ABSTRACT
Pathogen detection and genetic characterization has dramatically changed in recent years. Clinical laboratories are transitioning from traditional culture and primer-specific sequencing to more robust and rapid nucleic acid testing such as real-time PCR and meta-genomic characterization, respectively. Specimen collection is the first step in any downstream molecular diagnostic procedure. PrimeStore Molecular Transport Medium (MTM) is an optimized blend of nucleic acid stabilizing reagents that includes a non-specific internal positive control that can be amplified using real-time RT-PCR for tracking the integrity of a specimen from the point of collection to detection. PrimeStore MTM is shown here to effectively kill pathogens, including highly pathogenic H5 influenza virus, inactivate nucleases and to protect and preserve released RNA at ambient temperature for up to 30 days for downstream real-time and traditional RT-PCR detection and genetic characterization. PrimeStore MTM is also compatible with a variety of commercial extraction kits. PrimeStore is suited for routine clinical specimens and has added utility for field collection in remote areas, triage centres, border crossings and during pandemics where cold-chain, transport, and dissemination of potentially infectious pathogens are a concern.
Subject(s)
Pathology, Molecular/methods , Polymerase Chain Reaction/methods , Reagent Kits, Diagnostic , Specimen Handling/methods , Anti-Infective Agents/chemistry , Anti-Infective Agents/pharmacology , Bacteria/drug effects , Fungi/drug effects , Genomics , Humans , Influenza A Virus, H5N1 Subtype/drug effects , Influenza A Virus, H5N1 Subtype/genetics , Influenza A Virus, H5N1 Subtype/isolation & purification , Influenza, Human/diagnosis , Influenza, Human/virology , Laboratory Chemicals/chemistry , Laboratory Chemicals/pharmacology , RNA, Viral/analysis , RNA, Viral/chemistry , RNA, Viral/isolation & purification , Reference Standards , Virus Inactivation/drug effectsABSTRACT
We have developed a rapid and inexpensive approach to remove unconjugated protein from protein-polysaccharide conjugate vaccines, without using gel filtration or ultrafiltration. We employ porous particles that adsorb the protein, whether bound or free, but with a pore size that allows only the unconjugated protein to enter the particle. Using limited amounts of media there is preferential binding of the unconjugated protein over the high molecular weight protein-polysaccharide conjugate. Adsorption of the unconjugated protein is rapid, with greater than 90% recovery of the conjugate. The approach is applicable to both neutral and charged polysaccharides and is not dependent on the chemistry used to make the conjugate vaccine. We have used this method to prepare tetanus toxoid-polysaccharide conjugates and found their immunogenicity in mice comparable to conjugates prepared using gel filtration. The method described can be used to reduce the cost and increase the yields of protein-polysaccharide conjugate vaccines.
Subject(s)
Vaccines, Conjugate/isolation & purification , Adsorption , Animals , Cattle , Dextrans/isolation & purification , Female , Humans , Mice , Mice, Inbred BALB C , Molecular Weight , Particle Size , Polysaccharides, Bacterial/isolation & purification , Proteins/isolation & purification , Resins, Synthetic , Serum Albumin, Bovine/isolation & purification , Silicon Dioxide , Tetanus Toxoid/isolation & purificationABSTRACT
Covalently linking protein to polysaccharides converts the anti-polysaccharide immune response from a T-cell independent response to one which is T-cell dependent. The organic cyanylating reagent 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) (Vaccine 14:190, 1996) has been used to activate polysaccharides, which can then be reacted with spacer reagents or directly with protein. We wished to explore ways in which proteins could be linked to CDAP-activated polysaccharides to conjugate in a more controlled and selective fashion. To this end, we examined the reaction of nucleophilic amino acids with CDAP-activated polysaccharides under basic and acidic conditions. We found that lysine, cysteine and histidine but not methionine, serine or tyrosine conjugated to CDAP-activated dextran. We also examined the reaction of various spacer reagents with CDAP-activated dextran as a function of pH. The addition of hexanediamine was highly pH dependent and maximal at pH 9.3. In contrast, the addition of adipic dihydrazide, which has a pKa of ca 2.5 was essentially independent of pH. By performing the conjugation reaction at pH 5, we were able to selectively couple hydrazides even in the presence of high concentrations of amines. Proteins derivatized with limited numbers of hydrazides could be conjugated to CDAP-activated polysaccharides at pH5, where the native protein was not reactive. Proteins could be derivatized with hydrazides on carboxyls using adipic dihydrazide and a water soluble carbodiimide or on amines using a mild two-step reaction. Tetanus toxoid-pneumococcal type 14 conjugates produced by coupling hydrazide-derivatized tetanus toxoid under acidic conditions induced anti-polysaccharide antibodies at titers comparable to that stimulated by conjugates produced using a basic coupling pH. Our data suggest that crosslinking was occurring only with the limited number of hydrazides on the protein and that we achieved limited and selective crosslinking between the protein and CDAP-activated polysaccharide. This work also demonstrates that CDAP-mediated conjugation to polysaccharides can be applied even to very pH sensitive proteins and polysaccharides.
Subject(s)
Cross-Linking Reagents/chemistry , Nitriles/immunology , Polysaccharides, Bacterial/chemistry , Proteins/immunology , Pyridinium Compounds/immunology , Vaccines, Conjugate/chemistry , Amines/chemistry , Animals , Haptens/chemistry , Haptens/immunology , Hydrazones/chemistry , Hydrogen-Ion Concentration , Mice , Mice, Inbred BALB C , Nitriles/chemistry , Polysaccharides, Bacterial/immunology , Proteins/chemistry , Pyridinium Compounds/chemistry , Solubility , Structure-Activity Relationship , Vaccines, Conjugate/immunologyABSTRACT
A hybrid cell line (E-2) that secretes the enzyme acetylcholinesterase (AChE) has been prepared. The E-2 cell was the product of a fusion between primary mouse hepatocytes and a chemically transformed rat liver cell line (FRL), neither of which expresses AChE activity. The enzyme was determined to be AChE on the basis of its susceptibility to inhibition by BW284c51 but not by iso-OMPA, as well as its substrate specificity. Although the secreted enzyme was salt soluble and its activity not modified by the addition of the nonionic detergent, Triton X-100, the activity of the cellular enzyme (derived from homogenates of E-2 cells) was greatly enhanced in the presence of the detergent.
Subject(s)
Acetylcholinesterase/metabolism , Hybrid Cells/enzymology , Liver/enzymology , Animals , Cell Survival , Cells, Cultured , Culture Media , Detergents/pharmacology , Epidermal Growth Factor/pharmacology , Isoflurophate/pharmacology , Liver/cytology , Mice , Mice, Inbred BALB C , RatsABSTRACT
A new assay has been developed for detection of butyrylcholinesterase (EC 3.1.1.8) activity based upon the change in absorbance of phenol red, caused by the release of butyric acid from the substrate. Using commercially available enzyme prepared from horse serum, linear, dose-related decreases in absorbance were obtained, generally with correlation values of 0.965 or greater. The assay was modified and used to detect enzyme activity in the supernatants from primary cultures of mouse hepatocytes. The enzyme-mediated response was inhibited by NN'-diisopropylphosphorodiamidic anhydride, a specific inhibitor of butyrylcholinesterase.
Subject(s)
Butyrylcholinesterase/analysis , Cholinesterases/analysis , Liver/enzymology , Animals , Cholinesterase Inhibitors , Liver/cytology , Mice , Organophosphates/pharmacologyABSTRACT
Eighteen lots of fetal bovine serum were tested for their ability to support clonal growth and 3-methylcholanthrene-induced morphological transformation of hamster embryo cells in vitro. Most of them supported cloning efficiencies of over 11%. However, cloning efficiency alone was an inadequate criterion for selecting serum for transformation studies, since no transformation was observed with some lots, even though their cloning efficiencies were over 16%. This shows the importance of pretesting serum for its ability to support morphological transformation before it is used in mammalian cell carcinogenesis tests.
Subject(s)
Cell Transformation, Neoplastic/chemically induced , Clone Cells/drug effects , Fetal Blood , Methylcholanthrene , Animals , Cattle , Cell Division , Clone Cells/cytology , Cricetinae , Culture Techniques , Drug Evaluation, Preclinical , LungABSTRACT
Normal human embryonic cells were subcultured for over 100 population doublings without modification of the basic medium. The cells were evaluated for growth rate, confluent density, chromosome stability, growth in soft agar, ability to hydrolyze casein and tumorigenicity. The cells possessed the characteristics of normal cells. The batch of serum used to supplement the medium was found to be of primary importance in the long-term growth of this cell culture.
