Oral allergy syndrome and anaphylactic reactions in BALB/c mice caused by soybean glycinin and β-conglycinin

Background Soybean protein is used in a number of food products but is also a common cause of food allergy. Soybean glycinin and β‐conglycinin represent up to one‐third of protein in the soybean. Many reports have indicated that glycinin and β‐conglycinin have been characterized as major soybean allergens involved in food hypersensitivity. Objective To investigate oral allergy syndrome and anaphylactic reactions in BALB/c mice caused by soybean glycinin and β‐conglycinin with an intragastric feeding protocol without using an adjuvant. Methods BALB/c mice were sensitized by gavages with glycinin and β‐conglycinin, and allergen‐specific IgE and IgG1 responses were studied by a passive cutaneous anaphylaxis assay. Serum histamine release and blood pressure were measured according to other methods. Epithelium and mast cell dye used the method of light microscopy. Results Sensitization with soybean allergens induced high levels of antigen‐specific IgE and IgG1 and increased serum histamine in BALB/c mice. Percentiles of intact mast cell of small intestine in mice sensitized with glycinin and β‐conglyinin significantly decreased for 28 days. Degranulation of mast cells and damage of the epithelium in the small intestine of mice sensitized with globulins were observed. The level of blood pressure in sensitized mice reached a minimum at 3 h. Conclusion Soybean‐specific IgE and IgG1 antibodies increased, with high levels of histamine release, severe degranulation of mast cells and damage of the epithelium of small intestine in mice sensitized with glycinin and β‐conglyinin.     

Duration of clinical reactivity in cow's milk allergy is associated with levels of specific immunoglobulin G4 and immunoglobulin A antibodies to β‐lactoglobulin

Background The development of tolerance in IgE‐mediated allergies has been associated with lower cow's milk (CM)‐specific IgE levels, increasing levels of specific IgG4 and, more contestably, IgA. Objective We investigated whether specific antibody responses to CM proteins differ over time between patients who recovered from cow's milk allergy (CMA) by the age of 3 years and those who developed tolerance only after the age of 8 years. Methods The study population comprised of 83 patients with IgE‐mediated CMA. They belonged to a cohort of 6209 healthy, full‐term infants followed prospectively for the emergence of CMA. Serum samples were available at diagnosis (median age 7 months), 1 year later (median 19 months) and at follow‐up (median 8.5 years). Age‐matched control subjects with no history of CMA (n=76) participated in the follow‐up. Serum levels of IgE antibodies to CM were measured using UniCAP. Levels of IgA, IgG1 and IgG4 antibodies to β‐lactoglobulin and α‐casein were measured using ELISA. Results Patients with persistent CMA at the age of 8 years (n=18 at diagnosis, n=16 at later time‐points) had higher CM‐specific IgE levels at all three time‐points (P<0.001) compared with patients who became tolerant by 3 years (n=55 at diagnosis, n=54 a year later, n=40 at follow‐up). They had lower serum IgA levels to β‐lactoglobulin at diagnosis (P=0.01), and lower IgG4 levels to β‐lactoglobulin (P=0.04) and α‐casein (P=0.05) at follow‐up. Conclusion High CM‐specific IgE levels predict the persistence of CMA. Development of tolerance is associated with elevated levels of β‐lactoglobulin‐specific serum IgA at the time of diagnosis, and later increasing specific IgG4 levels to β‐lactoglobulin and α‐casein. Cite this as: E. M. Savilahti, K. M. Saarinen and E. Savilahti, Clinical & Experimental Allergy, 2010 (40) 251–256.     

Occupational sensitization to soy allergens in workers at a processing facility

Background Exposure to soy antigens has been associated with asthma in community outbreaks and in some workplaces. Recently, 135 soy flake processing workers (SPWs) in a Tennessee facility were evaluated for immune reactivity to soy. Allergic sensitization to soy was common and was five times more prevalent than in health care worker controls (HCWs) with no known soy exposure. Objective To characterize sensitization to soy allergens in SPWs. Methods Sera that were positive to soy ImmunoCAP (n=27) were tested in IgE immunoblots. Wild‐type (WT) and transgenic (TG) antigens were sequenced using nanoscale Ultra‐Performance Liquid Chromatography Tandem Mass Spectrometry (nanoUPLC MS/MS). IgE reactivity towards 5‐enolpyruvylshikimate‐3‐phosphate synthase (CP4‐EPSP), a protein found in TG soy, was additionally investigated. De‐identified sera from 50 HCWs were used as a control. Results Immunoblotting of WT and TG soy flake extracts revealed IgE against multiple soy antigens with reactivity towards 48, 54, and 62 kDa bands being the most common. The prominent proteins that bound SPW IgE were identified by nanoUPLC MS/MS analysis to be the high molecular weight soybean storage proteins, β‐conglycinin (Gly m 5), and Glycinin (Gly m 6). No specific IgE reactivity could be detected to lower molecular weight soy allergens, Gly m 1 and Gly m 2, in soybean hull (SH) extracts. IgE reactivity was comparable between WT and TG extracts; however, IgE antibodies to CP4‐EPSP could not be detected. Conclusions and Clinical Relevance SPWs with specific IgE to soy reacted most commonly with higher molecular weight soybean storage proteins compared with the lower molecular weight SH allergens identified in community asthma studies. IgE reactivity was comparable between WT and TG soy extracts, while no IgE reactivity to CP4‐EPSP was observed. High molecular weight soybean storage allergens, Gly m 5 and Gly m 6, may be respiratory sensitizers in occupational exposed SPWs. Cite this as: B. J. Green, K. J. Cummings, W. R. Rittenour, J. M. Hettick, T. A. Bledsoe, F. M.Blachere, P. D. Siegel, D. M. Gaughan, G. J. Kullman, K. Kreiss, J. Cox‐Ganser and D. H. Beezhold, Clinical & Experimental Allergy, 2011 (41) 1022–1030.     

Reduction of the allergenic protein in soybean meal by enzymatic hydrolysis

The effects of Alcalase and Trypsin hydrolysis on the allergenic protein and its solubility of soybean meal (SBM) were investigated by SDS-PAGE and ELISA. After 10 min of hydrolysis with Alcalase, the solubility of the protein was 87.19%, the α′, α and β-subunits of β-conglycinin disappeared and the acidic and basic subunits of glycinin decreased obviously by SDS-PAGE pattern. However, with Trypsin for 10 min, the solubility of the protein was 72.74%. The contents of α′, α subunits of β-conglycinin decreased slightly in SDS-PAGE, and the acidic and basic subunits of glycinin were almost not affected. For the insoluble hydrolysates prepared by Alcalase, the α′, α subunits of β-conglycinin and acidic subunit of glycinin decreased significantly, that by trypsin did not. The content of immune-reactive β-conglycinin obtained by ELISA was consistent with them. These results confirmed that the allergenic proteins of SBM were more sensitive to Alcalase than to Trypsin.     

Development of antibodies against the fluoroquinolone sarafloxacin and molecular modeling studies of cross‐reactive compounds

Polyclonal antibodies were prepared against the fluoroquinolone sarafloxacin. Sarafloxacin was conjugated directly to cationized bovine serum albumin (BSA) and ovalbumin. Balb/c mice were immunized with the sarafloxacin‐BSA conjugate (cBSA‐saraflox) and sarafloxacin‐reactive sera (1–5) were obtained from these mice. Serum from mouse 1 (Abl) exhibited the lowest IC₅₀ for free sarafloxacin using an indirect competitive inhibition ELISA (ci‐ELISA). Other structurally related quinolones, including difloxacin, enrofloxacin, norfloxacin, trovafloxacin and nalidixic acid, demonstrated cross‐reactivity with the sarafloxacin antibodies as determined by ci‐ELISA. In an effort to correlate antibody binding with three‐dimensional properties of the cross‐reactive compounds, all of the fluoroquinolones as well as nalidixic acid were modeled, and global energy minima were determined using molecular mechanical and quantum mechanical methods. The results demonstrate that the three‐dimensional models can yield information that explains observed cross‐reactivity data. These models are particularly helpful when the chemical structure of an analog varies greatly from the immunogen yet the IC₅₀ value for the compound is not vastly different. Furthermore, conformational and electronic data from this study can be used to predict whether other fluoroquinolones will exhibit good cross‐reactivity in this ELISA.     

The genetic basis of Brugada syndrome: A mutation update

Brugada syndrome (BrS) is a condition characterized by a distinct ST‐segment elevation in the right precordial leads of the electrocardiogram and, clinically, by an increased risk of cardiac arrhythmia and sudden death. The condition predominantly exhibits an autosomal dominant pattern of inheritance with an average prevalence of 5:10,000 worldwide. Currently, more than 100 mutations in seven genes have been associated with BrS. Loss‐of‐function mutations in SCN5A, which encodes the α‐subunit of the Na_v1.5 sodium ion channel conducting the depolarizing I_Na current, causes 15–20% of BrS cases. A few mutations have been described in GPD1L, which encodes glycerol‐3‐phosphate dehydrogenase‐1 like protein; CACNA1C, which encodes the α‐subunit of the Ca_v1.2 ion channel conducting the depolarizing I_L,Ca current; CACNB2, which encodes the stimulating β2‐subunit of the Ca_v1.2 ion channel; SCN1B and SCN3B, which, in the heart, encodes β‐subunits of the Na_v1.5 sodium ion channel, and KCNE3, which encodes the ancillary inhibitory β‐subunit of several potassium channels including the Kv4.3 ion channel conducting the repolarizing potassium I_to current. BrS exhibits variable expressivity, reduced penetrance, and “mixed phenotypes,” where families contain members with BrS as well as long QT syndrome, atrial fibrillation, short QT syndrome, conduction disease, or structural heart disease, have also been described. Hum Mutat 30:1–11, 2009. © 2009 Wiley‐Liss, Inc.     

Development and Validation of an ELISA for Hemoglobin‐A2: A Novel Method for β‐Thalassemia Screening in Developing Countries

Abstract

Hemoglobin‐A₂ (HbA₂) measurement in human hemolysates has great significance, since its level can indicate β‐thalassemia carrier status in other-wise healthy individuals. An ELISA for HbA₂ using antiserum mono‐specific to the δ chain of HbA₂ and affinity purified antirabbit gamma globulins (ARGG) conjugated to horseradish peroxidase (HRP) have been developed. The monospecific antiserum used does not cross react with other hemoglobins. Hemolysates from volunteers are used for measurement of HbA₂. In a limited trial for β ‐thalassemia carrier screening (n = 350), the results obtained with the developed ELISA are comparable with those obtained with a micro‐column chromatography method (r ≥ 0.89). The developed ELISA is simple, accurate, precise, inexpensive, and several samples can be processed simultaneously with ease, making this system a suitable candidate for transforming into a user friendly kit.     

Auto‐ and cross‐reactivity to thioredoxin allergens in allergic bronchopulmonary aspergillosis

Background: Thioredoxins are cross‐reactive allergens involved in the pathogenesis of atopic eczema and asthma. Cross‐reactivity to human thioredoxin can contribute to the exacerbation of severe atopic diseases. Methods: Human thioredoxin, Asp f28 and Asp f29, two thioredoxins of Aspergillus fumigatus, and thioredoxin of Malassezia sympodialis were cloned and produced as recombinant proteins. Allergenicity and cross‐reactivity to thioredoxins in allergic bronchopulmonary aspergillosis patients were assessed by enzyme‐linked immunosorbent assay (ELISA), inhibition ELISA, immunoblot analysis, proliferation assays and skin tests. Molecular homology modelling was used to identify conserved, surface‐exposed amino acids potentially involved in immunoglobulin E (IgE)‐binding. Results: All thioredoxins, including the human enzyme, bind IgE from patients with allergic bronchopulmonary aspergillosis and induce allergen‐specific proliferation in peripheral blood mononuclear cells and positive skin reactions in thioredoxin‐sensitized patients. Inhibition experiments showed that the thioredoxins are cross‐reactive indicating humoral immune responses based on molecular mimicry. To identify structural surface elements involved in cross‐reactivity, the three‐dimensional structures were modelled based on solved thioredoxin structures. Analysis of the molecular surfaces combined with sequence alignments allowed identification of conserved solvent exposed amino acids distantly located in the linear sequences which cluster to patches of continuous surface areas. The size of the surface areas conserved between human and fungal thioredoxins correlates well with the inhibitory potential of the molecules in inhibition ELISA indicating that the shared amino acids are involved in IgE‐binding. Conclusions: Conserved, solvent exposed residues shared between different thioredoxins cluster to continuous surface regions potentially forming cross‐reactive conformational B‐cell epitopes responsible for IgE‐mediated cross‐reactivity and autoreactivity.     

Activin‐βc reduces reproductive tumour progression and abolishes cancer‐associated cachexia in inhibin‐deficient mice

Activins are involved in the regulation of a diverse range of physiological processes including development, reproduction, and fertility, and have been implicated in the progression of cancers. Bioactivity is regulated by the inhibin α‐subunit and by an activin‐binding protein, follistatin. The activin‐β_C subunit was not considered functionally significant in this regard due to an absence of phenotype in knockout mice. However, activin‐β_C forms heterodimers with activin‐β_A and activin‐C antagonizes activin‐A in vitro. Thus, it is proposed that overexpression, rather than loss of activin‐β_C, regulates activin‐A bioactivity. In order to prove biological efficacy, inhibin α‐subunit knockout mice (α‐KO) were crossed with mice overexpressing activin‐β_C (ActC++). Deletion of inhibin leads to Sertoli and granulosa cell tumours, increased activin‐A, and cancer‐associated cachexia. Therefore, cachexia and reproductive tumour development should be modulated in α‐KO/ActC++ mice, where excessive activin‐A is the underlying cause. Accordingly, a reduction in activin‐A, no significant weight loss, and reduced incidence of reproductive tumours were evident in α‐KO/ActC++ mice. Overexpression of activin‐β_C antagonized the activin signalling cascade; thus, the tumourigenic effects of activin‐A were abrogated. This study provides proof of the biological relevance of activin‐β_C. Being a regulator of activin‐A, it is able to abolish cachexia and modulate reproductive tumour development in α‐KO mice.     

Cross‐reactivity of hepatitis C virus specific vaccine‐induced T cells at immunodominant epitopes

Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS3₁₀₇₃ and NS3₁₄₄₆, and multiple variants for epitope NS3₁₄₀₆ that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross‐reactivity of vaccine‐induced T cells was determined using variant peptides in IFN‐γ ELISPOT assays. Vaccine‐induced T cells targeted approximately 90% of NS3₁₀₇₃ genotype 1 sequences and 50% of NS3₁₄₄₆ genotype 1 and 3 sequences. For NS3_1406, 62% of subtype‐1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS3₁₄₀₆ that was maximally cross‐reactive. In vitro priming assays showed that of those tested the NS3₁₄₀₆ vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross‐reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross‐reactive T cells at immunodominant epitopes. The degree of cross‐reactivity varies between epitopes and may be HCV‐subtype specific.     

Beta‐adrenergic receptor gene polymorphisms and cardiovascular reactivity to stress in Black adolescents and young adults

Cardiovascular reactivity to stress and β‐adrenergic receptor (β‐AR) function may contribute to the development of hypertension. As Black Americans have an increased risk of hypertension, we evaluated associations between β₁‐AR (Arg389Gly) and β₂‐AR (Arg16Gly, Gln27Glu) gene variants and cardiovascular reactivity in 500 Black youth. Heart rate, preejection period, total peripheral resistance, and blood pressure reactivity were measured during cold and psychological stress. The Arg389Gly polymorphism in the β₁‐AR was associated with preejection period reactivity in males but not in females. The Arg16Gly polymorphism in the β₂‐AR was associated with diastolic blood pressure reactivity only during video game stress. An association between the Gln27Glu polymorphism in the β₂‐AR and vascular reactivity depended on sex. Thus, specific patterns of associations emerged between genetic variations in β‐ARs and cardiovascular reactivity in young Blacks.     

A study of CDR3 loop dynamics reveals distinct mechanisms of peptide recognition by T‐cell receptors exhibiting different levels of cross‐reactivity

T‐cell receptors (TCRs) can productively interact with many different peptides bound within the MHC binding groove. This property varies with the level of cross‐reactivity of TCRs; some TCRs are particularly hyper cross‐reactive while others exhibit greater specificity. To elucidate the mechanism behind these differences, we studied five TCRs in complex with the same class II MHC (1A^b)‐peptide (3K), that are known to exhibit different levels of cross‐reactivity. Although these complexes have similar binding affinities, the interface areas between the TCR and the peptide–MHC (pMHC) differ significantly. We investigated static and dynamic structural features of the TCR–pMHC complexes and of TCRs in a free state, as well as the relationship between binding affinity and interface area. It was found that the TCRs known to exhibit lower levels of cross‐reactivity bound to pMHC using an induced‐fitting mechanism, forming large and tight interfaces rich in specific hydrogen bonds. In contrast, TCRs known to exhibit high levels of cross‐reactivity used a more rigid binding mechanism where non‐specific π‐interactions involving the bulky Trp residue in CDR3β dominated. As entropy loss upon binding in these highly degenerate and rigid TCRs is smaller than that in less degenerate TCRs, they can better tolerate changes in residues distal from the major contacts with MHC‐bound peptide. Hence, our dynamics study revealed that differences in the peptide recognition mechanisms by TCRs appear to correlate with the levels of T‐cell cross‐reactivity.     

Production and characterization of antibodies against aflatoxin in laying hens

Polyclonal antibodies against aflatoxins were obtained from egg yolks of laying hens immunized with either aflatoxin B₁ (AFB₁) or aflatoxin M₁ (AFM₁) conjugated to bovine serum albumin (BSA). An indirect enzyme‐linked immunosorbent assay (ELISA) involving the use of AFB₁‐BSA or AFM₁‐BSA conjugate and anti‐chicken IgG‐horseradish peroxidase (HRP) conjugate, was developed for monitoring antibody titers and aflatoxin analysis. Production of the antibody in hens started as early as 10 days after immunization and reached a maximum in 20 days. Competitive indirect ELISA revealed that the antibodies were most specific for AFB₁. They were cross‐reactive with other aflatoxins in the following order: B₁ (100%)>G₁ (22–28%)>B₂ (6–16%)>M₁(3–9%)>G₂ (1–4%). Anti‐AFM₁‐BSA antibodies showed a similar pattern of cross‐reaction to the anti‐AFB₁‐BSA antibodies when AFB₁‐BSA was coated to the ELISA plate. The specificity of anti‐AFM₁‐BSA to AFM₁ was demonstrated when AFM₁‐BSA was coated to the ELISA plate. Cross‐reactivity with different aflatoxins was in the following order: M₁ (531%)>B₁ (100%)>G₂ (41%)>G₁ (19%)>B₂ (3%). The sensitivity for AFB₁ analysis in the indirect ELISA was 0.05–5 ng AFB₁/assay.     

The allergen profile of beech and oak pollen

Background Beech and oak pollen are potential allergen sources with a world‐wide distribution. Objective We aimed to characterize the allergen profile of beech and oak pollen and to study cross‐reactivities with birch and grass pollen allergens. Methods Sera from tree pollen‐allergic patients with evidence for beech and oak pollen sensitization from Basel, Switzerland, (n=23) and sera from birch pollen‐allergic patients from Vienna, Austria, (n=26) were compared in immunoblot experiments for IgE reactivity to birch (Betula pendula syn. verrucosa), beech (Fagus sylvatica) and oak (Quercus alba) pollen allergens. Subsequently, beech and oak pollen allergens were characterized by IgE inhibition experiments with purified recombinant and natural allergens and with allergen‐specific antibody probes. Birch‐, beech‐ and oak pollen‐specific IgE levels were determined by ELISA. Results Beech and oak pollen contain allergens that cross‐react with the birch pollen allergens Bet v 1, Bet v 2 and Bet v 4 and with the berberine bridge enzyme‐like allergen Phl p 4 from timothy grass pollen. Sera from Swiss and Austrian patients exhibited similar IgE reactivity profiles to birch, beech and oak pollen extracts. IgE levels to beech and oak pollen allergens were lower than those to birch pollen allergens. Conclusion IgE reactivity to beech pollen is mainly due to cross‐reactivity with birch pollen allergens, and a Phl p 4‐like molecule represented another predominant IgE‐reactive structure in oak pollen. The characterization of beech and oak pollen allergens and their cross‐reactivity is important for the diagnosis and treatment of beech and oak pollen allergy.     

nsLTP and profilin are allergens in mustard seeds: cloning,sequencing and recombinant production of Sin a 3 and Sin a 4

Background Patients allergic to mustard are frequently sensitized to peach. Objective To identify and analyse new yellow mustard allergens that could be involved in IgE cross‐reactivity. Methods Sera from mustard‐allergic patients with symptoms to peach were studied. Mass spectrometry analyses provided sequences of IgE‐reactive proteins. cDNAs encoding Sin a 3 and Sin a 4 were amplified by polymerase chain reaction, cloned and sequenced. The recombinant allergens were obtained in Pichia pastoris and Escherichia coli, respectively, and used for ELISA, immunoblotting and inhibition experiments. Sequence alignment was used to identify common IgE epitopes. Results Sin a 3‐ and Sin a 4‐specific cDNAs encode for mature proteins of 92 and 131 amino acids that belong to nsLTP and profilin protein families, respectively. Sin a 3 and Sin a 4 showed 54% and 80% identity with allergenic nsLTP from peach and profilin from melon, respectively. Both recombinant allergens were IgE‐reactive in ELISA and immunoblotting. Peach pulp and peel, and melon extracts nearly abolished the IgE binding to recombinant Sin a 3 or recombinant Sin a 4 in immunoblotting. Conclusion Sin a 3 (nsLTP) and Sin a 4 (profilin) were identified as new mustard allergens and showed IgE cross‐reactivity with fruits such as peach or melon, respectively. The knowledge of these two allergens will contribute towards better understand with cross‐reactivity between mustard and other plant food allergens, and their availability will provide physicians with useful tools for molecular diagnosis.     

Cloning and characterization of GABAA α subunits and GABAB subunits in Xenopus laevis during development

Gamma‐aminobutyric acid (GABA), the major inhibitory neurotransmitter in the adult nervous system, acts via two classes of receptors, the ionotropic GABA_A and metabotropic GABA_B receptors. During the development of the nervous system, GABA acts in a depolarizing, excitatory manner and plays an important role in various neural developmental processes including cell proliferation, migration, synapse formation, and activity‐dependent differentiation. Here we describe the spatial and temporal expression patterns of the GABA_A and GABA_B receptors during early development of Xenopus laevis. Using in situ hybridization and qRT‐PCR, GABA_A α2 was detected as a maternal mRNA. All other α‐subunits were first detected by tailbud through hatching stages. Expression of the various subunits was seen in the brain, spinal cord, cranial ganglia, olfactory epithelium, pineal, and pituitary gland. Each receptor subunit showed a distinctive, unique expression pattern, suggesting these receptors have specific functions and are regulated in a precise spatial and temporal manner. Developmental Dynamics 240:862–873, 2011. © 2011 Wiley‐Liss, Inc.     

IgG subclass reactivity to human cardiac myosin in cardiomyopathy patients is indicative of a Th1-like autoimmune disease

Studies performed in mice together with the demonstration of increased levels of heart-specific autoantibodies, cytokines and cytokine receptors in sera from cardiomyopathy (CMP) patients argued for a pathogenic role of autoimmune mechanisms in CMP. This study was designed to analyse the presence of IgG anti-heart antibodies in sera from patients suffering from hypertrophic and dilatative forms of CMP as well as from patients with ischaemic heart disease and healthy individuals. Patients' sera were analysed for IgG reactivity to Western-blotted extracts prepared from human epithelial and endothelial cells, heart and skeletal muscle specimens as well as from Streptococcus pyogenes. The IgG subclass (IgG1–4) reactivity to purified human cardiac myosin was analysed by ELISA. While sera from CMP patients and healthy individuals displayed comparable IgG reactivity to a variety of human proteins, cardiac myosin represented the prominent antigen detected strongly and preferentially by sera from CMP patients. Pronounced IgG anti-cardiac myosin reactivity was frequently found in sera from patients with dilatative CMP and reduced ventricular function. ELISA analyses revealed a prominent IgG2/IgG3 anti-cardiac myosin reactivity in CMP sera, indicating a preferential Th1-like immune response. Elevated anti-cytomegalovirus, anti-enterovirus IgG titres as well as IgG reactivity to nitrocellulose-blotted S. pyogenes proteins were also frequently observed in the group of CMP patients. If further work can support the hypothesis that autoreactivity to cardiac myosin represents a pathogenic factor in CMP, specific immunomodulation of this Th1- towards a Th2-like immune response may represent a promising therapeutic strategy for CMP.     

Preparation and characterization of a monoclonal antibody specific for the β‐agonist clenbuterol

A monoclonal antibody was obtained from a Balb/c mouse immunized with a conjugate prepared by the direct coupling of diazotized clenbuterol to bovine serum albumin. The antibody, characterized by γ₁,κ isotype and an affinity constant of 3.5 × 10⁸ l mol^‐1, is highly specific as its cross‐reactivity to structurally related molecules is less than 1%. A competitive ELISA for the detection of clenbuterol in horse and human urine was developed using this antibody.     

A soybean G2 glycinin allergen. 1. Identification and characterization

BACKGROUND: Multiple allergens have been documented in soybean extracts. IgE from individuals allergic to soybeans, but not to peanut, was shown by immunoblot analysis to bind to proteins with a molecular weight of approximately 21 kD. These findings suggested that unique proteins in soybeans might be responsible for soybean allergic reactivity. The objective of the present study was to identify unique proteins in soybean extracts that bind to specific IgE from soybean-sensitive individuals, and to characterize the allergen using physicochemical methods and IgE binding. METHODS: Two-dimensional and preparative SDS-PAGE/IgE immunoblot analysis was used to identify a 22-kD soybean-specific allergen from crude soybean extracts. N-terminal sequence analysis was used to determine the identification of the protein binding IgE from soybean-sensitive individuals. RESULTS: IgE immunoblot and amino acid sequence analysis identified the 22-kD protein as a member of the G2 glycinin soybean protein family. Further investigation revealed that the IgEs reacted with basic chains from each member of the glycinin family of soybean storage proteins. CONCLUSIONS: Each of the subunits from glycinin, the storage protein that is the most prevalent component of soybean, are major allergens.     

Monoclonal antibodies against the bitter sesquiterpene lactones,lactucin and lactucopicrin,from chicory (Cichorium intybus L.)

Monoclonal antibodies (MAbs) against lactucin and lactucopicrin (two of the bitter principles of chicory; Cichorium intybus L.) have been prepared. In competitive ELISA, the sera from mice immunized with keyhole limpet haemocyanin (KLH)‐lactucin and KLH‐ lactucopicrin have been screened for cross‐reactivity to various sesquiterpene lactones with a guaiane skeleton that are present in chicory, such as lactucin, 8‐deoxylactucin, and their 11β,13‐dihydro derivatives, and lactucopicrin. Mice showing low cross‐reactivity were used to produce hybridomas that were screened for cross‐reactivity in the same way. The screening resulted in a lactucopicrin‐specific hybridoma (no. 4H10) showing a low cross‐reactivity (?1%)for lactucin, 8‐deoxylactucin and their 11β,13‐dihydro derivatives. From KLH‐lactucin immunized mice, hybridoma no. 9F12 showed the best characteristics, i.e. 25% cross‐reactivity to 8‐deoxylactucin and its 11β,13‐dihydro derivative, and a 10% cross‐reactivity to lactucopicrin. The affinity constants of MAbs nos 9F12 and 4H10 were 2.2 X 10⁴ and 2.8 X 10⁵ M^‐1 respectively. Chicory extracts spiked with free sesquiterpene lactones showed an inhibition level equal to the sum of the inhibition level of the extract and the level of sesquiterpene lactones added.