Production of Reference Enteroviruses

Forty-five human enterovirus reagents of certified purity and quality were prepared for use as seed viruses and as immunizing antigens. One of the reagents was ampouled as "untreated" seed virus, whereas 14 were ampouled as "MgCl(2)-stabilized" reagents. The remaining 30 reagents were ampouled as "untreated" seed viruses and as "MgCl(2)-stabilized" reagents. Thirty of the reagents were propagated on primary African green monkey kidney cells, 3 on primary baboon kidney cells, 3 on primary rhesus monkey kidney cells, and the remaining 9 on human amnion cells. Forty-two of the viral antigens were concentrated for use in the production of high-titered specific antisera in large animals.     

Chemical modification of proteins: comments and perspectives

The use of chemical modification of proteins has increased exponentially during the past two decades. Today the many different uses of chemical modification include determination of relative reactivities of side chain groups, the quantitation of individual amino acids, development of affinity reagents, mechanism-based reagents for pharmaceutical uses, cross-linking reagents, special techniques for bioprostheses, blocking reagents for peptide synthesis, and reagents for specific cleavages of peptide bonds. Chemical modification should continue to be a primary tool in protein chemistry. It can supply information or products difficult or impossible to attain by the newer powerful technique of in vitro mutagenesis as well as serve as a supplementary procedure for the latter.     

Synthesis of bis- and tris-branched COOH-terminal pegylating reagents: conjugation to NH-terminal peptides.

In previous studies we reported an orthogonal protection scheme that was developed for the solution-phase synthesis of a family of bis- and tris-pegylating reagents which contain a free NH(2)-terminus. These pegylating reagents were coupled to the COOH-terminus of a model peptide. In the present study we report on the solution synthesis of a novel family of bis- and tris-pegylating reagents which contain a free COOH-terminus. To illustrate their general utility, conditions were developed for the coupling of these novel pegylating reagents to the NH(2)-function of a model pentapeptide. Taken together, our studies demonstrate that these pegylating reagents are well suited for conjugation to peptides and proteins that contain either free COOH- or NH(2)-functions. These reagents may have general utility in therapeutic development as branched pegylation has been shown to provide more effective protection of proteins from proteolysis by shielding the protein surface from approaching macromolecules.     

The application of phenylmethanethiol and benzenethiol derivatives as odorless organosulfur reagents in the synthesis of thiosugars and thioglycosides

p-octyloxyphenylmethanethiol and p-dodecylbenzenethiol were prepared as new odorless organosulfur reagents. Thiosugars and thioglycosides were synthesized using these reagents without encountering any malodorous procedures.     

Problems of selecting the reagent concentration in the solid-phase immunoenzyme method for determining antibody concentrations

The problems of selecting the concentration of reagents with the aim of increasing the accuracy and sensitivity of reactions, as well as decreasing the consumption of reagents, are shown as exemplified by the antigen-antibody reaction of the first order. A new approach to the ELISA reaction is proposed, which makes it possible to present the totality of consecutive interactions of the reagents as block diagrams described by known mathematical expressions. The limitations of the linear dependence of the results of the reaction are shown, and the methodological recommendations for overcoming these limitations are given.     

Electrospray mass spectra of three proprietary detergents

We have determined the major ingredients of the commercially available reagents M-PER, Y-PER, and B-PER from Pierce Chemical Co. using electrospray mass spectrometry. These three proprietary reagents have been widely used in the biochemical community as cell membrane dissolving tools during the initial step of protein purification. However, the identity and mechanism of these reagents remained unknown. In this paper, we identified these reagents as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, and n-octyl-beta-d-thioglucopyranoside, respectively. In addition, we wish to stress here the increasing importance of the role of electrospray mass spectrometry in the analysis of such proprietary biological preparations which are increasingly finding their way into the biochemical literature.     

Determination of relative protein abundance by internally normalized ratio algorithm with antibody arrays

In this paper, we report an experimental setup and mathematical algorithm for determination of relative protein abundance from directly labeled native protein samples applied to an array of antibodies. The application of the proposed experimental system compensates internally at each array element for a number of deficiencies in array experiments such as differential labeling efficiency in dual color assay systems, differential solubility of protein molecules in dual color assay systems, and differential affinity of capture reagents toward proteins labeled with two different fluorescent dyes. This system offers full compensation for variable amounts of capture reagents on separate array structures, as well as limited compensation for nonspecific interactions between capture reagents and analytes. The proposed experimental strategy enables the use of a large number of capture reagents to develop a true multiplex analysis system that will yield complete relative protein abundance information in two biological systems.     

Standardization: a Progress Report

The introduction of nucleic acid amplification technology (NAT) assays for the detection of viral contamination of blood and blood products requires the availability of well-characterized reference reagents. Working reagents for hepatitis C virus RNA, hepatitis B virus DNA, HIV-1 RNA and human parvovirus B19 DNA have been established at NIBSC and at many other laboratories (both official medicinal control laboratories and commercial laboratories). However, as these reagents have been characterised independently, it is difficult to compare results from assays using different working reagents. Recently, a WHO International Standard was established for HCV RNA NAT assays. This standard has been calibrated in International Units (IU) and provides a common standard against which all working reagents can be calibrated. Collaborative studies to characterise two further candidate International Standards for HBV DNA and HIV-1 RNA NAT assays have been completed.     

Efficient synthesis of functionalized oligodeoxyribonucleotides with base-labile groups using a new silyl linker

In this study, we developed new 3′-terminal deoxyribonucleoside-loading reagents 1 with a new silyl-type linker. These reagents could increase the efficiency of introduction of 3′-terminal deoxyribonucleoside components into polymer supports to a level of 17–29 μmol/g. The efficiency was higher than that of previous T-loading reagents because reagents 1 contain a 4-aminobutyryl residue as a spacer. Moreover, we could synthesize not only unmodified DNA oligomers but also a base-labile modified DNA oligomer using resins 9a–d in the activated phosphite method without base protection.     

Use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) for synthesis of phosphorothioate-containing oligodeoxyribonucleotides.

Previous methods for the preparation of phosphorothioate-containing oligodeoxyribonucleotides rely on the reaction of phosphite triesters with sulfurizing reagents such as tetraethylthiuram disulfide (TETD) and 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent). However, these and other sulfurizing reagents suffer from several disadvantages, and there is great impetus for the development of improved methods for sulfur transfer that are fully compatible with standard automated DNA synthesis. The present report describes the use of 1,2,4-dithiazolidine-3,5-dione (DtsNH) and 3-ethoxy-1,2,4-dithiazoline-5-one (EDITH) as effective sulfurizing reagents that meet these needs. Both reagents are easily prepared, and are stable upon prolonged room temperature storage in acetonitrile solution. The reagents are used at low concentrations (0.05 M) and for short reaction times (30 s). The methodology has been proven for the automated synthesis on 0.2-1.0 micromol scales of oligodeoxyribonucleotides, of length 6-20 bases, containing the phosphorothioate substitution at either a single site or at all positions.     

Evaluation of ready-to-use liquid reagents for clinical chemistry in laboratory animals.

We evaluated the ready-to-use liquid reagents for clinical chemistry (6 tests), to assess their suitability for use in the toxicology laboratory setting. Hitachi 736 automated analyzer was used for the analyses. The evaluation included the following studies: Precision, Linearity, Effects of interference substances such as hemolytic hemoglobin, bilirubin, turbidity to the analytical values and correlation to the solid reagents, which are prepared each time they are needed. The precision and linearity data were within the reagents' specifications. Results of comparison of the liquid reagents and the solid reagents in analyzing plasma samples of rats, dogs and monkeys were generally good except for a bias in results for GOT and GPT, regardless of the animal species tested. It is concluded that these types of liquid reagents can be used in clinical pathology examinations in animal studies.     

Use of colour reactions to identify lipid classes, including tocopherols, by thin-layer chromatography]

Colour reactions of different classes and individual lipids of plant origin with organic and inorganic compounds (used as reagents) were studied to identify them in thin-layer silica gel. Some of the reagents (p-dimethyl phenylene diamine, chromotropic acid in H2SO4, Ti(SO4)2 in H2SO4) helped to detect main lipid classes and to identify selectively individual compounds (alpha- and gamma-tocopherols). Hydrochloric p-nitrophenyl diazonium, p-dimethyl aminobenzaldehyde are of interest mainly as group reagents to lipid classes with common properties. The reaction sensitivity is measured in micrograms.     

The presence of non-isotype-specific antibodies in polyclonal anti-IgE reagents: demonstration of their binding to specifically selected Epstein-Barr virus-transformed cell lines

Polyclonal anti-human IgE reagents were earlier shown to contain variable amounts of nonisotype-specific antibodies depending on the strategy used for their preparation. The presence of these antibodies in two commercial anti-IgE reagents was demonstrated in this work by (a) their binding to human Ig-surface-positive lymphoblastoid cells specifically selected by one of the polyclonal anti-human IgE reagents and (b) their binding to the non-IgE immunoglobulins secreted by those lymphoblastoid cells. Peripheral blood B lymphocytes from two normal and two atopic patients were immortalized with Epstein-Barr virus (EBV) and then selected for cells that rosette with anti-IgE-coated erythrocytes. Selection was repeated four times and cells were then cloned. The cloned cells formed rosettes and their supernatants agglutinated erythrocytes coated with rabbit anti-IgE. The immunoglobulins of these clones were positive in an ELISA for IgE, using two different polyclonal anti-human IgE reagents. They were shown, however, to be 19 S IgMs. This discrepancy was due apparently to substantial contamination of anti-non-IgE-isotype-specific antibodies in the polyclonal anti-IgE reagents used both in the selection of cells and in the ELISA. The human monoclonal B-cell lines which were applied here as targets amplified the non-IgE-isotype specific antibody contamination present in the polyclonal anti-human IgE reagents. Because of the normally very low frequency of IgE-positive cells, the use of polyclonal anti-IgE reagents to detect these cells has to be carefully evaluated.     

Practical Synthesis of Cytidine-5-Carboxamide-Modified Nucleotide Reagents

Chemically-modified derivatives of cytidine, bearing a 5-(N-substituted-carboxamide) functional group, are new reagents for use in aptamer discovery via the SELEX process (Systematic Evolution of Ligands by EXponential enrichment). Herein, we disclose a practical synthesis of 5-(N-benzylcarboxamide)-2′-deoxycytidine, and the corresponding 5-(N-1-naphthylmethylcarboxamide)- and 5-(N-3-phenylpropylcarboxamide)-2′-deoxycytidine analogs, as both the suitably-protected 3′-O-cyanoethylphosphoramidite reagents (CEP; gram scale) and the 5′-O-triphosphate reagents (TPP; milligram-scale). The key step in the syntheses is a mild, palladium(0)-catalyzed carboxyamidation of an unprotected 5-iodo-cytidine. Use of the CEP reagents for solid-phase oligonucleotide synthesis was demonstrated and incorporation of the TPP reagents by KOD polymerase in a primer extension assay confirmed the utility of these reagents for SELEX. Finally, the carboxyamidation reaction was also used to prepare the nuclease-resistant sugar-variants: 5-(N-benzylcarboxamide)-2′-O-methyl-cytidine and 5-(N-3-phenylpropylcarboxamide)-2′-deoxy-2′-fluoro-cytidine.     

Aptamer mediated siRNA delivery

Nucleic acids that bind to cells and are subsequently internalized could prove to be novel delivery reagents. An anti-prostate specific membrane antigen aptamer that has previously been shown to bind to prostate tumor cells was coupled to siRNAs via a modular streptavidin bridge. The resulting conjugates could be simply added onto cells without any further preparation, and were taken up within 30 min. The siRNA-mediated inhibition of gene expression was as efficient as observed with conventional lipid-based reagents, and was dependent upon conjugation to the aptamer. These results suggest new venues for the therapeutic delivery of siRNAs and for the development of reagents that can be used to probe cellular physiology.     

Resolution of chiral drugs by liquid chromatography based upon diastereomer formation with chiral derivatization reagents

Chiral derivatization reagents for resolution of biologically important compounds, such as chiral drugs by high-performance liquid chromatography (HPLC), based upon pre-column derivatization and diastereomer formation, are reviewed. The derivatization reagents for various functional groups, i.e., amine, carboxyl, carbonyl, hydroxyl and thiol, are evaluated in terms of reactivity, stability, wavelength, handling, versatility, sensitivity, and selectivity. The applicability of the reagents to the analyses of drugs and bioactive compounds are included in the text.     

Bifunctional reagents as vapour- and liquid-phase fixatives for immunohistochemistry

Synopsis Out of a total of eleven bifunctional reagents tested as fixatives for light microscopic immunohistochemistry, four were found satisfactory when applied in the vapour phase to freeze-dried blocks. These were diethylpyrocarbonate, as observed in carlier studies, dimethyladipimidate,p-benzoquinone, and diacetyl. Adequate but less satisfactory liquid-phase fixation was provided by three reagents (dimethyladipimidate, dimethylsuberimidate,p-benzoquinone).None of the eleven reagents gave optimal preservation of antigenicity and structure when tested at the ultrastructural level. Encouraging results were obtained withp-benzoquinone, however.     

Crosslinking studies with different length dithiobisalkylimidates (I) solubilized erythrocyte spectrin

A series of different length dithiobisalkylimidate homologues was synthesized $\text{via}$ Bunte salts from corresponding ω-chloronitriles. And by using these reagents, crosslinking of human erythrocyte spectrin was examined. Shorter reagents formed three bands in the region expected for a dimer, all of which were identified as the bands 1–2 associate: bands 1 and 2 polypeptides were crosslinked only each to one another and not to itself. Longer reagents, however, produced higher multimers, which was enhanced by higher temperatures and divalent cations, and depressed by Triton X-100 and deoxycholate. The results suggest the validity of the chain length of the reagents in discriminating between dimer and multimer of spectrin.     

Evidence for the existence of a channel in the glucose-specific carrier EIIGlc of the Salmonella typhimurium phosphoenolpyruvate-dependent phosphotransferase system

The effect of membrane-impermeable sulfhydryl reagents on glucose-specific enzyme II (EIIGlc) activity has been studied in Salmonella typhimurium whole cells and in properly sealed inverted cytoplasmic membrane vesicles. Glutathione N-hexylmaleimide and N-polymethylenecarboxymaleimides inactivate methyl alpha-D-glucopyranoside (alpha-MeGlc) transport and phosphorylation in whole cell preparations at a dithiol that can be protected by oxidizing reagents, trivalent arsenicals, or phosphorylation of EIIGlc. Accessibility to this activity-linked site is restricted to small apolar reagents or to polar reagents with a hydrophobic spacer between the polar group and the reactive maleimide moiety. These same reagents inactivate alpha-MeGlc phosphorylation in inverted cytoplasmic membrane vesicles. Inhibition results from reaction at a dithiol that can be protected by nonpermeant mercurials, oxidants, and arsenicals as well as by phosphorylation of EII. The characteristics of this site are virtually identical with those of the activity-linked dithiol elucidated in intact cells. No evidence could be found for a second activity-linked site on the other side of the membrane when the permeable reagent N-ethylmaleimide was used. Since only one activity-linked dithiol can be detected with sealed inverted membrane vesicles or intact cells and it is accessible to membrane-impermeable sulfhydryl reagents from both sides of the cytoplasmic membrane, we suggest that it is located in a channel constructured by the carrier and that the channel spans the membrane. A second dithiol, not essential for activity, is located near the outer surface of the cytoplasmic membrane.     

A transfection compound series based on a versatile Tris linkage

The family of cationic lipid transfection reagents described here demonstrates a modular design that offers potential for the ready synthesis of a wide variety of molecular variants. The key feature of these new molecules is the use of Tris as a linker for joining the hydrophobic domain to a cationic head group. The molecular design offers the opportunity to conveniently synthesise compounds differing in charge, the number and nature of hydrophobic groups in the hydrophobic domain and the characteristics of the spacer between the cationic and hydrophobic moieties. We show that prototype reagents of this design can deliver reporter genes into cultured cells with efficiencies rivaling those of established cationic lipid transfection reagents. A feature of these reagents is that they are not dependent on formulation with a neutral lipid for activity.