Extensive in vitro cross-reactivity to seed storage proteins is present among walnut (Juglans) cultivars and species.

BACKGROUND: Tree nuts, including English walnuts (Juglans regia), are sources of food allergens often associated with life-threatening allergic reactions. It is unknown if seed storage proteins from other Juglans species have IgE epitopes similar to those of the important English walnut allergens, Jug r 1 (2S albumin) and Jug r 2 (vicilin-like). OBJECTIVE: To screen for potential germplasm sources of hypoallergenic seed storage proteins of relevance in walnut food allergy. We sought to identify English walnut cultivars (cvs) or other Juglans species that showed decreased IgE binding to major seed storage proteins or an inability to cross-react with Jug r 1 or Jug r 2. METHODS: We determined if IgE in sera of patients who have had life-threatening systemic reactions to English walnut bound protein extracts from all tested walnut cvs (57 cvs total) or species (six) by Western immunoblot. Further, we used immunoblot inhibition to determine the in vitro cross-reactivity of Jug r 1 and Jug r 2, native and recombinant, with several walnut species. RESULTS: All walnut cvs and species contain allergenic proteins. Furthermore, as shown by in vitro immunoblot inhibition, the major walnut allergens in the species tested cross-reacted with those in J. regia cv. Chandler and J. nigra cv. Thomas extracts. CONCLUSIONS: Based on our findings, it is unlikely that a composite hypoallergenic walnut could be bred from available germplasm. In addition, patients with severe allergy to English walnut are likely to be clinically allergic to all commercial English walnut cvs and other closely related Juglans species.

Identification and cloning of a complementary DNA encoding a vicilin-like proprotein, jug r 2, from english walnut kernel (Juglans regia), a major food allergen.

BACKGROUND: Walnuts and other tree nuts are important food-allergen sources that have the potential to be associated with life-threatening, IgE-mediated systemic reactions in some individuals. OBJECTIVE: The purpose of this study was to characterize a complementary (c)DNA clone encoding one of the walnut food allergens. METHODS: A cDNA expression library prepared from walnut somatic embryo was screened for IgE reactivity with patient serum. A reactive clone of 2060 bp, which encoded a protein of 593 amino acids in length, was subcloned by excision into the pGEX expression vector. IgE-binding inhibition experiments were performed. RESULTS: A recombinant fusion protein was induced and shown to bind serum IgE from 9 of 15 patients tested, thus identifying a major allergen. This clone, named Jug r 2, exhibited significant homology with genes encoding the vicilin group of seed proteins. An IgE-binding inhibition experiment suggested that the encoded protein undergoes posttranslational modification into at least one major polypeptide (47 kd) and possibly several others, which is similar to the vicilin-like proteins characterized in cocoa bean (Theobroma cacao) and cottonseed (Gossypium hirsutum). N-terminal sequencing of the 47-kd band, Jug r 2, identified it as a mature protein obtained from the precursor. A second IgE-binding inhibition experiment showed that there is minimal or no cross-reactivity between Jug r 2 and pea vicilin, peanut proteins, or cacao proteins. CONCLUSION: Jug r 2 is the third vicilin food allergen identified in addition to vicilins from soy and peanut. The availability of recombinant food allergens should help advance studies on the immunopathogenesis and possible treatment of IgE-mediated food hypersensitivity.

Systemic allergic reaction to coconut (Cocos nucifera) in 2 subjects with hypersensitivity to tree nut and demonstration of cross-reactivity to legumin-like seed storage proteins: new coconut and walnut food allergens.

BACKGROUND: Two patients with tree nut allergy manifested by life-threatening systemic reactions reported the subsequent onset of systemic reactions after the consumption of coconut. OBJECTIVE: Herein, the IgE-binding proteins from coconut are described, and in vitro cross-reactivity with other nuts is investigated. METHODS: The IgE-binding profile of coconut endosperm tissue extract was analyzed by SDS-PAGE followed by immunoblotting. Immunoblot inhibition studies with walnut, almond, peanut, and coconut were performed. RESULTS: Sera IgE from both patients recognized reduced coconut allergens with molecular weights of 35 and 36.5 kd. IgE from 1 patient also bound a 55-kd antigen. Preabsorption of sera with nut extracts suppressed IgE binding to coconut proteins. Preabsorption of sera with coconut caused the disappearance of IgE binding to protein bands at 35 and 36 kd on a reduced immunoblot of walnut protein extract in 1 patient and suppression of IgE binding to a protein at 36 kd in the other patient. CONCLUSION: The reduced coconut protein at 35 kd was previously shown to be immunologically similar to soy glycinin (legumin group of seed storage proteins). The clinical reactivity in these 2 patients is likely due to cross-reacting IgE antibodies primarily directed against walnut, the original clinical allergy reported, and most likely to a walnut legumin-like protein. Coconut allergy in patients with tree nut allergy is rare; these are the first 2 patients ever reported, and therefore there is no general indication to advise patients with tree nut allergy to avoid coconut.

Cloning and sequencing of a gene encoding a 2S albumin seed storage protein precursor from English walnut (Juglans regia), a major food allergen.

BACKGROUND: Walnuts rank third in per capita consumption of tree nuts in the United States and can be associated with systemic IgE-mediated reactions in some individuals. OBJECTIVE: The objectives of the study were to clone a gene encoding one of the major food allergens in the walnut kernel and to characterize the recombinant allergen. METHODS: A cDNA expression library in the lambda vector Uni-ZAP, which was prepared from walnut somatic embryos, was screened by using a patient's sera that reacted with multiple protein bands on immunoblotting. RESULTS: A cDNA clone containing an insert of 663 bp was identified and named Jug r 1. DNA sequence analysis of this clone revealed that it encoded a protein 142 amino acids in length. Comparison of the encoded protein sequences with protein databases revealed that this clone exhibits a 46.1% identity with the Brazil nut (Bertholletia excelsa) methionine-rich 2S albumin seed storage protein precursor, Ber e 1. Jug r 1 appears to be an important walnut food allergen; 12 of 16 sera from patients allergic to walnuts demonstrated IgE binding to the 2S albumin seed storage protein precursor fusion protein. An IgE-binding inhibition study suggests that the walnut 2S protein precursor undergoes posttranslational modification into a large and small subunit that is similar to castor seed, cottonseed, mustard seed, and Brazil nut 2S seed storage protein allergens. Interestingly, the gene encoding this allergenic protein in Brazil nuts has recently gained notoriety because of its experimental use as a transgene to enhance the nutritional quality of legumes. CONCLUSION: This is now the sixth definitive 2S albumin seed storage protein demonstrated to bind IgE, suggesting that this class of proteins is inherently allergenic.

Solid-phase time-resolved fluorescence detection of human immunodeficiency virus polymerase chain reaction amplification products.

A new assay system for the detection of polymerase chain reaction (PCR) amplification products is presented. This single-pot sandwich assay system employs solid-support oligonucleotide-coated capture beads, a rare earth metal chelate-labeled probe, and a time-resolved fluorescence detection. The new assay system was evaluated for various reaction conditions including, DNA denaturation time, hybridization salt concentration, probe concentration, and hybridization time, all of which are important in designing an assay with a high level of sensitivity for the detection of duplex DNA. This nonisotopic assay system was applied to the detection of purified human immunodeficiency virus (HIV) DNA and sensitivity was compared with agarose gel electrophoresis and slot blot hybridization using a 32P-labeled probe. We were able to detect the amplified product from one copy of HIV DNA after 35 cycles of PCR amplification in less than 30 min using this assay, which compared with one copy by gel electrophoresis after 40 cycles of PCR amplification and one copy by slot blot hybridization after 35 cycles of PCR amplification and an overnight exposure of the autoradiogram. Thus, this assay is rapid, sensitive, and easy to use.

Detection of human immunodeficiency virus type 1 RNA in plasma samples from high-risk pediatric patients by using the self-sustained sequence replication reaction.

There is an urgent need for rapid and sensitive methods to assess human immunodeficiency virus (HIV) infection in infants and children. We evaluated an approach by using the self-sustained sequence replication reaction (3SR) to amplify HIV type 1 (HIV-1) RNA directly. The amplified RNA product was then detected by bead-based sandwich oligonucleotide capture hybridization and rare earth metal chelate time-resolved fluorescence. The sensitivity of this technology was determined to be less than 12 HIV-1 RNA copies with an amplification level of 10(10)-fold with purified HIV-1 RNA. Plasma samples from 19 high-risk pediatric patients younger than 5 years of age were examined, and results were compared with viral culture of patient plasma. Results from plasma culture and 3SR amplification agreed for 14 of these patients and disagreed for 5. Of the five samples which did not agree, four were positive by 3SR and negative by culture and one was positive by culture and negative by 3SR but became positive by 3SR at a subsequent testing. We conclude that 3SR amplification coupled with time-resolved fluorescence is a promising technology for investigating the relationship between the presence of HIV-1 RNA in plasma and progression of disease in HIV-infected pediatric patients. This technology should be important in the assessment of HIV-1 infection, in evaluating drug therapies, and in understanding the pathogenesis and transmission of the virus.

Detection of Escherichia coli rRNA using target amplification and time-resolved fluorescence detection.

The development of technology to increase the sensitivity and speed of detection of bacterial pathogens in samples is important for diagnosis and monitoring of illness. We have developed a sensitive and rapid method for the detection of bacteria, using Escherichia coli as a model, which combines transcription-based target amplification with a bead-based sandwich hybridization assay using rare earth metal chelate labelled probes and time-resolved fluorescence detection. Using these methods as little as 100 copies (0.00016 attomoles) of purified native Escherichia coli rRNA or just one bacterial cell in a spiked sample could be detected. These results demonstrate that amplification of rRNA by transcription-based amplification and detection by time-resolved fluorescence provide a sensitive technology for the direct detection of micro-organisms without the requirement for prior cultivation.

In situ detection of human immunodeficiency virus (HIV) nucleic acid in H9 cells using nonradioactive DNA probes and an image cytophotometry system.

Rapid and sensitive nonradioactive methods to detect human immunodeficiency virus (HIV)-infected cells are needed in clinical medicine. We developed an in situ hybridization test using 2-acetylaminofluorene (AAF)-labeled HIV DNA as a hybridization probe. Hybridized probe was detected using rabbit anti-AAF antibody, followed by alkaline phosphatase-conjugated goat anti-rabbit, and the bromochloroindolyl phosphate-nitroblue tetrazolium reaction. An image cytophotometry system was used to quantitate the percentage of HIV-infected cells. These methods were used to determine the percentage of H9 cells infected with HIV. HIV was detected in 0% of cells on day 1 post infection, 7% on day 4, 41% on day 8, and 5% on day 15. These results paralleled those of the reverse transcriptase assay and an antigen capture ELISA assay for HIV antigen. Thus the AAF modified HIV DNA probe detected HIV nucleic acid in infected H9 cells and the image cytophotometry system improved the sensitivity and objectivity of detection.

Comparison of non-radioactive DNA hybridization probes to detect human immunodeficiency virus nucleic acid.

Simple and sensitive methods to directly detect the human immunodeficiency virus (HIV) are needed for routine use in the clinical laboratory. In this study, we compared DNA probes prepared by: (1) nick translation with biotinylated dATP; (2) direct covalent biotinylation with photobiotin; (3) direct covalent reaction with 2-acetylaminofluorene (AAF); and (4) a standard radioactive (32P) nick translation procedure. These four DNA probes were hybridized with dilutions of purified target HIV DNA blotted onto nitrocellulose strips. Hybridization was detected using a complex of strepavidin-alkaline phosphatase [for (1) and (2)], alkaline phosphatase-tagged antibodies [for (3)] and by autoradiography [for (4)]. Alkaline phosphatase was detected colorimetrically using nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate. After 1 h, AAF probes were most sensitive (amount detected less than 5 pg), followed by biotin (10 pg), photobiotinylated probes (20 pg) and the radioactive probe (10 pg). The AAF probes were then used to detect HIV DNA in infected CEM cells. We conclude that non-radioactive DNA labelling methods can be used to directly detect HIV DNA under conditions compatible with present clinical laboratory procedures.