IndiaA specific and sensitive method to quantitate aflatoxin B1-N7-guanine adduct in urine samples by immunoaffinity chromatography coupled with indirect competitive enzyme-linked immunosorbent assay (ELISA) is reported. A novel in vitro method to synthesize an antigen (bovine serum albumin-guanine-aflatoxin B1) and its use to produce polyclonal antibodies specific to the hapten aflatoxin B-N7-guanine are discussed. An indirect competitive ELISA developed to quantitate aflatoxin adduct showed a 50% inhibition at 15.6 pmol aflatoxin B1-N7-guanine (y=66.73+ (-19.8)x, r=-0.997). Interference by aflatoxin B1 was less than 5%, and no interference by guanine, aflatoxin Gι and aflatoxin M1 was observed. An immunoaffinity column was also developed, by using these polyclonal antibodies, for single-step purification of aflatoxin B1-N7-gua-nine. The immunoaffinity column and the indirect competitive ELISA were evaluated and validated by quantitation of aflatoxin B1-N7-guanine in urine from rats dosed with aflatoxin B1 (1 mg/kg body weight). Spiking studies with standard aflatoxin B1-N7-guanine adduct at 2 and 4 μg/mL phosphate- buffered saline gave 96 and 100% recoveries, respectively, for immunoaffinity cleanup column. The method also was tested successfully for quantitating aflatoxin B1-N7-guanine adduct in spiked human urine. Recoveries of the adduct were 79-90%.
Objective: To study clinical, morphological and molecular characteristics in a Swiss family with autosomal dominant familial neurohypophyseal diabetes insipidus (adFNDI). Participants and methods: A 15-month-old girl presenting with symptoms of polydipsia and polyuria was investigated by water deprivation test. Evaluation of the family revealed three further family members with symptomatic vasopressin-de®cient diabetes insipidus. T1-weighted magnetic resonance images of the posterior pituitary were taken in two affected adult family members and molecular genetic analysis was performed in all affected individuals. Results: The water deprivation test in the 15-month-old child con®rmed the diagnosis of vasopressinde®cient diabetes insipidus and the pedigree was consistent with autosomal dominant inheritance. The characteristic bright spot of the normal vasopressin-containing neurophypophysis was absent in both adults with adFNDI. Direct sequence analysis revealed a new deletion (177±179DCGC) in exon 2 of the AVP-NP II gene in all affected individuals. At the amino acid level, this deletion eliminates cysteine 59 (C59D) and substitutes alanine 60 by tryptophan (A60W) in the AVP-NP II precursor; interestingly, the remainder of the reading frame remains unchanged. According to the threedimensional structure of neurophysin, C59 is involved in a disulphide bond with C65. Conclusions: Deletion of C59 and substitution of A60W in the AVP-NP II precursor is predicted to disrupt one of the seven disulphide bridges required for correct folding of the neurophysin moiety and thus disturb the function of neurophysin as the vasopressin transport protein. These data are in line with the clinical and morphological ®ndings in the reported family with adFNDI.
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