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Biomedical subjects

M Malmqvist

Publications and source records attributed to M Malmqvist.

14 recordsLinked to original sources
PubMed1992

By-passing immunization: building high affinity human antibodies by chain shuffling.

Diverse antibody libraries can be displayed on the surface of filamentous bacteriophage, and selected by panning of the phage with antigen. This allows human antibodies to be made directly in vitro without prior immunization, thus mimicking the primary immune response. Here we have improved the affinity of one such "primary" antibody by sequentially replacing the heavy and light chain variable (V) region genes with repertoires of V-genes (chain shuffling) obtained from unimmunized donors. For a human phage antibody for the hapten 2-phenyloxazol-5-one (phOx) (Kd = 3.2 x 10(-7) M), we shuffled the light chains and isolated an antibody with a 20 fold improved affinity. By shuffling the first two hypervariable loops of the heavy chain, we isolated an antibody with a further 15-fold improved affinity. The reshuffled antibody differed in five of the six hypervariable loops from the original antibody and the affinity for phOx (Kd = 1.1 x 10(-9) M) was comparable to that of mouse hybridomas from the tertiary immune response. Reshuffling offers an alternative to random point mutation for affinity maturation of human antibodies in vitro.

Amino Acid Sequence↗
PubMed1987

Characterization of monoclonal anti-DNA antibodies and visualization of binding to chromosomes and cell nuclei.

Monoclonal antibodies against DNA from two hybridoma cell lines were produced and characterized. One had specificity for single stranded (ss) DNA with some cross-reactivity to RNA, while the other was specific for both single (ss) and double stranded (ds) DNA. The latter ds and ss DNA-binding antibody was used as a model for analysing the distribution of the epitope in chromosomes and cell nuclei. A linear correlation between antibody binding and propidium iodide counterstaining was found on flow cytometric analysis of suspended chromosomes. Immunofluorescence of rat myoblast cells showed a speckled distribution of the antibody in the nucleus with a variability between the cells. Using electron microscopy to visualize antibody binding with gold particles, codistribution with uranyl acetate staining of leucocytes was found. These results suggested that the antibody preferentially binds to condensed chromatin in cells and chromosomes.

Animals↗
PubMed1987

Regional hyperthermia combined with blockade of the hepatic arterial blood flow by degradable starch microspheres in pigs.

The benefit of hepatic arterial microembolization by degradable starch microspheres (DSM) was investigated in regional hyperthermia of the liver. Hyperthermia with and without blood flow blockade of the hepatic artery using degradable starch microspheres was performed on six pigs. Heat was given for 30 min in each treatment by 8 MHz radiofrequency capacitive heating equipment. To maintain blood flow blockade during hyperthermia, 10 mg/kg of degradable starch microspheres was administered into the hepatic artery as an initial dose and 5 mg/kg of the drug was added periodically under the measurement of hepatic arterial blood flow by an electromagnetic flowmeter. To evaluate the effect of degradable starch microspheres, the temperature increase in the liver and rectum was compared between the treatment with and without DSM. All pigs showed a larger increase in intrahepatic temperature when heated in combination with degradable starch microspheres than without. On the other hand, temperature increase in the rectum as a result of hyperthermia to the liver was suppressed by DSM as compared with hyperthermia alone. These results indicate that hepatic arterial embolization by degradable starch microspheres potentiates radiofrequency capacitive heating of the liver. Although this study was not made with liver tumors, regional hyperthermia may be effective in the control of liver tumors when heat is given after the blockade of the hepatic artery by DSM.

Animals↗
PubMed1985

Immobilization of immunoglobulins on silica surfaces. Stability.

The development of new immunosensors based on surface-concentration-measuring devices requires a stable and reproducible immobilization of antibodies on well-characterized solid surfaces. We here report on the immobilization of immunoglobulin G (IgG) on chemically modified silica surfaces. Such surfaces may be used in various surface-oriented analytical methods. Reactive groups were introduced to the silica surfaces by chemical-vapour deposition of silane. The surfaces were characterized by ellipsometry, contact-angle measurements and scanning electron microscopy. IgG covalently bound by the use of thiol-disulphide exchange reactions, thereby controlling the maximum number of covalent bonds to the surface, was compared with IgG adsorbed on various silica surfaces. This comparison showed that the covalently bound IgG has a superior stability when the pH was lowered or incubation with detergents, urea or ethylene glycol was carried out. The result was evaluated by ellipsometry, an optical technique that renders possible the quantification of amounts of immobilized IgG. The results outline the possibilities of obtaining a controlled covalent binding of biomolecules to solid surfaces with an optimal stability and biological activity of the immobilized molecules.

Cross-Linking Reagents↗
PubMed1985

Immobilization of immunoglobulins on silica surfaces. Kinetics of immobilization and influence of ionic strength.

The kinetics of, and the influence of ionic strength on, the immobilization of rabbit immunoglobulin G (IgG) on different types of well-characterized silica surfaces were investigated. Adsorptive immobilization was compared with covalent attachment via thiol-disulphide exchange reactions. The amount of immobilized IgG on five different types of silica surfaces as a function of IgG concentration, at two different ionic strengths, was determined. The IgG-solid-surface interaction involved different types of interaction forces, depending on the surface chemistry of the solid surface. The solid-surface chemistry is an important parameter determining the immobilized amount of IgG. When conditions for covalent attachment of IgG to the surfaces were fulfilled, the IgG showed high affinity and the immobilized amount of IgG showed a fast saturation. Changes in ionic strength showed no significant influence on the kinetics of immobilization on these surfaces. The amount of covalently attached IgG was partially ionic-strength-dependent, indicating that adsorptive interactions were involved. The results are of fundamental interest for the development of new immunosensors based on surface-concentration-measuring devices.

Adsorption↗
PubMed1978

Purification and characterization of two different agarose-degrading enzymes.

Agarase was concentrated and purified from culture filtrates of an agar-degrading Pseudomonas-like bacteria by affinity chromatography on divinyl sulphone cross-linked Sepharose 4B. By fractionation on Sephadex G-200 three fractions were obtained two of which, agarases I and II, had agarase activity. Agarase II was further purified by isoelectric focusing, and the main peak, agarase IIb, was isoelectric at pH 5.1. Molecular weight determinations indicated agarase I to be a dimer with Mr approximately 210 000. The Mr of agarase IIb was 63 000 as determined by analytical ultra-centrifugation, polyacrylamide gel electrophoresis in sodium dodecyl sulphate and molecular sieve chromatography on Sepharose 4B in 6M Gdn-HCl. The amino acid compositions of the two proteins were very similar and both were found to be glycoproteins. The pH optimum of the enzymes was 6.7 and the optimal temperature was 38 degree C for agarase I and 43 degree C for agarase IIb. Melted agarose and agarose gel were used as substrates for the enzymes. The ratio of the activities towards the different substrates was 4.3 for agarase I and 1.0 for agarase IIb. Agarase I hydrolyzed the beta-linkages in neoagarooctaose so as to produce two moles of neoagarotetraose or one mole of neoagarohexaose and one mole of neoagarobiose. Agarase IIb hydrolyzed only the central beta-linkage to form two moles of neoagarotetraose.

Amino Acids↗
PubMed1977

Effects of bacterial agarase on agarose gel in cell culture.

Bacterial agarase, concentrated and purified from culture filtrate of agar-degrading bacteria, has been used to clean cells cultured in soft agarose from gel residues. The enzyme also has been used to liquefy the gel directly in the dishes to facilitate the removal of cells. The sufaces of glioma cells from agarase-treated colonies could not be distinguished in the scanning electron microscope from surfaces of cells which had never been in contact with agarose or agarase. This implies that most agarose residues had been removed, and also that the treatment did not seriously alter the cell surfaces. The influence of the agarase treatment also was tested by comparison of the mitotic index and the incorporation of [3H]thymidine in agarase-treated and untreated cells. No effects of the treatment could be seen in these tests.

Bacteria↗
PubMed1975

Degradation of agar by a gram-negative bacterium.

An agar-degrading bacterium, having a guanine-cytosine content of 50-5 mol% has been isolated from sewage. This Gram-negative rod grew well in a simple salts medium containing various carbohydrates. Growing bacteria dissolved gels and suspensions of agar and agarose rapidly, but did not attack cross-linked agars. Agarase was cell-bound in exponentially growing cultures but was released into the medium at stationary phase. Both cell extracts and culture filtrates released reducing sugars from agar solutions and prevented them from gelling. Gels were not dissolved by enzyme solutions, but the turbidity and iodine-binding properties of the agar were decreased.

Agar↗
PubMedDate not supplied

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.

Amino Acid Sequence↗