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84901970965
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note
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A chemiluminescence, solid-phase multiallergen immunoassay (Magic Lite, ALK, Copenhagen-CIBA-Corning, Medfield, MA) was used to measure IgE Ab concentrations. The solid phase comprised standardized quality (SQ) extracts of the following 20 common aeroallergens: grass pollens (Cynodon dactylon, Lolium perenne, and Phleum pratense), tree pollens (Betula verrucosa, Ouercus alba, Olea europeae, and Cryptomeria japonica), weed pollens (Ambrosia artemisiifolia, Artemisia vulgaris, Plantago lanceolata, and Parietaria officinalis), house-dust mites (D. pteronyssinus and D. farinae), animal danders (cat, dog, and horse), cockroach (Blattella germanica), and molds (Alternaria alternata, Cladosporium herbarum, and Aspergillus fumigatus). The 20 extracts (each containing 20 or more distinct allergens) were coupled separately to activated, paramagnetic particles, and the immunosorbents were standardized against controls. The immunosorbents were then blended, and the mixture was further standardized. Serum or plasma samples were allowed to incubate with the multiallergen particles and washed, and then with monoclonal Ab to human IgE labeled with acridinium ester. The particles were washed and transferred to an autoanalyzer, and the measured relative light units (RLUs) were compared with the RLUs of a standard serum pool of known relative Ab content. Two further similar immunoassays were performed with solid-phase SQ standardized extracts of the house-dust mites, D. pteronyssinus (Der p) and D. farinae (Der f). The IgE Ab concentrations were expressed in terms of arbitrary units per milliliter that were internally consistent for the different IgE Abs. The lower limits of detectability were similar for the multiallergen Ab and for each of the mite Abs. Assays were performed in duplicate and values differing by ≥10% were repeated until the coefficient of variation was within 10%. All subjects for whom IgE Abs were not detectable were assigned the lowest limit of detectability
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0024582686
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DNA microsatellite analysis was performed essentially according to J. L. Weber and P. E. May [Am. J. Hum. Genet. 44, 388 (1989)] with B cell lines or lysed white blood cells (WBCs) separated by the Ficoll-Hypaque density gradient or phytohemagglutinin techniques
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2, 0.25 U of Taq polymerase (Promega) in tris buffer at pH 9.0, and the mixture was overlayed with 20 μl of mineral oil. The samples were heated for 5 min at 94°C, and PCRs were carried out (Hybaid thermocycter) with 40 cycles of denaturation (45 s, 94°C), which was followed by primer-annealing (45 s at the temperatures indicated) and extension (45 s, 72°C), and then a final extension for 10 min at 72°C. Electrophoresis was performed in urea-formamide acrylamide gels at 60 W. for 2 to 4 hours. The gels were dried and exposed to Kodak XAR-5 film.
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Biotechniques
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Ehrtich-Kautzky, E.1
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We also analyzed marker IL4-R1 by agarose gel electrophoresis in 4.5% gels (GTG agarose, NuSieve) without radiolabeling, using primers described (18) and the primers of R. Mout, R. Willemze, and J. E. Landegent [Nucleic Acids Res. 19, 3763 (1991)]. For the analysis of the IL4-R1 alleles we utilized data from both acrylamide and agarose gel electrophoresis. IL4-R2, a dimorphic minisatellite also described by R. Mout et al., was studied by agarose gel electrophoresis but was of little value (heterozygosity = 0.09).
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Mout, R.1
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84901970966
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note
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Total IgE concentrations were measured in duplicate on serum or plasma samples from all 170 subjects by immunoassay (IMx Assay, Abbott Labs). Total IgE concentrations are expressed in nanograms per milliliter, relative to a set of standards calibrated against the International Union of Immunological Societies (IUIS) standard, taking 1 IU = 2.42 ng. The sensitivity of the assay was 0.5 ng/ml; values <1.0 ng/ml were set at 1.0 ng/ml for the subsequent statistical analyses. Values differing by ≥10% were repeated until the coefficient of variation was within 10%, except for total IgE concentrations <10 ng/ml, where the coefficient of variation was within 20%.
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41
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J. K. Haseman and R. C. Elston, Behav. Genet. 2, 3 (1972); S.A.G.E. (Statistical Analysis for Genetic Epidemiology) 2.1, Department of Biometry and Genetics, Louisiana State University Medical Center, New Orleans (1992). Though this method was originally developed for noninbred families, the impact of inbreeding on our results is likely to be minimal. First, inbreeding will increase by a modest amount the probability of sharing marker alleles IBD, but because both parents (and some grandparents) were typed as part of this study, the reliance on Hardy-Weinberg assumptions is reduced. All 20 sibships were found to be moderately inbred, with a mean inbreeding coefficient of 0.0158 (slightly more than second cousins). In assessing IBD, parental marker information was incorporated into the analyses Data were missing in three small nuclear families who were the first and second generation of three-generation pedigrees: one parent was deceased in two families and both parents were missing in one family.
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Elston, R.C.2
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Department of Biometry and Genetics, Louisiana State University Medical Center, New Orleans
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J. K. Haseman and R. C. Elston, Behav. Genet. 2, 3 (1972); S.A.G.E. (Statistical Analysis for Genetic Epidemiology) 2.1, Department of Biometry and Genetics, Louisiana State University Medical Center, New Orleans (1992). Though this method was originally developed for noninbred families, the impact of inbreeding on our results is likely to be minimal. First, inbreeding will increase by a modest amount the probability of sharing marker alleles IBD, but because both parents (and some grandparents) were typed as part of this study, the reliance on Hardy-Weinberg assumptions is reduced. All 20 sibships were found to be moderately inbred, with a mean inbreeding coefficient of 0.0158 (slightly more than second cousins). In assessing IBD, parental marker information was incorporated into the analyses Data were missing in three small nuclear families who were the first and second generation of three-generation pedigrees: one parent was deceased in two families and both parents were missing in one family.
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(1992)
S.A.G.E. (Statistical Analysis for Genetic Epidemiology) 2.1
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43
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+ subjects from consideration, the underlying genetic effect on total IgE should be more clearty evident in the sib-pair analyses.
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m repeat in intron 2 of IL4 (35) was also analyzed by agarose gel electrophoresis (19). The length for the shortest, most common allele of 1L4-R1 corresponds to that reported (35).
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note
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We wish to thank the Amish families for their willingness to participate in this study, W. Bias for providing cell and plasma samples from certain Amish families, R. Hamilton for help with the total IgE analyses, J. York-Blasser, RN, and S. Beiler for fieldwork, C. Stewart and G. Hansen for technical assistance, S.-K. Huang and B. Catipovic for discussions, and CIBA-Corning for provision of equipment. This research was supported by NIH grant AI20059. The program package S.A.G.E. is supported by USPHS resource grant 1 P41 RR03655 from the Division of Research Resources.
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