Detection and characterization of oligosaccharides in column effluents using surface plasmon resonance.

Surface plasmon resonance was used to detect oligosaccharides derived from glycoproteins. No sample derivatization or labeling is required for this technique. Sensor surfaces were prepared by immobilizing lectins. In the case of Sambucus nigra agglutinin reactive to terminal sialic acid or Ricinus communis agglutinin preferring terminal beta-linked galactose, femtomole levels of oligosaccharides could be detected. Using this affinity detector system, oligosaccharides and glycopeptides from a chromatographic separation on a gel filtration column were detected either by on-line monitoring or by analyzing the fractions individually.

Analysis of active antibody concentration. Separation of affinity and concentration parameters.

Antibody binding to surfaces with differing amounts of immobilised antigen was measured in a biosensor system using surface plasmon resonance detection. Binding rates obtained during the initial binding phase on high density antigen surfaces were proportional to antibody concentration and independent of antigen-antibody affinity. One antibody calibration curve covering the range from 0.5 to 160 nM (0.08-25 micrograms/ml) antibody was valid for IgG antibodies with different antigen specificities. To illustrate the use of this methodology active antibody concentrations were analysed in culture media and in rabbit serum.

Lactose repressor-operator DNA interactions: kinetic analysis by a surface plasmon resonance biosensor.

Lactose repressor binding to operator DNA and subsequent dissociation of the complex was monitored continuously by a biosensor, measuring surface plasmon resonance. In this analysis a synthetic, double-stranded oligonucleotide containing the operator site was immobilized on the sensor surface and repressor protein was passed over the surface. The formation of the repressor-operator complex was specific and could be inhibited by isopropyl-beta-D-thiogalactopyranoside inducer. From the association curve, the apparent kass was determined to be 1.8 x 10(6) M-1 s-1. Dissociation of the complex was, for the first time for the lac repressor, determined as an uncatalyzed reaction and the kdiss was determined to be 3.4 x 10(-4) s-1. As a reference, the repressor-operator interaction was analyzed by electrophoretic mobility shift assay under similar reaction conditions. With this method the equilibrium binding constant was calculated to be 2.4 (+/- 0.2) x 10(8) M-1. The corresponding value calculated from biosensor data was 5.1 x 10(9) M-1.

Introducing a biosensor based technology for real-time biospecific interaction analysis.

This report describes a system for real-time biospecific interaction analysis, using biosensor technology based on the optical phenomenon surface plasmon resonance. The biospecific interface is a sensor chip consisting of a thin gold film deposited on a glass support and covered with a hydrogel matrix. One component of the interaction being studied is attached covalently to the hydrogel, and other interactants are passed over the chip in solution. The interaction is followed in real time in terms of changes in the mass concentration of biomolecules at the sensor surface. Surface concentrations down to 10 pg/mm2 can be measured. The technique does not require molecular labels such as isotopes or spectroscopic markers, and purification of interacting components can often be avoided. Repeated analyses can be performed on the same sensor chip. With this system, the same general procedure can be used for a wide range of different applications, including concentration determination, kinetic measurements and multi-site binding studies. The sensitivity of the technique can be adjusted by choice of reagents and experimental procedure: determination of specific proteins in serum down to 20 ng/ml and macromolecular association constants from 10(7) M-1 up to 4 x 10(11) M-1 are documentated examples. No other single analytical system has the same versatility and general applicability to biospecific interaction analysis. The system is developed and marketed by Pharmacia Biosensor AB, Sweden.

Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis.

A system for real-time biospecific interaction analysis using biosensor technology based on the optical phenomenon surface plasmon resonance is described. The biospecific interface is a sensor chip covered with a hydrogel matrix. One component of the interaction to be studied is immobilized covalently to the hydrogel and other interactants are passed over the chip in solution. The mass change at the sensor surface, reflecting the progress of the interaction studied, is monitored in real time. The technique, which does not require molecular labels for detection, can measure mass changes down to 10 pg/mm2. Repeated analyses can be performed on the same sensor chip. Applications shown include kinetic measurements, binding site analysis and concentration determination.

Real-time biospecific interaction analysis using surface plasmon resonance and a sensor chip technology.

We report here the development and application of a biosensor-based technology that employs surface plasmon resonance for label-free studies of molecular interactions in real time. The sensor chip interface, comprising a thin layer of gold deposited on a glass support, is derivatized with a flexible hydrophilic polymer to facilitate the attachment of specific ligands to the surface and to increase the dynamic range for surface concentration measurements. The sensor can be used to measure surface concentrations down to 10 pg/mm2. Typical coefficients of variation are from two to five percent. We anticipate that the ability to monitor multi-molecular complexes as they form will greatly contribute to the understanding of biorecognition and the structural basis of molecular function.

Detection of antigen-antibody interactions by surface plasmon resonance. Application to epitope mapping.

Surface plasmon resonance (SPR) detection requires no labeling of antigen or antibodies and allows quantification of two or more interacting molecular species. The automated SPR instrument used here consists of an optical detection unit, an integrated liquid handling unit, and an autosampler. A first molecule is immobilized to the dextran modified surface of the sensor chip. By sequential introduction, the stepwise formation of multimolecular complexes can then be monitored. A two-site binding assay which allows characterization of MoAb epitope specificities is described. A polyclonal rabbit anti-mouse IgG1 (RAMG1) immobilized to the dextran surface is used to capture the first MoAb from unprocessed hybridoma culture supernatants. After introducing the antigen, the ability of a second MoAb to bind to the antigen is tested. The analysis cycle which is fully automated can be performed more than 100 times using the same RAMG1 surface. Since the detection principle allows monitoring of each reactant in the consecutive formation of a multimolecular complex, multi-site binding experiments can be performed. Five MoAbs recognizing different epitopes on an antigen were shown to bind sequentially, forming a hexamolecular complex. MoAbs were further characterized by inhibition analysis using synthetic peptides derived from the primary structure of their antigen. As a model system MoAbs against recombinant HIV-1 core protein p24 were used in all experiments.

Fast protein liquid chromatography scale-up procedures for the preparation of low-molecular-weight proteins from urine.

A system for the rapid isolation of low molecular weight proteins from urine has been devised, and illustrated by alpha 1-microglobulin, beta 2-microglobulin, retinol binding protein, lysozyme and monoclonal light chains. Urine proteins from patients with tubular dysfunction were concentrated, either by ultrafiltration or ammonium sulphate precipitation. This was followed by gel chromatography on Sephadex G-50. The appropriate fractions were then separated by chromatography on Pharmacia monobead columns. A Mono Q strong anion exchanger was used for beta 2-microglobulin, retinol binding protein, alpha 1-microglobulin and free monoclonal light chains. Lysozyme was separated on a Mono S cation exchanger. The chromatography was first optimized on HR 5/5 columns and then scaled up to HR 16/10 columns.

Fractionation of human red cell membrane proteins by ion-exchange chromatography in detergent on Mono Q, with special reference to the glucose transporter.

The anion exchanger Mono Q has been used for rapid and efficient fractionation of human red cell membrane proteins in the easily removable detergents n-octyl-beta-D-glucopyranoside or nonanoyl-N-methylglucamide. In practice the chromatographic resolution of membrane proteins was lower than for water-soluble proteins, perhaps due to protein-protein interactions and microheterogeneity, but several components, or groups of components, separated well upon salt gradient elution. The glucose transporter (or transportase) was eluted early, glycophorins later, and the anion transporter still later. The detergents Berol 185 and the zwitter-ionic derivatives of cholate, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonat e and 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propan esulphonate, gave similar chromatographic results but differed in solubilization selectivity. A relatively pure material was also fractionated; viz., a glucose transportase which had been prepared by DEAE-cellulose chromatography. Mono Q, in the presence of octyl glucoside, afforded additional purification, which made automatic sequence determination possible for eighteen amino acid residues. The results indicate that two polypeptides were present in about equimolar amounts.

Hb-Linköping (beta 36 Pro----Thr): a new hemoglobin mutant characterized by reversed-phase high-performance liquid chromatography.

Preparative separation of alpha- and beta-chains from a newly identified abnormal hemoglobin has been performed on a new C1/C8 column. Tryptic peptides from the abnormal beta-chain were subjected to peptide mapping on a new C2/C18 column with a reversed-phase system including potassium dihydrogen phosphate (pH 2.9) as a hydrophilic ion-pairing reagent. A hydrophobic substitution, beta 36 proline-threonine, was evident after amino acid analysis and Edman degradation of the isolated mutant peptide. Replacement of proline as the second amino acid in the C-helix represents an important structural change in the alpha 1 beta 2-contact.

Fast chromatofocusing of human serum proteins with special reference to alpha 1-antitrypsin and Gc-globulin.

A new chromatofocusing medium, MonoP, was used for fast (60 min or less) separations of human serum proteins. Separations in the broad pH interval 6.0-3.8 were analysed by fused rocket immunoelectrophoresis to identify a number of proteins, and by gradient gel electrophoresis to determine the molecular weight distribution of the eluted material. To illustrate further the high resolving power of chromatofocusing, narrow pH intervals of about 0.5 pH units were used to study the microheterogeneity of alpha 1-antitrypsin and Gc-globulin. Due to its high resolving power and preparative capacity, chromatofocusing is attractive as the first dimension in two-dimensional techniques for the resolution of complex protein mixtures.

A 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414. Purification, characterization and preparation of an immunoadsorbent for the enzyme.

A 1,4-beta-glucan glucanohydrolase (EC 3.2.1.4) was isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by molecular-sieve chromatography on Bio-Gel P-30, ion-exchange chromatography on DEAE-Sephadex A-50 and isoelectric focusing in a density gradient. Polyacrylamide-gel electrophoresis at two different pH values, analytical isoelectric focusing in a polyacrylamide-gel slab and molecular-sieve chromatography of the reduced and alkylated enzyme in a denaturing medium indicated a homogeneous protein. The enzyme has a mol.wt. of 51,000 and is not a glycoprotein. The pI was found to be 4.66 at 23 degrees C. Antiserum against the purified enzyme was prepared and the amount of enzyme in the original filtrate was determined by rocket immunoelectrophoresis to be about 50mg/liter. An immunoadsorbent made from CNBr-activated sepharose 4B and antiserum affords a rapid and highly specific purification of the enzyme.

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414.

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.