Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Immunoblotting Research Articles

In Vitro and In Vivo Cross-reactivity Studies of Legume Allergy in a Mediterranean Population

Martínez San Ireneo M, Ibáñez MD, Fernández-Caldas E, Carnés J. Int Arch Allergy Immunol. 2008;147(3): 222–230PURPOSE OF THE STUDY. Legume allergy, mainly to lentils and chickpeas, is the fifth most common cause of food allergy in Spanish children. Serological cross-reactivity among legumes is frequent, but its clinical relevance is controversial. The aim of this study was to investigate the cross-reactivity among lentils, chickpeas, peas, white beans, and peanuts and its clinical relevance in pediatric patients.STUDY POPULATION. Fifty-four children with clinical allergy to legumes were included.METHODS. Cross-reactivity was evaluated with enzyme-linked immunosorbent assay inhibition experiments and oral food challenges to legumes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblots were conducted with raw and boiled legume extracts.RESULTS. Enzyme-linked immunosorbent assay inhibition experiments demonstrated >80% inhibition with lentil, chickpea, and pea extracts. Immunoblots performed with raw legume extracts (lentil, chickpea, and pea) and individual sera revealed that >50% of the sera identified an allergen of ∼50 kDa in all 3 legume extracts. In all 3 boiled extracts, an intense band at ∼50 kDa was visualized by using a serum pool. The oral legume challenges demonstrated that 37 children (69%) were allergic to ≥2 legumes (median: 3 legumes). The most frequent associations were allergy to lentils and chickpeas (57%), allergy to lentils and peas (54%), and allergy to lentils, chickpeas, and peas (43%).CONCLUSIONS. In vitro inhibition experiments demonstrated a high degree of cross-reactivity among lentils, chickpeas, and peas. Food challenges confirmed that clinical allergy to all 3 legumes is frequent in this cohort of Spanish children.REVIEWER COMMENTS. Although legumes are not major allergens in the United States and some European countries, they are a common cause of food allergies in Mediterranean countries. The authors demonstrated that, in their group of Spanish children, there was a high degree of in vitro and in vivo cross-reactivity among legumes, which is in contrast to North American children, in whom clinical reactivity to >1 legume is considered to be infrequent (eg, children with peanut allergy typically tolerate most legumes). These contrasting results highlight the fact that genetic and dietary influences (among other factors) can have significant influences on food allergy. Additional studies are needed to elucidate the contribution of dietary habits and genetics to food allergy.

Vine pollen allergy in areas with a high density of vineyards

In Castilla-La Mancha, Spain, the area with the highest density of vineyards in the world, 2 cases of Vitis vinifera pollen allergy have been previously reported. To determine the clinical relevance and biochemical characteristics of vine pollen in the Spanish province of Ciudad Real. We designed a prospective study of patients treated in the allergy units from Puertollano and Ciudad Real for respiratory symptoms of 4 months' duration in the year 2000. Skin prick tests with a standard aeroallergen battery and V vinifera pollen extract were performed on all patients. We also performed conjunctival and bronchial provocation tests and serum specific IgE and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting on the patients who tested positive for V vinifera pollen. We included 200 patients, 98 sensitized to any pollen and 9 to V vinifera pollen. We found 8 of 9 positive conjunctival provocation test results and a positive bronchial provocation result to vine pollen in a vine grower. Serum specific IgE against V vinifera pollen was detected in 9 of 9 patients. Immunoblotting with V vinifera pollen extract showed IgE-binding bands at 45 and 67 kDa. Vine pollen could be the cause of pollinosis in exposed patients living in areas with a high density of vineyards.

Occupational rhinitis and asthma due to crickets

Insects may cause airborne hypersensitivity reactions. However, few reports exist on specific allergy to crickets. To report a case of occupational rhinitis and bronchial asthma in a cricket farm worker. A 28-year-old woman developed rhinitis and bronchial asthma related to her job in a farm where she was exposed to crickets: Gryllus campestris, Gryllus bimaculatus, and Acheta domestica. Extracts were prepared from whole and crushed bodies and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Skin prick tests, specific IgE assays (enzyme allergosorbent test [EAST], immunoblotting, EAST inhibition assays), serial peak expiratory flow monitoring at work, and specific (A domestica) and nonspecific bronchial challenge tests were performed. Skin prick test results were positive for G campestris, G bimaculatus, and A domestica. Levels of specific IgE were 2.9, 2.4, and 5.4 kU/L, respectively. The total IgE level was 131 kU/L. Serial peak expiratory flow monitoring at work was consistent with occupational asthma. The result of a bronchial challenge test with A domestica was positive with a dual response and elicited an increase in nonspecific bronchial hyperresponsiveness. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting revealed a similar pattern of IgE-binding bands with the 3 cricket extracts (bands of 78 and 64 kDa appeared in nonreducing conditions, whereas bands of 107 to 80, 58, and 52 kDa appeared in reducing conditions). None of these bands was detected by control sera. EAST inhibition studies showed a high degree of cross-reactivity among the 3 species. Crickets are responsible for occupational rhinitis and asthma by an IgE mechanism. Cross-reactivity among the crickets tested in our study was found.

Anaphylaxis to Hippobosca equina (louse fly)

IgE-mediated allergy to insects different from Hymenoptera species is seldom reported. To describe and study the case of a previously nonatopic man with an anaphylactic reaction (grade III, Müeller) caused by a bite from a louse fly (Hippobosca equina). In vivo (skin prick tests) and in vitro (enzyme-linked immunosorbent assay and sodium dodecyl sulfatepolyacrylamide gel electrophoresis immunoblotting) tests were used for diagnosis. Cross-reactivity between H. equina and different insects was investigated. Results of skin prick tests and serum specific IgE were positive to H. equina. Immunoblot inhibition studies identified common bands in H. equina, Apis mellífera, and Musca domestica, but this cross-reactivity did not affect a band of 16 or 15 kDa. This molecular weight is similar to that of phospholipase A2 in A mellifera venom. We describe a case of IgE-mediated allergy to H. equina in which specific molecular band proteins seemed to be responsible for the reaction.

Identification of a 7S globulin as a novel coconut allergen

Coconut (Cocos nucifera) is a monocotyledonous plant of the Arecaceae family. Allergy to coconut is infrequent, with only 5 cases reported so far in the medical literature. To identify coconut allergens in 2 patients allergic to this food. We describe 2 patients allergic to coconut: an adult pollen-allergic patient monosensitized to coconut who presented with severe oropharyngeal symptoms and a child with a previous allergy to walnut, not allergic to pollen, who developed anaphylaxis on coconut ingestion. Both patients had positive skin prick test results and serum specific IgE (CAP) to coconut. IgE sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting was performed to identify the allergens involved, and a strong IgE binding band detected in both patients was further analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS). Stability to pepsin digestion of the coconut extract and its cross-reactivity with tree nuts were studied. An immunoblot showed an almost identical profile of IgE binding proteins in the coconut extract in both patients who reacted strongly to a band of approximately 29 kDa. The peptide analysis by MALDI-TOF MS of this band obtained the sequence GHGKREDPEKR. The protein with the highest correlation with this peptide was found to be a 7S globulin from Elaeis guineensis, another oil palm species also belonging to the Arecaceae family. The 29-kDa band was digested by pepsin in less than 1 minute. Cross-reactivity among coconut, walnut, and hazelnut was demonstrated by CAP inhibition in patient 2. We have identified a 7S storage protein as a novel coconut allergen.

Tobacco as an allergen in bronchial disease

Skin testing and sera measurements have verified the existence of tobacco specific IgE. However, the few published studies on this matter report conflicting results concerning their clinical significance. To verify if a specific clinical allergenic response against tobacco might be possible in allergenic and nonallergenic bronchial diseases. We performed a cross-sectional observational case-control analysis on 180 patients with asthma, chronic obstructive pulmonary disease (COPD), and bronchial carcinoma and controls who were randomly chosen. Skin prick tests and serum specific IgE to tobacco and related allergens, bronchial challenge with cigarettes and tobacco extract, patch tests with tobacco and nicotine, sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting, and Enzyme AllergoSorbent Test (EAST) inhibition were performed. Twenty-eight patients had positive tobacco skin prick test results. The association among positive skin prick test results, IgE, and bronchial challenge was strong (P < .001). Tobacco sensitivity was higher in patients with pollen asthma than in patients with COPD and carcinoma and negative in patients with intrinsic asthma and controls. A positive bronchial challenge result was related to the length of habit (P < .001) and the tobacco index in patients who had stopped smoking (P < .001). Delayed bronchial and patch response was more common in patients with COPD (P < .001). Tobacco IgE response (EAST) was related to sensitivity to Lolium perenne (rye grass) pollen (P < .001) but not to other vegetables that belong to the Solanaceae family. EAST inhibition showed cross-reactivity between tobacco and Lolium pollen. Tobacco may be responsible for a specific IgE response. Patients with pollen asthma were those with more positive responses to tobacco due to cross-reactivity between Lolium and tobacco allergens.

Occupational rhinitis and bronchial asthma due to artichoke ( Cynara scolymus)

The artichoke is a perennial horticultural plant that belongs to the Compositae family. To present case studies of 2 vegetable warehouse workers who developed occupational rhinitis and bronchial asthma by sensitization to artichoke. Skin prick tests with common inhalants and foods were performed. Specific IgE to artichoke, Parietaria judaica pollen, and Olea europaea pollen extracts was measured by a specific IgE enzyme immunosorbent assay kit. Molecular mass of the allergens was studied by the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting technique. Patients underwent a nasal challenge test, and one patient provided peak expiratory flow rate (PEFR) measurements in her workplace. In both patients, results of skin prick tests to artichoke were positive. Levels of specific IgE for artichoke were 0.68 kU/L in patient 1 and 2.14 kU/L in patient 2. The protein composition of the artichoke extract, studied by SDS-PAGE, showed that most bands ranged from 30 to 14 kDa. The IgE-binding bands with the serum samples of patient 1 showed apparent molecular masses of 56, 48, 38, 31, 27, 25, 16, and 15 kDa; however, the serum samples of patient 2 showed IgE bands of 21 and 19 kDa. Western blotting of artichoke extract showed a complete inhibition of IgE-binding bands when serum samples were preincubated with P. judaica pollen extract. Nasal challenge with artichoke extract triggered a peak nasal inspiratory flow decrease of 81% and 85% in patient 1 and patient 2, respectively. Finally, patient 1 recorded a PEFR decrease of up to 36% after exposure to artichoke in her workplace. SDS-PAGE immunoblotting inhibition performed for the artichoke extract showed a total disappearance of the specific IgE binding bands when serum samples were previously incubated with P. judaica pollen extract, thus establishing the existence of a serologic cross-reactivity between artichoke and P. judaica pollen.

Characterization of recombinant monoclonal IgA anti—PDC-E2 autoantibodies derived from patients with PBC

Primary biliary cirrhosis (PBC) is an autoimmune liver disease characterized by the presence of autoantibodies to mitochondria (AMA). Recent evidence suggests that PBC develops after a locally driven response in the mucosa, where immunoglobulin A (IgA) is the dominant antibody isotype. In this study, we produced recombinant pyruvate dehydrogenase complex (PDC-E2)—specific dimeric human IgA monoclonal antibodies (mAbs) in a baculovirus expression system. By using 2 anti—PDC-E2 IgG mAbs derived from patients with PBC, we constructed 2 recombinant baculoviruses, each containing heavy chains with the Cα constant region. These were simultaneously co-infected into Sf9 insect cells with recombinant baculovirus containing the J chain. A sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) immunoblotting profile of the IgA using a 6% nonreducing gel verified the dimeric nature of the autoantibodies. Both recombinants retained their original specificity for PDC-E2. In addition, the antibody showed a mitochondrial staining pattern in HEp2 cells and apically stained the biliary epithelial cells (BECs) in the liver of a patient with PBC but not a normal patient. Transcytosis experiments performed using human polymeric immunoglobulin receptor (pIgR) expressing Madine-Darby canine kidney (MDCK) cells showed that one of the recombinants showed a high degree of colocalization with PDC-E2. In conclusion, these data provide further support of the hypothesis that PDC-E2-specific IgA may enter biliary epithelial cells of PBC patients via the pIgR and complex with PDC-E2, thereby potentially contributing to the pathology of BECs. Moreover, this recombinant PDC-E2-specific mAb provides a tool for further determination of the role of anti-PDC-E2 IgA in the pathogenesis of PBC. (Hepatology2002;36:1383-1392).

IgE-reactive proteins from Stachybotrys chartarum

Stachybotrys chartarum has been associated with idiopathic pulmonary hemorrhage in infants. This is thought to be mycotoxin-related. There are increasing numbers of reports linking this fungus to the indoor environment of patients with other pulmonary problems, including allergies and asthma. Given the potential significance of this fungus as a pulmonary pathogen, this work evaluates the antigenic proteins of S. chartarum as to their molecular size and the prevalence of immunoglobulin (Ig)E and IgG directed against them in the general population. S. chartarum was isolated from a local home. S. chartarum for extract production was grown on minimum salts and glucose. Plasma from 132 healthy individuals was evaluated for IgE and IgG directed against S. chartarum using direct and inhibition enzyme immunoassay. The number and molecular size of those proteins that were bound by IgE from pooled sera known to contain IgE to S. chartarum were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting. Enzyme immunoassay indicated 65 of 132 (49.2%) sera tested contained IgG against S. chartarum and 13 of 139 (9.4%) sera tested contained IgE against S. chartarum. Pooled sera identified two IgE-binding proteins from extracts of S. chartarum spores and mycelia. These proteins are 34 and 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot. We conclude sensitivity to S. chartarum is potentially much more widespread than previously appreciated. This fungus may impact the asthmatic and allergic population through both immunologic and toxic mechanisms. Its significance in the milieu of allergenic fungi may need to be re-evaluated.

Occupational rhinoconjunctivitis and food allergy because of aniseed sensitization

Aniseed is a spice frequently used in Mediterranean cooking and, as with other Umbelliferae, it has been involved in clinical allergy. This investigation was undertaken to study the allergens implicated in a case of occupational allergy to aniseed associated with rhinoconjunctivitis and gastrointestinal symptoms. Skin prick tests were performed to inhalant allergens, spices used in the patient's workplace (aniseed and cinnamon), and 12 other Umbelliferae spices, birch, and mugwort. A nasal challenge test to aniseed and cinnamon and a double-blind placebo-controlled oral food challenge test to aniseed were also performed. The molecular weights of the allergens were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting and cross-reactivity among Umbelliferae species by enzyme immunoassay inhibition. Skin prick tests showed a positive immediate response to aniseed, asparagus, caraway, coriander, cumin, dill, and fennel extracts, and an intense late response to aniseed. Skin prick tests to celery, carrot, birch pollen, and mugwort pollen extracts were negative. Results of a nasal challenge test were positive to aniseed and negative to cinnamon; an aniseed oral food challenge test yielded a positive response. The molecular weights of the main immunoglobulin (Ig)E-binding proteins in aniseed extracts were approximately 48, 42, 39, 37, 34, 33, and 20 kD. Caraway, fennel, cumin, and coriander extracts showed similar IgE-binding patterns. Enzyme immunoassay inhibition studies with the patient's serum revealed cross-reactivity among the IgE components from aniseed, caraway, coriander, fennel, and dill extracts. We demonstrate the presence of aniseed allergens in a case of occupational rhinoconjunctivitis and food allergy, with molecular weights for this spice that differed from those previously reported.

Identification of Sunflower Seed IgE-Binding Proteins

Background: Sunflower seed can cause severe anaphylactic reactions in some susceptible individuals. It is conceivable that the 2S sunflower seed protein is an allergen based on its high degree of homology (34%) with the allergenic mature 2S albumin protein of the Brazil nut. The first step in determining the allergenicity of sunflower seed proteins is to identify IgE-binding proteins. Methods: Sera from sunflower seed-sensitive individuals were evaluated by radioallergosorbent test (RAST), isoelectric focusing (IEF) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting with sunflower seed proteins. Results: Positive RAST scores (>2) were observed in 3 individuals and immunoblotting demonstrated IgE-binding to 2–7 distinct proteins ranging in size from 10 to 50 kD. Two out of 3 sera recognized two proteins between 16 and 17 kD. The lower molecular weight protein (16 kD) approximates to the prepo region of the precursor methionine-rich 2S albumin protein found in sunflower seed (SFA-8/SSA). IEF followed by immunoblotting demonstrated several IgE-binding proteins, including two proteins with isoelectric points of 5.97 and 5.3, respectively, which are consistent with the mature and immature forms of the SFA-8/SSA region. Conclusions: Sunflower seed contains several IgE-binding proteins, including regions of the high-methionine 2S albumin SFA-8/SSA.

High-Affinity Folate Receptor in Human Ovary, Serous Ovarian Adenocarcinoma, and Ascites: Radioligand Binding Mechanism, Molecular Size, Ionic Properties, Hydrophobic Domain, and Immunoreactivity

High-affinity folate receptors are expressed in normal ovaries and ovarian carcinomas. Binding of [3H]folate in human ovary, serous ovarian carcinoma tissue, and ascites is a complex process that has not been well characterized. This study shows changes in binding affinity and mechanism of binding with decreasing receptor concentration, inhibition by folate derivatives, and a slow radioligand dissociation at pH 7.4 becoming rapid and complete at pH 3.5. The receptor seems to be positively charged since it elutes in the front effluent of a DEAE-Sepharose CL-6B ion-exchange column at pH 6.3. The gel filtration profile of Triton X-100-solubilized tissue and ascites contained two peaks of radioligand-bound receptor (25 and 100 kDa). Exposure of ascites to cleavage by phosphatidylinositol-specific phospholipase C resulted in a partial conversion of the 100-kDa peak to a 25-kDa peak. This suggests that the receptor may be anchored to the membrane by a glycosylphosphatidyl residue that inserts into Triton X-100 micelles, resulting in a large molecular size on gel filtration. The receptor in ovarian carcinoma tissue immunoreacts with antibodies against purified human milk folate receptor protein as shown by enzyme-linked immunosorbent assay, immunoprecipitation, sodium dodecyl sulfate–polyacrylamide gel electrophoresis immunoblotting (a single band of 45 kDa), and immunohistochemistry. In only three of seven ovarian carcinomas did expression of radioligand-bound receptors exceed levels found in five normal ovaries. However, only receptors in ovarian carcinoma specimens showed a high degree of immunoreactivity. Hence, even without elevations of the total receptor level, a folate receptor isoform homologous to human milk folate receptor protein seemed to prevail in serous ovarian carcinomas.

Expression of LTC4 Synthase During the Development of Eosinophils In Vitro From Cord Blood Progenitors

AbstractThe expression of leukotriene C4 synthase (LTC4S) was examined during the development of eosinophils in vitro from cord blood mononuclear cells. At 7 days, the cells contained mRNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot signals for cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and 5-lipoxygenase-activating protein (FLAP), but lacked LTC4S and did not generate cysteinyl leukotrienes when stimulated with 20 µmol/L calcium ionophore. At 14 days, 94% of the cells were of eosinophil lineage, both LTC4S mRNA transcript and protein were present, and ionophore stimulation resulted in the generation of 23.9 ± 6.0 pmol cysteinyl leukotrienes/106 eosinophil-lineage cells (mean ± SEM, n = 6). At 28 days, progressive eosinophil maturation was accompanied by further increments in 5-LO, FLAP, and LTC4S proteins, and by the ionophore-induced production of 94.6 ± 9.0 pmol cysteinyl leukotrienes/106 eosinophil-lineage cells (n = 6). Cells selected for CD34 expression lacked detectable 5-LO/LTC4S pathway proteins, and with culture generally expressed immunodetectable cPLA2 and 5-LO proteins by 3 days, FLAP protein by 7 days, and LTC4S protein by 10 days. Thus, during the development of eosinophils in vitro, cPLA2, 5-LO, and FLAP are expressed before LTC4S. Once the lineage is established by morphologic criteria, the eosinophilopoietic cytokines mediate upregulation of FLAP and LTC4S, members of a newly recognized gene family, and of 5-LO, during ongoing cell maturation.

Expression of LTC4 synthase during the development of eosinophils in vitro from cord blood progenitors

The expression of leukotriene C4 synthase (LTC4S) was examined during the development of eosinophils in vitro from cord blood mononuclear cells. At 7 days, the cells contained mRNA and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot signals for cytosolic phospholipase A2 (cPLA2), 5-lipoxygenase (5-LO), and 5-lipoxygenase-activating protein (FLAP), but lacked LTC4S and did not generate cysteinyl leukotrienes when stimulated with 20 mumol/L calcium ionophore. At 14 days, 94% of the cells were of eosinophil lineage, both LTC4S mRNA transcript and protein were present, and ionophore stimulation resulted in the generation of 23.9 +/- 6.0 pmol cysteinyl leukotrienes/10(6) eosinophil-lineage cells (mean +/- SEM, n = 6). At 28 days, progressive eosinophil maturation was accompanied by further increments in 5-LO, FLAP, and LTC4S proteins, and by the ionophore- induced production of 94.6 +/- 9.0 pmol cysteinyl leukotrienes/10(6) eosinophil-lineage cells (n = 6). Cells selected for CD34 expression lacked detectable 5-LO/LTC4S pathway proteins, and with culture generally expressed immunodetectable cPLA2 and 5-LO proteins by 3 days, FLAP protein by 7 days, and LTC4S protein by 10 days. Thus, during the development of eosinophils in vitro, cPLA2, 5-LO, and FLAP are expressed before LTC4S. Once the lineage is established by morphologic criteria, the eosinophilopoietic cytokines mediate upregulation of FLAP and LTC4S, members of a newly recognized gene family, and of 5-LO, during ongoing cell maturation.

Genetic and carbon source regulation of phosphorylation of Sip1p, a Snf1p-associated protein involved in carbon response in Saccharomyces cerevisiae.

The SIP1 gene of Saccharomyces cerevisiae is a carbon-catabolite-specific negative regulator of GAL gene transcription and acts as a multicopy suppressor of growth defects associated with impaired Snf1p protein kinase activity. The Sip1 protein is known to undergo phosphorylation when associated in vitro with the Snf1 protein kinase. We have carried out in vivo studies of the genetic and carbon control of Sip1p phosphorylation. Metabolic labeling reveals phosphorylation of Sip1p under both carbon catabolite-repressing and non-repressing conditions and in both SNF1 wild-type and snf1-deletion cells. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblot assay, we detect apparent changes in Sip1p phosphorylation states in response to changes in carbon source. At least one dephosphorylation of Sip1p occurs with a shift from non-repressing carbon source to repressing carbon source. The MIG1 gene, acting through SNF1-dependent and SNF1-independent pathways, is required for some Sip1p phosphorylations. REG1 appears to be required for at least one dephosphorylation of Sip1p, whereas SSN6 appears to be required for at least one phosphorylation of Sip1p. These results reveal new complexities in carbon response signaling, and may reflect the involvement of the Sip1 protein in the same complex as the Mig1 and Ssn6 proteins.

High-Purity Factor IX Concentrates for Treatment of Hemophilia B: Relative Purity and Thrombogenic Potential

Constituents other than factor IX have been implicated as etiologic agents for thrombotic complications in patients receiving prothrombin complex concentrates (PCCs). In vitro studies, in vivo animal models, and clinical evaluations in patients with hemophilia B indicate that high-purity factor IX concentrates contain significantly fewer potentially thrombogenic contaminants than PCCs. A recent in vitro study from our laboratory used highly sensitive assays to analyze the relative purity of these newer products. The following products were studied using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting: Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC (now known as Replinine). The mean specific activity of the high-purity factor IX products ranged from 68 IU factor IX/mg (Aimafix) to 246 IU factor IX/mg (Mononine). SDS-PAGE analysis under reducing and nonreducing conditions showed that Mononine had the fewest contaminating bands. The immunoblot to detect factor IX showed a dominant factor IX band for all products, visible light chain of factor IX for all products except Aimafix, and another contaminating band visible at 49,500 daltons for all products except 9MC. High molecular weight contaminants were apparent for some products. Factor VIIa was detected in some lots of Immunine, Nanotiv and 9MC. Factor X and prothrombin contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv and 9MC. Thus, Mononine, Nanotiv and 9MC demonstrated the highest purity but no product was totally free of contaminants.

In Vitro Characterization of High Purity Factor IX Concentrates for the Treatment of Hemophilia B

This study employed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoblotting to assess the purity of seven high purity factor IX concentrates: Aimafix (Aima), AlphaNine-SD (Alpha Therapeutic), Factor IX VHP (Biotransfusion), Immunine (Immuno), Mononine (Armour Pharmaceutical), Nanotiv (Kabi Pharmacia), and 9MC (Blood Products Laboratory). The mean specific activity of these products ranged from 68 U factor IX/mg (Aimafix) to 246 U factor IX/mg (Mononine). SDS-PAGE analysis showed that the highest purity product, Mononine, had a single contaminating band under non-reducing conditions. Two additional bands were detected when this product was analyzed under reducing conditions. All other products had multiple contaminating bands that were more apparent under reducing than non-reducing conditions. The immunoblot for factor IX showed a dominant factor IX band for all products. In addition, visible light chain of factor IX was detected for AlphaNine-SD, Factor IX VHP, Immunine, Mononine, Nanotiv, and 9MC, suggesting that the factor IX in these products had undergone partial activation to factor IXa. Another contaminating band was visible at 49,500 for all of the products except 9MC. In addition to this band, high molecular weight contaminants were apparent for some products, most notably AlphaNine-SD. The identity of these bands is unknown. Immunoblotting failed to demonstrate factor VII as a contaminant of any of the high purity products, although factor VIIa could be detected in some lots of Immunine, Nanotiv, and 9MC by a clot-based assay. Factor X contaminated Aimafix, AlphaNine-SD, Factor IX VHP, Immunine, Nanotiv, and 9MC, but activation products of factor X were not detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparison of Psilocybe cubensis spore and mycelium allergens

Background: Basidiospores are an important cause of respiratory allergy in mold-sensitive atopic subjects. Collection of the large amounts of spores required for extract preparation is tedious and difficult. A desirable alternative could be mycelium grown in vitro if it is allergenically similar to spores. Methods: Therefore this study compared the allergen contents of Psilocybe cubensis spore and mycelium extracts by different techniques with the use of pooled sera from subjects who had skin test and RAST results that were positive to P. cubensis spores. Results: Isoelectric focusing immunoprints revealed six common IgE-binding bands at isoelectric points 4.7, 5.0, 5.5, 5.6, 8.7, and 9.3. Two additional bands at isoelectric points 3.9 and 5.7 were detected only in the spore extract. Sodium dodecylsulfate polyacrylamide gel electrophoresis immunoblots exhibited six common IgE-binding bands at 16, 35, 487, 52, 62, and 76 kd, 20 and 40 kd bands were present only in the spore extract. Although RAST and isoelectric focusing inhibition demonstrated that P. cubensis spore and mycelium extracts share many allergens, spores were allergenically more potent than mycelium. Conclusion: The results indicate that mycelium is a useful source of P. cubensis allergen, even though several spore allergens were not detected in mycelium.

Fish allergy: Evaluation of the importance of cross-reactivity

Fish constitute one of the most important groups of allergens in the induction of immediate (type I) food hypersensitivity. In our environment, fish allergy is present in 22% of all patients with a diagnosis of food hypersensitivity. We studied the allergenic significance of the fish species considered most representative because of their greater consumption in our environment (flatfishes: Pleuronectiformes such as sole, whiff, and witch; Gadiformes such as hake; and Scombriformes such as albacore) or because of the results of previous studies of Gadiformes such as cod. Through the use of isoelectric focusing and sodium dodecyl sulfate-polyacrylamide gel electrophoresis immunoblotting, we have observed that several allergens common to all these species are able to bind specific IgE from the sera of sensitized patients. This allergenic community has been confirmed by radioallergosorbent inhibition. Another group of species-specific allergens focuses in the regions at about pH 5 and with molecular weights less than 14 kilodaltons; these allergens correspond to sarcoplasmic parvoalbumins. From the results observed, which have been confirmed by various procedures and techniques, we conclude that hake is the fish with the capability to induce the strongest IgE response, followed by whiff; the witch seems to be the least allergenic of all flatfishes. Among all species studied, albacore was the least allergenic. These results may be considered when one introduces supplementary feeding with fish in infants, most particularly in infants at high risk for atopy.

Prevalence of specific IgE to the storage mite, Aleuroglyphus ovatus

Aleuroglyphus ovatus (Ao) is a storage mite that has a worldwide distribution and has been found in stored bran, wheat, chicken meal, and dried fish products. Ao was isolated from a chicken meal sample, cultured on whole wheat flour at 75% relative humidity, and 1 gm of pure Ao bodies were extracted, 1:20 wt/vol, in 0.2 mol/L of ammonium bicarbonate. Der p I, major allergen of Dermatophagoides pteronyssinus (Dp), or Der f I, major allergen to Dermatophagoides farinae (Df), was not detected in extracts of Ao. Sodium dodecyl sulfate—polyacrylamide gel electrophoresis revealed 25 bands (6 to 110 kd), and thin-layer isoelectric focusing revealed 19 bands (isoelectric point, 3.5 to 9.3). Sodium dodecyl sulfate—polyacrylamide gel electrophoresis immunoblots with six different sera identified nine IgE binding bands (16 to 100 kd). Thin-layer isoelectric focusing immunoblots identified 16 IgE binding bands (isoelectric point, 4.5 to 9.3). An aliquot of the Ao extract was diluted in bicarbonate buffer (pH 9.2) and adsorbed to plastic microtiter plates. RAST to Ao and Dp were performed with sera of 163 atopic individuals (88 with allergic rhinitis and 75 with allergic asthma with or without allergic rhinitis). All individuals had appropriately positive skin tests that correlated with a history of exacerbation secondary to allergen exposure. RAST results to Dp and Ao were analyzed by regression analysis ( r = 0.64; p = 0.0001). Fifty-five individuals were RAST positive to Dp alone, 32 to Ao and Dp, and three to Ao alone; 73 individuals were negative to Ao and Dp. RAST inhibition demonstrated moderate cross-reactivity between Ao and Dp.