SUMMARY Public health protection requires an indicator of fecal pollution. It is not necessary to analyse drinking water for all pathogens. Escherichia coli is found in all mammal faeces at concentrations of 10 log 9−1, but it does not multiply appreciably in the environment. In the 1890s, it was chosen as the biological indicator of water treatment safety. Because of method deficiencies, E. coli surrogates such as the ‘fecal coliform’ and total coliforms tests were developed and became part of drinking water regulations. With the advent of the Defined Substrate Technology in the late 1980s, it became possible to analyse drinking water directly for E. coli (and, simultaneously, total coliforms) inexpensively and simply. Accordingly, E. coli was re‐inserted in the drinking water regulations. E. coli survives in drinking water for between 4 and 12 weeks, depending on environmental conditions (temperature, microflora, etc.). Bacteria and viruses are approximately equally oxidant‐sensitive, but parasites are less so. Under the conditions in distribution systems, E. coli will be much more long‐lived. Therefore, under most circumstances it is possible to design a monitoring program that permits public health protection at a modest cost. Drinking water regulations currently require infrequent monitoring which may not adequately detect intermittent contamination events; however, it is cost‐effective to markedly increase testing with E. coli to better protect the public's health. Comparison with other practical candidate fecal indicators shows that E. coli is far superior overall.

In this work, we present studies of the covalent structure of human IgG2 molecules. Detailed analysis showed that recombinant human IgG2 monoclonal antibody could be partially resolved into structurally distinct forms caused by multiple disulfide bond structures. In addition to the presently accepted structure for the human IgG2 subclass, we also found major structures that differ from those documented in the current literature. These novel structural isoforms are defined by the light chain constant domain (C L ) and the heavy chain C H 1 domain covalently linked via disulfide bonds to the hinge region of the molecule. Our results demonstrate the presence of three main types of structures within the human IgG2 subclass, and we have named these structures IgG2-A, -B, and -A/B. IgG2-A is the known classic structure for the IgG2 subclass defined by structurally independent Fab domains and hinge region. IgG2-B is a structure defined by a symmetrical arrangement of a (C H 1-C Lhinge) 2 complex with both Fab regions covalently linked to the hinge. IgG2-A/B represents an intermediate form, defined by an asymmetrical arrangement involving one Fab arm covalently linked to the hinge through disulfide bonds. The newly discovered structural isoforms are present in native human IgG2 antibodies isolated from myeloma plasma and from normal serum. Furthermore, the isoforms are present in native human IgG2 with either or light chains, although the ratios differ between the light chain classes. These findings indicate that disulfide structural heterogeneity is a naturally occurring feature of antibodies belonging to the human IgG2 subclass.

While the literature documents the universal occurrence of heterotrophic plate count (HPC) bacteria in soils, foods, air, and all sources of water, there is a lingering question as to whether this group of organisms may signal an increased health risk when elevated populations are present in drinking water. This paper reviews the relevant literature on HPC bacteria in drinking water, the lack of clinical evidence that elevated populations or specific genera within the HPC flora pose an increased health risk to any segment of the population, and the appropriate uses of HPC data as a tool to monitor drinking water quality changes following treatment. It finds no evidence to support health-based regulations of HPC concentrations.

In this communication we present the detailed disulfide structure of IgG2 molecules. The consensus structural model of human IgGs represents the hinge region positioned as a flexible linker connecting structurally isolated Fc and Fab domains. IgG2 molecules are organized differently from that model and exhibit multiple structural isoforms composed of (heavy chain-light chain-hinge) covalent complexes. We describe the precise connection of all the disulfide bridges and show that the IgG2 C H1 and C-terminal C L cysteine residues are either linked to each other or to the two upper hinge cysteine residues specific to the IgG2 subclass. A defined arrangement of these disulfide bridges is unique to each isoform. Mutation of a single cysteine residue in the hinge region eliminates these natural complexes. These results show that IgG2 structure is significantly different from the conventionally accepted immunoglobulin structural model and may help to explain some of the unique biological activity attributed only to this subclass.

In this paper, a wide range of antibodies from various subclasses and subfamilies are employed to evaluate the creation of generic separation processes using Protein A chromatography. The reasons for elution pH differences amongst several IgG1s, IgG2s, antibody fragments, and Fc-fusion proteins during Protein A chromatography are investigated using several complimentary techniques. The results indicate that variable region interactions play a major role in determining elution pH for V H 3 subfamily antibodies while using traditional protein A chromatographic materials. On the other hand, experiments with a resin which employs a ligand consisting solely of B domain of Protein A indicate that variable region interactions can be mitigated, enabling the use of a single elution pH for a range of antibodies. Finally, the moderation of elution conditions associated with this engineered ligand are shown to minimize problems associated with low pH induced aggregation. It is expected that the findings reported in this paper will facilitate faster process development cycle times for this important class of human therapeutics.