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Biomedical subjects

M Suter

Publications and source records attributed to M Suter.

At least 145 records · Page 8Linked to original sources
PubMed1988

Production of streptavidin in a synthetic medium.

A simple, inexpensive procedure for producing streptavidin has been described. The biotin-binding protein was produced by growing Streptomyces avidinii in a synthetic liquid culture medium containing L-asparagine as the sole nitrogen source. With this procedure, extraneous proteinaceous substances inherently present in culture media prepared with yeast extract or with peptones were not present to interfere with isolation and purification of streptavidin. When harvested after 7-8 days of incubation, the culture fluid was relatively free of contaminating cell breakdown products. Maximal production of streptavidin (100-120 mg/l) was obtained in 8-10 day cultures. For some applications, the culture fluid can be used directly as a source of streptavidin. Under the same conditions used to grow S. avidinii, 11 other actinomycete strains and 134 eumycetes were found to lack the capacity to produce detectable amounts of an extracellular biotin-binding protein.

Bacterial Proteins↗
PubMed1988

Isolation and characterization of highly purified streptavidin obtained in a two-step purification procedure from Streptomyces avidinii grown in a synthetic medium.

A method is described for isolation of streptavidin from cultures of Streptomyces avidinii grown in a synthetic culture medium for 6-10 days. Streptavidin is precipitated directly from culture supernatant fluid using 80% ammonium sulfate, and the precipitate is dialyzed against water and centrifuged at 40,000 X g for 60 min. The absorbency coefficient at 280 nm of purified streptavidin was estimated to be 31.7142 +/- 0.1806 for a 1% solution. The protein appeared to be greater than 90% homogeneous by gel permeation chromatography and polyacrylamide gel electrophoresis. No biotin-binding molecules less than 70 kDa in size were detected at any step during the purification of streptavidin. Streptavidin was able to maintain a stable crosslink between two biotinylated molecules in a solid-phase assay. Streptavidin purified by this method was stable in 50% glycerol/water at -20 degrees C for more than 1 year. Lyophilization or iodination did not produce apparent damage to the protein.

Bacterial Proteins↗
PubMed1988

Genetic engineering of bovine Ig. Construction and characterization of hapten-binding bovine/murine chimeric IgE, IgA, IgG1, IgG2, and IgG3 molecules.

A bovine recombinant phage library was constructed and screened with rabbit S mu and human C gamma probes. IgM, IgA, and IgE H chain constant region (CH) genes were isolated with the rabbit S mu probe and three IgG CH genes were isolated with the C gamma probe. The CH genes were individually cloned into an expression vector which contained a murine VDJ gene cloned from a hybridoma producing anti-dansyl hapten antibody. The resulting constructs were transfected into murine hybridoma cells producing L chain of the anti-dansyl antibody and stable transfectomas secreting chimeric bovine-murine IgA, IgE, or IgG subclass anti-dansyl antibodies were obtained. The chimeric antibodies, immunoprecipitated with Ag or with anti-bovine H chain antibodies, were analyzed by SDS-PAGE and were shown to contain H and L chains of expected size. Of the three chimeric antibodies derived from the C gamma genes, one reacted with anti-IgG1 antibody, another reacted with anti-IgG2 antibody and the third did not react with either anti-IgG1 or anti-IgG2. This third IgG appears to represent a "new" subclass of bovine IgG, IgG3. Southern blot analysis indicated that the bovine genome contains a fourth C gamma gene. These experiments demonstrate the usefulness of molecular genetic techniques for the isolation and characterization of Ig which are not readily purified from biologic fluids. These techniques will be useful for isolation and characterization of Ig genes from other outbred mammals.

Animals↗
PubMed1988

The rib gap anomaly in partial or mosaic trisomy 8.

Gaps in the first ribs were observed in two children, one with partial and the other with mosaic trisomy for chromosome 8. The sign may be considered in conjunction with other features as a relative indication for chromosomal studies.

Bone and Bones↗
PubMed1987

The use of biotinylated monoclonal antibodies and streptavidin affinity chromatography to isolate herpesvirus hydrophobic proteins or glycoproteins.

A streptavidin/biotin-based immunoaffinity system was optimized to isolate herpesvirus (human cytomegalovirus) immediate early proteins or late glycoproteins from crude infected cell lysates. Biotinylation of the primary antibody by biotin substitution of epsilon amino groups was superior to biotin substitution of sugar residues. Biotinylation of the primary antibody was superior to that of a secondary antibody. A biotin substitution of approximately 8 M biotin/M antibody allowed for maximal recovery of viral antigens. The streptavidin/biotin-based immunoaffinity system can allow for relatively pure preparations of viral antigens that may be used for functional, immunological, or structural studies.

Antibodies, Monoclonal↗
PubMed1987

Long-term cultivation of canine keratinocytes.

The growth characteristics and morphology of canine keratinocytes grown in vitro were studied. Keratinocytes from canine oral mucosa, ear skin, and ventral abdominal skin were grown in culture either as explants or as dispase/trypsin-derived suspensions in the absence of a feeder cell layer. Cholera toxin and epidermal growth factor were essential to the successful long-term growth and propagation of the cells during multiple passages. Keratinocytes from all tissue sources, either as primary cultures or subcultivated for up to 10 passages, had growth characteristic and morphology similar to that reported in other species. The use of cultured canine keratinocytes should provide a suitable model for comparative in vitro studies of the pathogenesis of dermatologic diseases.

Abdomen↗
PubMed1987

The immunochemistry of solid-phase sandwich enzyme-linked immunosorbent assays.

Multivalent antigens (Ags) such as membrane proteins can be quantitated by using sandwich enzyme-linked immunosorbent assays (ELISAs), which typically show sensitivity from 0.1 to 50 ng/ml. The percentage of antigen that binds in the log-log linear region reflects the affinity of the capture antibody (CAb), and the range of linearity for assays conducted with a particular CAb is proportional to the antibody (Ab) concentration. The sandwich ELISA titration plot reflects the actual amount of Ag bound when asymmetrical configurations are used; steric hindrance that occurs with certain symmetrical configurations, especially when enzyme-Ab conjugates of greater than or equal to 10(6) daltons are used, can alter this relationship. Monoclonal CAbs bind less Ag than polyclonal CAbs. Immobilization of monoclonal CAbs by using a modified avidin-biotin system can result in greater antigen capture capacity (AgCC) than when the Abs are directly adsorbed on plastic. Adsorption of proteins on polystyrene is noncovalent and proportional to the amount added for up to 150 ng/200 microliter in a microtiter well. Adsorption can result in substantial loss of antigenic or antibody activity. Desorption is continuous at a low level and can negatively influence the results of an immunoassay. Data from microtiter sandwich ELISAs can be readily acquired and analyzed by using a computer-based analysis system (ELISANALYSIS) written for the IBM PC. This analytical system considers the immunochemical principles of sandwich ELISAs predicted theoretically and demonstrated empirically.

Adsorption↗
PubMed1986

The immunochemistry of sandwich ELISAs. II. A novel system prevents the denaturation of capture antibodies.

The studies reported here describe a model system by which the effect of surface adsorption on the antigen capture capacity (AgCC) of monoclonal antibodies (MoAbs) can be quantitated. Three mouse anti-fluorescein (FLU) MoAbs were biotinylated and iodinated with [125I]Na. The AgCC/ng of these MoAbs was compared when they were adsorbed directly on plastic or immobilized via a Protein Avidin Biotin Capture (PABC) system. In this system, biotinylated MoAbs were allowed to adsorb on biotinylated carrier proteins through a "bridging" process using Streptavidin. This permits MoAbs to complex with antigen at some distance from the plastic surface. The AgCCs of the MoAbs immobilized using the PABC system were 5-400-fold higher than when they were directly adsorbed on plastic.

Antibodies, Monoclonal↗
PubMed1986

The immunochemistry of sandwich ELISAs--I. The binding characteristics of immunoglobulins to monoclonal and polyclonal capture antibodies adsorbed on plastic and their detection by symmetrical and asymmetrical antibody-enzyme conjugates.

Radiolabelled bovine IgG1, IgG2, SIgA and IgM and heavy-chain specific polyclonal and monoclonal antibodies to these isotypes were employed as models to investigate immunochemical aspects of sandwich enzyme immunoassays (ELISAs). The titration plots obtained by measuring enzyme activity paralleled those obtained when the binding of radiolabelled immunoglobulins to solid-phase capture antibodies was quantitated. As predicted from the Mass Law, the percentage of labelled immunoglobulin which was bound remained constant over the range in which the sandwich ELISA titration was linear on a log-log plot. Also as predicted from the Mass Law, increasing the solid-phase concn of polyclonal antibodies by affinity purification increased the linear region of the log-log ELISA plot and the corresponding region over which a constant percentage of immunoglobulin binding was observed. When used as capture antibodies adsorbed on plastic at equal concns, the best monoclonal antibodies were 1/8- less than 1/16 as effective as their polyclonal counterparts in binding iodinated bovine immunoglobulins; these differences can be directly interpreted to result from an 8 and greater than 16-fold higher functional, relative affinity of the polyclonal reagents. Steric hindrance was shown to occur when symmetrical sandwich ELISAs, i.e. capture and detection antibody are both heavy-chain specific, are used to measure monomeric but not IgM immunoglobulins. The use of an asymmetrical configuration, i.e. anti-Fab antibody-enzyme conjugates, avoids this problem. Symmetrical conjugates based on the avidin-biotin system, horseradish peroxidase or alkaline phosphatase, were less effective than their asymmetrical (anti-Fab) counterparts. Evidence that the lower activity of symmetrical conjugates was due to steric hindrance was illustrated using horseradish peroxidase-antibody conjugates of different sizes. Sandwich assays using affinity-purified, polyclonal solid-phase antibodies and an asymmetrical conjugate were judged to be immunochemically and economically optimal. Using an asymmetrical configuration, the non-linear nature of sandwich ELISA titration plots is the predictable result of changing antibody to antigen ratios in an antibody-limiting system, and not the result of steric hindrance of the detection system.

Animals↗
PubMed1985

An ELISA assay efficiently detects clonal antibody formation by single, hapten-specific B lymphocytes.

Spleen cells from non-immunized adult mice were fractionated on thin layers of fluorescein (FLU)-gelatin to yield FLU-specific B cells. These B cells were cultured either singly or in very small numbers in 10 microliter microcultures with 0.1 microgram/ml of the T cell-independent (TI) antigen FLU-E. coli lipopolysaccharide (FLU-LPS). Cultures were either filler cell-free, or supported by the addition of 10(5) CBA/N thymus cells per well. At 4-6 days, culture supernatants were assayed for the presence of anti-FLU antibody either by an enzyme-linked immunosorbent assay (ELISA) or a radioimmunoassay (RIA). With the filler cell-free cultures, B cell proliferation was scored microscopically before removal of culture supernatant. The cultured cells from each well were assayed for their capacity to form directly hemolytic FLU-specific plaques. In the filler cell-free system, the ELISA was much more sensitive than the plaque assay in identifying antibody-forming cell (AFC) clones, with over 10% of fractionated B cells yielding clones secreting detectable antibody, though with a low mean optical density (OD). This value represented over 80% of the proliferating clones. In the more efficient, filler cell-supported system, the difference between the 2 read-out methods was smaller. Here, one half of the hapten-specific B cells formed AFC clones, the highest cloning efficiency yet reported for an antigen-driven system. Comparative studies showed the RIA to be only marginally more sensitive than the ELISA, and not nearly as convenient for routine use.

Animals↗
PubMed1985

A study of optical zone size and incision redeepening in experimental radial keratotomy.

The effects of optical clear zone size and incision redeepening following four- and eight-incision radial keratotomy were studied in a series of human cadaver eyes. Keratometry was measured with both the Terry keratometer and corneascope photographs. After eight incisions, induced corneal flattening for each clear zone (Terry keratometry readings) was 3.0 mm, 9.06 diopters; 4.0 mm, 6.44 D; 5.0 mm, 5.23 D; and 6.0 mm, 2.29 D. Differences were statistically significant, except for the 4.0-mm and 5.0-mm zones. Induced corneal flattening following eight incisions (corneoscopy) was 3.0 mm, 9.83 D; 4.0 mm, 8.87 D; 5.0 mm, 6.04 D; and 6.0 mm, 2.59 D. Differences were statistically significant, except for the 3.0-mm and 4.0-mm zones. Peripheral redeepening of the incisions did not significantly increase the amount of corneal flattening for any optical zone, as measured by the Terry keratometer. However, the 3.0-mm optical zone flattened an additional 1.23 D after the redeepening incisions, as measured by corneoscopy. The redeepening incisions were complicated by perforations in 38% of the eyes. Titrating the effect of radial keratotomy by varying the size of the optical zone appears to be valid, but the value of peripheral redeepening in the acute laboratory situation is not supported by these data.

Cornea↗
PubMed1985

Stimulation of immunoglobulin-containing cells and isotype-specific antibody response in experimental Mycoplasma hyopneumoniae infection in specific-pathogen-free pigs.

The development of immunoglobulin-containing cells and antigen-specific plasma cells in retropharyngeal and bronchial lymph nodes, in lung tissue, and in nasal mucous membrane was followed during an experimental infection of pigs with Mycoplasma hyopneumoniae. An enzyme-linked immunosorbent assay was used to follow stimulation of the immunoglobulin isotypes (classes) M, G, and A directed against the infectious agent in tracheobronchial secretions and systemically. A significant increase of antigen-specific plasma cells in lymph nodes and lung tissue as well as antibodies in tracheobronchial secretions was demonstrated 2 weeks after infection. This specific reaction was followed by a marked increase of immunoglobulin-containing cells in lymph nodes and lung tissue. The data are discussed in terms of autoimmune reactions in the pathogenesis of enzootic pneumonia.

Animals↗
PubMed1984

Regulation of Sulfate Assimilation by Nitrogen Nutrition in the Duckweed Lemna minor L.

The effect of nitrate and ammonium on the extractable activity of two enzymes of assimilatory sulfate reduction, ATP sulfurylase (EC 2.7.7.4) and adenosine 5'-phosphosulfate sulfotransferase (APSSTase), was examined in Lemna minor L. cultivated under steady state conditions. Nitrate reductase (EC 1.6.6.1) was measured for comparison. Low nitrate concentrations (0.2 and 0.04 millimolar) caused a decrease in the specific activity of all three enzymes measured. Twenty-four hours after transfer to medium without a nitrogen source, the specific activity of APSSTase and nitrate reductase was at less than 30% of the original level, whereas ATP sulfurylase was still at about 80%. NH(4) (+) added to the nutrient solution caused a 50 to 100% increase in the specific activity of APSSTase within 24 hours, followed by a slow decrease. After 72 hours with NH(4) (+), the specific activity was still 25% higher than originally. During the same period, the extractable protein increased by 30% on a fresh weight basis, and total protein by 55 to 60%. Nitrate reductase activity decreased to less than 5%. After omission of NH(4) (+) from the nutrient solution extractable APSSTase activity rapidly decreased to the level of cultures with NO(3) (-) as a nitrogen source. Using [(35)S]SO(4) (2-) as a sulfur source, an increased incorporation of label into the protein fraction could be detected when NH(4) (+) was added to the nutrient solution. This indicated that more sulfate was assimilated and used for protein synthesis. The higher extractable activity of APSSTase with NH(4) (+) may be a regulatory mechanism involved in the formation of sufficient sulfur amino acids during a period of increased protein synthesis.

Journal Article↗