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PubMedDate not supplied

Evaluation of enzyme-linked immunosorbent assay of cleanup for thin-layer chromatography of aflatoxin B1 in corn, peanuts, and peanut butter.

A simple, rapid enzyme-linked immunoassay (ELISA) was used to evaluate the performance of each step (extraction, filtration, solvent partition, and silica gel column chromatography) of a solvent-efficient thin-layer chromatographic (TLC) method which is undergoing interlaboratory collaborative study for the determination of aflatoxin B1 in corn, raw peanuts, and peanut butter. The apparent average recoveries using the ELISA method were about 30 to 50% higher than those using the TLC method if only the amount of B1 added to the samples was used in the calculations. After the cross-reaction of the antibody with other aflatoxins added to the samples was considered, the amounts recovered approached the levels of aflatoxins added in all 3 commodities tested. With no cleanup treatment, ELISA recoveries at aflatoxin B1 levels above 7.5 ng/g were 84, 79, and 103% for corn, raw peanuts, and peanut butter, respectively. The coefficients of variation were between 5.2 and 25.2%. With each cleanup step in the TLC method, ELISA detected a progressive decrease in recovery from 150.5 to 105.3% (before correction for the presence of other aflatoxins) or from 93.5 to 65.4% (after correction for other aflatoxins) of B1 added to the samples. The ELISA data support the conclusion obtained from previous studies that cleanup treatments were not necessary in the ELISA. When large amounts of other aflatoxins are present, an understanding of the cross-reactivity of antibody with other aflatoxins in the ELISA is essential for final interpretation of the data.

Aflatoxin B1↗
PubMed1982

International mycotoxin check sample program: Part I. Report on laboratory performance for determination of aflatoxins B1, B2, G1, and G2 in raw peanut meal, deoiled peanut meal, and yellow corn meal.

Three aflatoxin-contaminated samples (raw peanut meal, deoiled peanut meal, and yellow corn meal) were analyzed by 121 laboratories in 31 countries. Sufficient data were obtained to permit a statistical comparison of the performance of laboratories using the BF, CB, and EEC methods and those using high performance liquid chromatography (HPLC) for quantitation. No significant differences were found between means for laboratories using these 4 methods for the analysis of raw peanut meal or yellow corn meal. However, for deoiled peanut meal, means were significantly different for laboratories using the BF method compared with the CB or EEC methods for B1 and B2, and for laboratories using the CB method compared with HPLC methods for G2.

Aflatoxin B1↗
PubMed2005

Influence of the maillard reaction on the allergenicity of rAra h 2, a recombinant major allergen from peanut (Arachis hypogaea), its major epitopes, and peanut agglutinin.

The influence of thermal processing and nonenzymatic browning reactions on the IgE-binding activity of rAra h 2 was studied and compared to findings recently reported for the allergen's natural counterpart. ELISA experiments as well as inhibition assays revealed that thermal treatment of rAra h 2 in the presence of reactive carbohydrates and carbohydrate breakdown products induces a strong increase of the IgE-binding activity, thus collaborating with the data reported for the natural protein isolated from peanuts. To localize the Ara h 2 sequences responsible for the formation of highly IgE-affine glycation sites, model peptides have been synthesized mimicking sequences which contain possible targets for glycation as well as the immunodominant epitopes. Immunological evaluation of these peptides heated in the absence or presence of reducing sugars and carbonyls, respectively, revealed that neither the two lysine residues of Ara h 2 nor its N-terminus are involved in the formation of IgE-affine structures by Maillard reaction. Also, the cysteine-containing major epitope 3 (aa 27-36) was found to lose its IgE-binding capacity upon heating. By contrast, the overlapping major epitopes 6 and 7, which do not contain any lysine or arginine moieties, showed a distinct higher level of IgE binding when subjected to Maillard reaction, thus giving the first evidence that nonbasic amino acids might be accessible for nonenzymatic glycation reactions and that these posttranslational modifications might induce increased IgE binding of the glycated Ara h 2. Analogous experiments were performed with peanut agglutinin, considered in the literature as a minor allergen. ELISA experiments revealed that the majority of tested sera samples from peanut-sensitive patients showed a high level of IgE binding to the lectin even after heat treatment. In contradiction to published data, nonenzymatic browning reactions seem to deteriorate the IgE affinity of the lectin.

2S Albumins, Plant↗
PubMed2003

Persistent protective effect of heat-killed Escherichia coli producing "engineered," recombinant peanut proteins in a murine model of peanut allergy.

BACKGROUND: Peanut allergy (PNA) is a life-threatening food allergy for which there is no definitive treatment. OBJECTIVE: We investigated the long-term immunomodulatory effect of heat-killed Escherichia coli producing engineered (mutated) Ara h1, 2, and 3 (HKE-MP123) administered rectally (pr) in a murine model of PNA. METHODS: Peanut-allergic C3H/HeJ mice received 0.9 (low dose), 9 (medium dose), or 90 (high dose) microg HKE-MP123 pr, HKE-containing vector (HKE-V) alone, or vehicle alone (sham) weekly for 3 weeks. Mice were challenged 2 weeks later. A second and third challenge were performed at 4-week intervals. RESULTS: After the first challenge, all 3 HKE-MP123 and HKE-V-treated groups exhibited reduced symptom scores (P <.01,.01,.05,.05, respectively) compared with the sham-treated group. Interestingly, only the medium- and high-dose HKE-MP123-treated mice remained protected for up to 10 weeks after treatment accompanied by a significant reduction of plasma histamine levels compared with sham-treated mice (P <.05 and.01, respectively). IgE levels were significantly lower in all HKE-MP123-treated groups (P <.001), being most reduced in the high-dose HKE-MP123-treated group at the time of each challenge. IL-4, IL-13, IL-5, and IL-10 production by splenocytes of high-dose HKE-MP123-treated mice were significantly decreased (P <.01;.001,.001, and.001, respectively), and IFN-gamma and TGF-beta production were significantly increased (P <.001 and.01, respectively) compared with sham-treated mice at the time of the last challenge. CONCLUSIONS: Treatment with pr HKE-MP123 can induce long-term "downregulation" of peanut hypersensitivity, which might be secondary to decreased antigen-specific T(H)2 and increased T(H)1 and T regulatory cytokine production.

2S Albumins, Plant↗
PubMed2006

Oral sensitization to peanut is highly enhanced by application of peanut extracts to intact skin, but is prevented when CpG and cholera toxin are added.

BACKGROUND: CpG oligonucleotides might offer an alternative to conventional immunotherapy in preventing and potentially reversing Th2-biased immune deregulation which leads to allergy. However, non-invasive ways of administration, especially in peanut-allergic patients, should be explored. METHODS: One hundred micrograms of whole peanut protein extract (PE) alone, or mixed with cholera toxin (CT, 50 microg) plus CpG (100 microg) as adjuvant, was applied on intact skin of mice (40 min, twice). Initiation of an immune response was monitored by detection of specific antibodies in sera. The effect of this pretreatment on a further oral sensitization by PE was then evaluated by assaying antibodies and cytokines specific for PE and purified allergens. Cytokine production in liver 40 min after skin application was also assayed. RESULTS: Two brief skin applications of PE alone highly potentiated further oral sensitization, as demonstrated by very intense specific IgE, IL-4 and IL-5 productions. Conversely, skin pretreatment with PE and CT + CpG efficiently prevented further sensitization via gastro-intestinal exposure. In both cases, the specificity of the antibodies and cytokines was the same as in control mice. CT + CpG treatment allowed the rapid production of IL-12 and TGFbeta in liver and of specific IgG2a in sera, suggesting the activation of Th1 and/or regulatory T cells. CONCLUSIONS: Oral sensitization to peanut is highly enhanced by a previous short exposure of allergens to intact skin. Conversely, the use of CT + CpG adjuvant for skin application efficiently prevents further oral sensitization. The potential of such treatment in specific immunotherapy needs to be evaluated.

Administration, Oral↗
PubMed1981

Receptors for peanut agglutinin on a high percentage of human cord-blood lymphocytes: phenotype characterization of peanut-positive cells.

The results of this study show that a high percentage of human cord-blood lymphocytes bears receptors for peanut agglutinin. Determination of the presence of other lymphocyte markers (E rosettes, membrane immunoglobulins, alpha-naphthyl-acetate esterase, Ia antigens) in the peanut-positive cells indicated that they include both T and B cells. The peanut receptor, also expressed by blast cells of most acute, but not chronic, leukemias, seems to be a marker of immaturity for both T- and B-lymphocyte subsets in man as it is in the mouse.

Aging↗
PubMed1994

Cloning and sequence analysis of the coat protein genes of an Australian strain of peanut mottle and an Indonesian 'blotch' strain of peanut stripe potyviruses.

We have analysed the coat protein gene sequences of two potyviruses infecting peanut. The 3' terminal 1247 nucleotides (nt) of an Australian strain of peanut mottle virus (PeMoV-AU) and the 3' terminal 1388 nt of an Indonesian 'blotch' strain of peanut stripe virus (PStV-Ib) were cloned and sequenced. Those regions included the 861 and 864 nt encoding the respective putative coat proteins as well as the 285 and 253 nt, respectively of 3' non-coding sequences. Comparison of the nucleotide sequences of PeMoV-AU and PStV-Ib revealed a sequence similarity of 64.4% for the coat protein gene and 34.6% for the 3' non-coding region. The deduced amino acid sequences of PeMoV-AU and PStV-Ib coat proteins are 66.7% identical. These results provide further evidence that PeMoV and PStV are distinct viruses. Comparisons of the 3' terminal sequences of PeMoV-AU and PStV-Ib with those of the genomic RNA of other strains of PeMoV and PStV and with other potyviruses are discussed.

Amino Acid Sequence↗
PubMed2004

The major peanut allergen Ara h 1 and its cleaved-off N-terminal peptide; possible implications for peanut allergen detection.

Ara h 1 was purified from raw peanuts (Arachis hypogaea L.) in the presence or absence of protease inhibitors. N-Terminal amino acid sequences were determined after western blotting. Both purification procedures proved to be very consistent and resulted in identical chromatographic and electrophoretic behavior of Ara h 1 and in the isolation of identical proteins of approximately 64 kDa with RS/H_PPGERTRG as the N-terminal amino acid sequence. Consequently, purified Ara h 1 appears to be truncated at the N-terminal side. The observations strongly suggest that Ara h 1 occurs physiologically as a protein of which the first 84 and 78 amino acids, respectively, are cleaved off in planta upon maturation of the protein. On the basis of epitope mapping, the cleaved-off N-terminal peptide contains three allergenic epitopes, of which two are major. These truncated epitopes will go undetected in assays when purified Ara h 1 from peanuts is used as reference material. Patients' sera, however, contain IgE-type antibodies against the epitopes that are contained in the cleaved-off peptide, implying that the peptide, or part of it, is still present in peanuts that are consumed. Possible consequences of this exposure to these three epitopes are discussed. On the basis of literature data the cleaved-off peptide is hypothesized to have antifungal activity.

Allergens↗
PubMed1979

Comparative study of two methods for extraction of aflatoxin from peanut meal and peanut butter.

The difference between the CB and Best Foods methods in extracting aflatoxins from peanut products has been studied. The CB method yields 60, 121, 35, and 22% higher results for aflatoxins B1, B2, G1, and G2, respectively for 4 samples of peanut meal and 6 samples of peanut butter studied. Both reverse phase liquid chromatography and thin layer chromatography were used to quantitate the extracted aflatoxins.

Aflatoxins↗
PubMed2006

Structure and stability of 2S albumin-type peanut allergens: implications for the severity of peanut allergic reactions.

Resistance to proteolytic enzymes and heat is thought to be a prerequisite property of food allergens. Allergens from peanut (Arachis hypogaea) are the most frequent cause of fatal food allergic reactions. The allergenic 2S albumin Ara h 2 and the homologous minor allergen Ara h 6 were studied at the molecular level with regard to allergenic potency of native and protease-treated allergen. A high-resolution solution structure of the protease-resistant core of Ara h 6 was determined by NMR spectroscopy, and homology modelling was applied to generate an Ara h 2 structure. Ara h 2 appeared to be the more potent allergen, even though the two peanut allergens share substantial cross-reactivity. Both allergens contain cores that are highly resistant to proteolytic digestion and to temperatures of up to 100 degrees C. Even though IgE antibody-binding capacity was reduced by protease treatment, the mediator release from a functional equivalent of a mast cell or basophil, the humanized RBL (rat basophilic leukaemia) cell, demonstrated that this reduction in IgE antibody-binding capacity does not necessarily translate into reduced allergenic potency. Native Ara h 2 and Ara h 6 have virtually identical allergenic potency as compared with the allergens that were treated with digestive enzymes. The folds of the allergenic cores are virtually identical with each other and with the fold of the corresponding regions in the undigested proteins. The extreme immunological stability of the core structures of Ara h 2 and Ara h 6 provides an explanation for the persistence of the allergenic potency even after food processing.

2S Albumins, Plant↗
PubMed2004

Isoforms of the major peanut allergen Ara h 2: IgE binding in children with peanut allergy.

BACKGROUND: The major peanut allergen Ara h 2 consists of two isoforms, namely Ara h 2.0101 and Ara h 2.0201. The recently identified Ara h 2.0201 isoform contains an extra 12 amino acids including an extra copy of the reported immunodominant epitope DPYSPS. This study aimed to evaluate the IgE binding of the two Ara h 2 isoforms. METHODS: Ten clones of Ara h 2 were sequenced to assess the relative frequency of the Ara h 2 isoforms and to identify whether there was further variation in the Ara h 2 sequence. IgE binding to Ara h 2.0101 and Ara h 2.0201 was measured for 70 peanut-allergic children using an IgE DELFIA assay to quantitate specific IgE binding. A competition assay was used to measure whether Ara h 2.0201 contained IgE epitopes other than those found for Ara h 2.0101. RESULTS: The original Ara h 2.0101 sequence was found for 6/10 clones and Ara h 2.0201 was found for 2/10 clones. Ara h 2.0201 had the expected insertion of 12 amino acids as well as substitutions at positions 40 (40G) and 142 (142E). Two new isoforms were identified as different polymorphisms of position 142. One Ara h 2.01 clone (Ara h 2.0102) contained 142E and one Ara h 2.02 clone (Ara h 2.0202) contained 142D. A polymorphism that was previously identified by other investigators at position 77 (77Q or 77R) was not found for any of the 10 sequences. Although the level of IgE binding to Ara h 2.0201 of individual patients was frequently higher than the binding to Ara h 2.0101 (p < 0.01), there was a strong correlation in binding to both isoforms (r = 0.987, p < 0.0001) and when analyzed as a group the means were similar. Ara h 2.0101 was not as efficient at blocking reactivity to Ara h 2.0201 indicating there is an additional IgE specificity for the Ara h 2.0201 isoform. CONCLUSIONS: Ara h 2.0201 has similar but higher IgE binding than the originally sequenced Ara h 2.0101 isoform and contains other IgE specificities.

2S Albumins, Plant↗
PubMed2001

Hypogin, a novel antifungal peptide from peanuts with sequence similarity to peanut allergen.

A protein designated hypogin, with a prominent suppressive action on the growth of the fungi Mycosphaerella arachidicola, Fusarium oxysporum and Coprinus comatus, was isolated from seeds of the peanut Arachis hypogaea. The protein inhibited human immunodeficiency virus (HIV) reverse transcriptase and enzymes associated with HIV infection including alpha-glucosidase and beta-glucosidase. The proliferative response of mouse splenocytes was attenuated in the presence of the protein. The protein exhibited a molecular mass of 7.2 kDa in tricine gel electrophoresis and gel filtration on Superdex 75 and an N-terminal sequence resembling peanut allergen Ara H1. The isolation procedure involved affinity chromatography on Affi-gel blue gel and ion-exchange chromatography on CM-Sepharose. The protein was adsorbed in both chromatographic media.

Allergens↗
PubMed2003

Engineered recombinant peanut protein and heat-killed Listeria monocytogenes coadministration protects against peanut-induced anaphylaxis in a murine model.

Peanut allergy (PNA) is the major cause of fatal and near-fatal anaphylactic reactions to foods. Traditional immunotherapy using peanut (PN) protein is not an option for PNA therapy because of the high incidence of adverse reactions. We investigated the effects of s.c. injections of engineered (modified) recombinant PN proteins and heat-killed Listeria monocytogenes (HKLM) as an adjuvant on anaphylactic reactions in a mouse model of PN allergy. PN-allergic C3H/HeJ mice were treated s.c. with a mixture of the three major PN allergens and HKLM (modified (m)Ara h 1-3 plus HKLM). The effects on anaphylactic reactions following PN challenge and the association with Ab levels and cytokine profiles were determined. Although all mice in the sham-treated groups exhibited anaphylactic symptoms with a median symptom score of 3, only 31% of mice in the mAra h 1-3 plus HKLM group developed mild anaphylaxis, with a low median symptom score of 0.5. Alterations in core body temperature, bronchial constriction, plasma histamine, and PN-specific IgE levels were all significantly reduced. This protective effect was markedly more potent than in the mAra h 1-3 protein alone-treated group. HKLM alone did not have any protective effect. Reduced IL-5 and IL-13, and increased IFN-gamma levels were observed only in splenocytes cultures from mAra h 1-3 plus HKLM-treated mice. These results show that immunotherapy with modified PN proteins and HKLM is effective for treating PN allergy in this model, and may be a potential approach for treating PNA.

2S Albumins, Plant↗
PubMedDate not supplied

Immunoaffinity column coupled with solution fluorometry or liquid chromatography postcolumn derivatization for determination of aflatoxins in corn, peanuts, and peanut butter: collaborative study.

An AOAC/IUPAC (International Union of Pure and Applied Chemistry) collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column for the determination of aflatoxin. The test portion is extracted with methanol-water (7 + 3), filtered, diluted to less than 30% methanol with water, and applied to the affinity column. The column is washed with water and the concentrated aflatoxins are eluted with methanol. Total aflatoxins are determined by solution fluorometry with bromine (SFB), and individual toxins are determined by reverse-phase liquid chromatography with postcolumn derivatization with iodine (PCD). Corn naturally contaminated with aflatoxins, and peanuts, peanut butter, and corn containing added aflatoxins (B1:B2:G1:G2 = 7:1:3:1) were sent to 24 collaborators in the United States, France, Canada, and the Republic of South Africa. Twelve collaborators used the SFB method, 9 used the PCD method, and 3 used both SFB and PCD methods. Twenty collaborators completed the study (10 used the SFB method, 7 used the PCD method, and 3 used both SFB and PCD methods). Test portions were spiked at 10, 20, and 30 ng/g. For SFB analyses, recoveries of total aflatoxins were 123, 105, and 107%, respectively; the relative standard deviation for repeatability (RSDr) ranged from 11.75 to 16.57%, and the relative standard deviation for reproducibility (RSDR) ranged from 10.97 to 33.09%. For PCD analyses, recoveries were 81, 81, and 83%, respectively; the RSDr ranged from 5.20 to 17.22%, and the RSDR ranged from 4.68 to 50.77%. The RSDr for aflatoxins B1 and G1 for spiked test portions ranged from 5.45 to 23.55%, and the RSDR ranged from 4.21 to 57.28%.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxins↗
PubMedDate not supplied

Enzyme-linked immunosorbent assay of aflatoxins B1, B2, and G1 in corn, cottonseed, peanuts, peanut butter, and poultry feed: collaborative study.

A direct competitive enzyme-linked immunosorbent assay (ELISA) screening method for aflatoxins at 20 ng/g was studied by 12 collaborators. Test samples of peanut butter were extracted by blending with methanol-water-hexane (55 + 45 + 100) and heating the test extracts on a steam bath; test samples of the other commodities were extracted by blending with methanol-water (80 + 20). All test extracts were filtered and the filtrates were diluted with buffer to a final methanol concentration of less than 30%. Each diluted filtrate was applied to a cup containing a filter with immobilized polyclonal antibodies specific to aflatoxins B1, B2, and G1. Aflatoxin B1-peroxidase conjugate was added, the cup was washed with water, and a mixture of hydrogen peroxide and tetramethylbenzidine was added. The test sample was judged to contain greater than or equal to 20 ng aflatoxins/g when, after exactly 1 min, no color was observed on the filter; when a blue or gray color developed, the test sample was judged to contain less than 20 ng aflatoxins/g. All collaborators correctly identified naturally contaminated corn and raw peanut positive test samples. No false positives were found for controls containing less than 2 ng aflatoxins/g. The correct responses for positive test samples spiked at levels of 10, 20, and greater than or equal to 30 ng aflatoxins/g (the ratio of B1:B2:G1 was 10:1:3) were 52, 86, and 96%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Aflatoxin B1↗
PubMed1987

Differentiation of peanut lectin positive suppressor T-cells from peanut lectin negative precursors in bovine cells by 12-O-tetradecanoylphorbol-13-acetate.

Although 12-O-tetradecanoylphorbol-13-acetate (TPA) can synergize with lectins to enhance lymphocyte proliferation, pretreatment of lymphocytes with TPA decreases their response. Pretreatment also inhibits the response to allogeneic cells in the mixed lymphocyte culture. In this study we determined that at least part of this inhibition was due to the generation of T-lymphocytes with the ability to suppress proliferation in vitro. By using populations enriched in lymphocytes or macrophages we determined that the interaction of TPA with lymphocytes, but not macrophages, was required to mediate the suppression. The number of macrophages present in culture (range, 0.5-10%) was irrelevant to the generation of inhibitory activity. Moreover, the TPA-induced suppressor activity copurified with T-cells. Furthermore, when peanut lectin (agglutinin) (PNA) was used to separate T-cells after treatment with TPA, essentially all of the activity copurified with the PNA positive cells. When PNA separations were carried out before treatment with TPA, the suppressor activity arose from the PNA negative fraction. Therefore, TPA appeared to cause phenotypically PNA negative T-cells to gain the PNA positive marker, as well as to function as suppressor cells in vitro. Suppressor activity was also found in the culture medium. Thus the suppression observed may be mediated through a soluble factor released by the TPA-treated cells. Although the suppressor cell activity induced by TPA can only partially account for its in vitro inhibition of lymphocyte proliferation, the development of suppressor cells merits further study with respect to lymphocyte phenotypic and functional differentiation. The results also suggest the possibility that similar processes could occur in vivo, possibly during the course of tumor promotion.

Animals↗
PubMed1982

The acquisition of receptors for peanut agglutinin by peanut agglutinin-negative thymocytes and peripheral T cells.

Using fluorescein-conjugated peanut agglutinin (PNA) and the fluorescence-activated cell sorter, we have shown that the cytotoxic T cells generated by culturing thymocytes (about 85% PNA+ cells) with Con A and medium conditioned by Con A-stimulated spleen cells were mostly PNA+. Cytotoxic T cells generated in parallel cultured of PNA- thymocytes or normal lymph node cells (in which the T cells are PNA-), however, were also mostly PNA+. The development of PNA+ cells from PNA- mature peripheral T cells was confirmed by using cultures of PNA- T cells separated on a fluorescence-activated cell sorter from lymph node cells. Immunization in vivo also resulted in the appearance of PNA+ T cells in the lymph node. These results indicate that receptors for PNA can no longer be regarded as unambiguous markers of T cell immaturity, or of the origin of a T cell from a PNA+ precursor. The possibility that a variable proportion of the PNA+ cells in the thymus may be generated by intrathymic activation of PNA- medullary thymocytes is discussed.

Animals↗
PubMed1979

Peanut agglutinin. II. Characterization of the Thy-1, Tla and Ig phenotype of peanut agglutinin-positive cells in adult, embryonic and nude mice using double immunofluorescence.

The Thy-1, Tla and Ig phenotype of peanut agglutinin (PNA)-binding cells was characterized in various strains of mice. In the thymus, PNA was found to bind principally but not exclusively to the Thy-1+ Tla + Ig-- steroid-sensitive cortical thymocytes. Thy1 + Tla -- Ig -- steroid-resistant cells are not labeled with PNA. In other lymphoid organs, PNA bound to a minority of T or null cells but generally not to B cells. During ontogeny, PNA + and PNA -- T lineage cells appear simultaneously in the liver at day 10 of gestation, in the thymus at day 11 and in the spleen at day 18. No evidence was found for a maturation from PNA + to PNA -- cells. Prethymocytes present in nude mice were also divided into a PNA + and PNA -- population.

Animals↗