ABSTRACT
AIM/HYPOTHESIS: Radiobinding assays (RBA) are unable to differentiate insulin autoantibodies (IAA) from insulin antibodies (IA). We sought to establish whether random peptide phage display might generate reagents with which to distinguish IAA idiotopes from IA idiotopes. METHODS: Two insulin-binding sera were used to select phagotopes from a phage library. The first, designated IAS, came from an insulin-treated patient with the insulin autoimmune syndrome, and was known to contain both IA and a high titre of human insulin specific (B30 threonine dependent) IAA. The second, designated IDD, was taken from a newly-diagnosed IAA(+) Type 1 diabetic patient. Phage colonies selected by insulin-purified IgG extracts of IAS and IDD were selected at random for DNA sequencing, and tested for their reactivity with insulin antibodies and ability to distinguish disease-associated idiotopes. RESULTS: Seven phagotopes bound IAS and the phagotope designated IAS-9, corresponding to sequence KRSRLDV, gave the highest binding standard deviation (SD) score. Seven phagotopes bound IDD and the phagotope designated IDD-10, corresponding to sequence LGRGGSK, bound most strongly. IAS-9 was able to displace insulin binding in IAS and all of ten insulin-treated Type 2 diabetic patients, but not the IAA present in any of the eight patients with newly-diagnosed Type 1 diabetes. IDD-10, on the other hand, could displace insulin binding detected in the sera of eight patients with untreated Type 1 diabetes (IAA), but not in IAS or sera of the insulin-treated Type 2 diabetics. CONCLUSION: Phagotopes provide reagents which between them can distinguish positively as well as negatively diabetes-associated IAA from non-diabetes associated IAA and from IA.
Subject(s)
Autoantibodies/blood , Immunoglobulin Idiotypes/blood , Insulin Antibodies/blood , Insulin/genetics , Insulin/immunology , Amino Acid Sequence , Humans , Insulin/blood , Peptide Fragments/chemistry , Peptide LibrarySubject(s)
Fish Diseases/immunology , Gram-Positive Bacterial Infections/veterinary , Hemolysin Proteins/immunology , Micrococcaceae/pathogenicity , Oncorhynchus mykiss , Salmo salar , Animals , Blotting, Western/veterinary , Disease Outbreaks/veterinary , England/epidemiology , Fish Diseases/epidemiology , Fish Diseases/microbiology , Gram-Positive Bacterial Infections/epidemiology , Gram-Positive Bacterial Infections/immunology , Hemolysin Proteins/genetics , Hemolysis , Kidney Diseases/immunology , Kidney Diseases/microbiology , Kidney Diseases/veterinary , Micrococcaceae/immunologyABSTRACT
Environmental management is inevitably complicated by the large variation in susceptibility to chemical toxicity exhibited by the living components of ecosystems, a significant proportion of which is determined by genetic factors. This paper examines the concept of genetic susceptibility in ecosystems and suggests the existence of two distinct forms reflecting genetic changes at the level of the individual and at the level of population and community. The influence of genetic susceptibility on exposure-response curves is discussed and the consequent accuracy of data used for toxicity test-based risk assessments examined. The paper concludes by describing a possible biomarker-based approach to future studies of susceptibility in ecosystems, suggesting the use of modern molecular genetic methods.
Subject(s)
Ecosystem , Hazardous Substances/toxicity , Animals , Biomarkers , Genetic Variation , HumansABSTRACT
A gene encoding haemolytic activity from Renibacterium salmoninarum (strain PPD) was cloned into Escherichia coli using the cosmid vector pHC79, and subsequently subcloned on a 1.6 kbp SAlI fragment into pBR328. Southern blot hybridisation revealed that a homologous sequence is found in other strains of R. salmoninarum.
