In vitro antigen ELISA for quality control of tetanus vaccines

Consistency of production is recognised as an important aspect of vaccine manufacture and suitably validated in vitro assays are required for quality control testing of these products. For the manufacture and batch release of tetanus vaccines, antigen content and integrity, and degree of adsorption of antigen to the adjuvant are critical parameters that should be monitored for consistency. Here we describe the development and use of an Enzyme Linked Immunosorbent Assay (ELISA) to quantify tetanus antigen in combined vaccine products and to measure the degree of adsorption of antigen to adjuvant. Whilst the antigen assay cannot be assumed to predict potency for different products, it can be used as part of a panel of in vitro methods to provide a more informative product profile and to monitor trends in production. The antigen assay is particularly valuable for providing quantitative information on every final lot when modifications of in vivo potency tests, such as single dilution assays, are used.     

Opinions of UK Rescue Shelter and Rehoming Center Workers on the Problems Facing Their Industry

Animal shelters exist worldwide to care for and rehome unwanted or straying pets. Previous studies have examined why owners breed unwanted animals, or relinquish their pets to shelters. However, the views of shelter workers, who receive and care for these animals, have previously been largely unexplored. The aim of this study was to investigate the perceptions of animal shelter workers on the problems facing their industry. A sampling frame was constructed, consisting of every identified shelter in the UK, and a postal questionnaire sent to each. This included two open questions, soliciting respondents’ views on their biggest problems, and inviting further comments. A total of 661 respondents replied to at least one question. Thematic analysis on the free text content was carried out, and basic and global themes identified. Respondents’ main concerns centered on a mismatch between the continuous demand for their services and their limited resources, which has worsened during the recent financial crisis. Respondents perceived a need for increased public awareness of the commitment involved in keeping a pet, and of controlling breeding by neutering. Points of intervention, such as education programs, were suggested. Coordinating efforts with others, including local authorities, landlords, and housing associations, and a potential role for veterinary professionals working in shelter medicine were all explored by respondents. Rehoming organizations play an important role in the management of pet overpopulation, and the views and beliefs of their workers form an important contribution to the dialogue surrounding this issue. Consideration of these perspectives may suggest alternative routes to address underlying causes and management of pet overpopulation.     

Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein α-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not

Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.     

Identification of a gene, closely linked to dnaK, which is required for high-temperature growth of Escherichia coli.

We have constructed four deletion derivatives of the cloned dnaK gene. Plasmid pDD1, in which the last 10 amino acids of the DnaK protein have been replaced by three different amino acids derived from the pBR322 vector, was as effective as plasmid pKP31, from which it was derived, in restoring the ability of a dnaK null mutant, Escherichia coli BB1553, to plate lambda phage and to grow at high temperatures. The other three mutations, involving much larger deletions of the dnaK gene, did not restore the ability to plate lambda phage or the ability to grow at high temperatures. Plasmid pKUC2, which contains the whole dnaK gene and its promoters, was capable of restoring the ability of E. coli BB1553 to plate lambda phage but, surprisingly, it did not restore the ability to grow at high temperatures, even though it was shown that the DnaK protein was efficiently expressed in these cultures. By transposon mutagenesis and sub-cloning, we have shown the presence of a second gene in plasmid pKP31 which is required for high-temperature growth of E. coli BB1553. This gene, which we call htg A, is presumably also defective in the dnaK null mutant E. coli BB1553. We have also demonstrated that the inability of E. coli K756 to grow above 43.5 degrees C is complemented by sub-clones which contain the htg A gene, but not by plasmid pKUC2.     

Classical Analysis of Variance Methods and Nonparametric Counterparts

The present investigation is concerned with the use of classical and nonparametric techniques in the analysis of experiments. Occasionally biometricians are confronted with results arising from an experiment with data which do not necessarily satisfy the assumption of Normality, the basis of the classical analysis of variance methods. Sometimes the biometrician handles such a situation by transformation of the observations and applying the classical techniques. Although this attack may lead to a satisfactory analysis, the results may be questionable in a number of cases. In analyzing continuous data without the Normality assumption nonparametric methods provide realistic alternatives. In this paper a number of problems will be discussed with and without the Normality assumption resulting in the well-known classical analysis and its nonparametric counterparts. Most of the nonparametric procedures to be discussed are based on ranks.