the adsorption of bovine serum albumin (BSA) or lysozyme by a commercial aluminium hydroxide or aluminium phosphate adjuvant was studied at pH 7.4 and 25 degrees C. The adsorption of BSA by aluminium hydroxide adjuvant and lysozyme by aluminium phosphate adjuvant was found to be inversely related to ionic strength. This indicates that electrostatic attractive forces contribute to adsorption. The adsorption of lysozyme by aluminium phosphate adjuvant was reduced by the addition of ethylene glycol. However, no change in the adsorption of BSA by aluminium hydroxide adjuvant was noted when up to 40% ethylene glycol was present. This behaviour indicates that hydrophobic forces contribute to the adsorption of lysozyme but not of BSA. However, virtually no adsorption was observed when the protein and the adjuvant had the same surface charge. Thus, attractive forces may not be sufficient to produce adsorption of an antigen by an aluminium-containing adjuvant if electrostatic repulsive forces are present.

Adjuvants, Immunologic↗
PubMed1994

Selectivity in electrophoretically mediated microanalysis by control of product detection time.

Differential electrophoretic mobility between an analyte and its product offers electrophoretically mediated microanalysis (EMMA) a unique capability to selectively control the detection time of the product of a reaction-based chemical analysis. If an analyte and its product differ in migration velocity under the influence of an applied electric field, the apparent velocity and, consequently, the detection time of the product are dependent upon the relative amounts of time the product effectively traverses the capillary with the respective mobilities of the analyte and the product. Consequently, by controlling when the analytical reaction is allowed to occur, the detection time of the reaction product can be selectively maneuvered within a time window defined by the mobilities of the analyte and the product. This paper describes the use of this technique to manipulate the detection time of product profiles independently of nonreacting matrix interferants for the determination of both enzymes and substrates by EMMA. Furthermore, the ability to manipulate product detection times allows for simultaneous EMMA determinations of multiple enzymes or substrates.

Alcohol Dehydrogenase↗
PubMed1994

Rapid, automated, two-dimensional high-performance liquid chromatographic analysis of immunoglobulin G and its multimers.

It is important to determine the amount of IgG multimers in immunoglobulin-containing pharmaceuticals because these aggregates can cause adverse reactions in patients. Previous methods for determining aggregates either suffered from interference of other proteins or required fraction collection and sample purification. A new, automated two-dimensional approach has been developed in which size-exclusion chromatography is performed in the first dimension followed by protein A affinity chromatography in the second dimension. This method is robust in that the aggregates are not disturbed by a preliminary purification step. Further, the presence of contaminating proteins has no effect on the analysis since affinity chromatography is used to determine the presence of IgG in the second dimension. The entire automated two-dimensional analysis can be performed in ca. 1 h.

Autoanalysis↗
PubMed1994

Electrophoretically mediated micro-assay of alkaline phosphatase using electrochemical and spectrophotometric detection in capillary electrophoresis.

Electrophoretically mediated micro-assays of alkaline phosphatase (ALP) are demonstrated in capillary electrophoretic systems using both electrochemical (ED) and spectrophotometric (UV) detection. In the ED mode, p-aminophenylphosphate was used as the substrate and p-aminophenol (pAP) was monitored at a carbon fiber electrode held at +180 mV vs. Ag/AgCl. Spectrophotometric detection was achieved using the substrate p-nitrophenylphosphate, and monitoring the product p-nitrophenol (pNP) at 405 nm. The detection limit for pAP by ED was determined to be 100-fold lower than for pNP using UV detection. In the determination of ALP, both methods were found to be linear. The detection limit for ALP using zero potential assays with UV detection was determined to be 1.8.10(-6) mg/ml compared to 1.8.10(-7) mg/ml using ED.

Alkaline Phosphatase↗
PubMed1994

Electrophoretically mediated microanalysis of calcium.

Electrophoretically mediated microanalysis (EMMA) was used for the determination of calcium. The faster analyte zone containing the calcium was injected spatially behind a slower zone of o-cresolphthalein complexone in a capillary electrophoresis based system. Upon application of the electric field the calcium zone was electrophoretically mixed with the reagent and product was formed. The bulk electroosmotic flow carried the product to the detector where the absorbance of the resulting complex at 575 nm was measured. Quantitation using an internal standard yielded a linear response with an R.S.D. of 8.1%. An inter-method comparison was performed with the standard bulk method and yielded results that did not significantly differ. The advantages of EMMA with respect to traditional methods were addressed.

Calcium↗
PubMed1994

Ensemble averaging and digital filtering in chromatography and electrophoresis.

Ensemble averaging and digital filtering were implemented for signal-to-noise ratio improvement in the separation techniques of size-exclusion chromatography, immunoaffinity chromatography, capillary zone electrophoresis and capillary ion analysis. Results of ensemble averaging were always greater than statistically predicted. Techniques included five to nine replicate separations and yielded signal-to-noise improvement factors of 2.5 to 9.3. Running-average and time constant (RC)-convolution digital filters yielded increases in the signal-to-noise ratio ranging from zero to twelve. This paper will discuss and illustrate the usage of ensemble averaging and digital filtering in liquid-phase separation techniques.

Algorithms↗