Wheat α-gliadin and high-molecular-weight glutenin subunit accumulate in different storage compartments of transgenic soybean seed.

Wheat seed storage proteins (prolamins) are important for the grain quality because they provide a characteristic texture to wheat flour products. In wheat endosperm cells, prolamins are transported from the Endoplasmic reticulum to Protein storage vacuoles through two distinct pathways-a conventional pathway passing through the Golgi apparatus and an unconventional Golgi-bypassing pathway during which prolamins accumulate in the ER lumen, forming Protein bodies. Unfortunately, transport studies conducted previously achieved limited success because of the seed-specificity of the latter pathway and the multigene architecture of prolamins. To overcome this difficulty, we expressed either of the two families of wheat prolamins, namely α-gliadin or High-molecular-weight subunit of glutenin, in soybean seed, which naturally lacks prolamin-like proteins. SDS-PAGE analysis indicated the successful expression of recombinant wheat prolamins in transgenic soybean seeds. Their accumulation states were quite different-α-gliadin accumulated with partial fragmentation whereas the HMW-glutenin subunit formed disulfide-crosslinked polymers without fragmentation. Immunoelectron microscopy of seed sections revealed that α-gliadin was transported to PSVs whereas HMW-glutenin was deposited in novel ER-derived compartments distinct from PSVs. Observation of a developmental stage of seed cells showed the involvement of post-Golgi Prevacuolar compartments in the transport of α-gliadin. In a similar stage of cells, deposits of HMW-glutenin surrounded by membranes studded with ribosomes were observed confirming the accumulation of this prolamin as ER-derived PBs. Subcellular fractionation analysis supported the electron microscopy observations. Our results should help in better understanding of molecular events during the transport of prolamins in wheat.

Management of temporomandibular joint diseases: a rare case report of coexisting calcium pyrophosphate crystal deposition and synovial chondromatosis.

BACKGROUND: The coexistence of calcium pyrophosphate dihydrate crystal deposition (CPP) and synovial chondromatosis (SC) in the temporomandibular joint (TMJ) is rarely reported. CPP disease (CPPD) is complex arthritis synonymous with excessive pyrophosphate production and variable aberrations in mineral and organic phase metabolism of the joint cartilage, leading to local inundated CPP and crystal deposition of partially deciphered predispositions. Meanwhile, SC is a rare benign synovial joint proliferative disease of unclear etiology and has a low risk of malignant transformation. However, SC manifests severe joint disability and dysfunction because of connective tissue metaplasia of the synovial membrane, which forms cartilaginous nodules with or without calcifications or ossifications. These nodules often detach and form intra-articular loose bodies and very rarely within extraarticular spaces. CASE PRESENTATION: We report the case of a 61-year-old man to expand the body of literature on these unusual coexisting arthropathies of the TMJ. The patient presented to our hospital in 2020 with complaints of pain in the right TMJ and trismus for over 6 months. Radiographic assessments of the TMJ provided a preoperative provisional diagnosis of SC. However, the histopathology of the open biopsy revealed tumor-like lesions comprising several deposits of rhomboid and rod-shaped crystals that displayed positive birefringence in polarized light, confirming a coexistence of CPPD. A second-stage operation was performed for the complete removal of the loose bodies and chalk-like lesions including synovectomy. No evidence of recurrence was recorded after a follow-up of nearly 1.5 years. CONCLUSIONS: Isolated CPPD and SC of the TMJ are prevalent in the literature however, monoarticular coexistence of these diseases is rare, due to the lack of consistency in the diagnostic criteria in clinical practice. Moreover, optimal treatment depends on several considerations. This report delineated the molecular etiopathology and underscored the need for continued deciphering of the causal mechanisms of coexisting CPPD and SC of the TMJ. In addition, the importance of confirmatory testing for accurate diagnosis, and appropriate management of these diseases were discussed.

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

Components of plant-derived food allergens: Structure, diagnostics, and immunotherapy.

A large number of plant-derived food allergen components have been identified to date. Although these allergens are diverse, they often share common structural features such as numerous disulfide bonds or oligomeric structures. Furthermore, some plant-derived food allergen components cross-react with pollen allergens. Since the relationship between allergen components and clinical symptoms has been well characterized, measurements of specific IgE to these components have become useful for the accurate clinical diagnosis and selection of optimal treatment methods for various allergy-related conditions including allergy caused by plant-derived foods. Herein, I have described the types and structures of different plant allergen components and outlined the diagnosis as well as treatment strategies, including those reported recently, for such substances. Furthermore, I have also highlighted the contribution of allergen components to this field.

Simultaneous induction of mutant alleles of two allergenic genes in soybean by using site-directed mutagenesis.

BACKGROUND: Soybean (Glycine max) is a major protein crop, because soybean protein has an amino acid score comparable to that of beef and egg white. However, many allergens have been identified among soybean proteins. A decrease in allergenic protein levels would be useful for expanding the market for soybean proteins and processed foods. Recently, the CRISPR/Cas9 system has been adopted as a powerful tool for the site-directed mutagenesis in higher plants. This system is expected to generate hypoallergenic soybean varieties. RESULTS: We used two guide RNAs (gRNAs) and Agrobacterium-mediated transformation for simultaneous site-directed mutagenesis of two genes encoding the major allergens Gly m Bd 28 K and Gly m Bd 30 K in two Japanese soybean varieties, Enrei and Kariyutaka. We obtained two independent T0 Enrei plants and nine T0 Kariyutaka plants. Cleaved amplified polymorphic sequence (CAPS) analysis revealed that mutations were induced in both targeted loci of both soybean varieties. Sequencing analysis showed that deletions were the predominant mutation type in the targeted loci. The Cas9-free plants carrying the mutant alleles of the targeted loci with the transgenes excluded by genetic segregation were obtained in the T2 and T3 generations. Variable mutational spectra were observed in the targeted loci even in T2 and T3 progenies of the same T0 plant. Induction of multiple mutant alleles resulted in six haplotypes in the Cas9-free mutants derived from one T0 plant. Immunoblot analysis revealed that no Gly m Bd 28 K or Gly m Bd 30 K protein accumulated in the seeds of the Cas9-free plants. Whole-genome sequencing confirmed that a Cas9-free mutant had also no the other foreign DNA from the binary vector. Our results demonstrate the applicability of the CRISPR/Cas9 system for the production of hypoallergenic soybean plants. CONCLUSIONS: Simultaneous site-directed mutagenesis by the CRISPR/Cas9 system removed two major allergenic proteins from mature soybean seeds. This system enables rapid and efficient modification of seed components in soybean varieties.

Gly m 5/Gly m 8 fusion component as a potential novel candidate molecule for diagnosing soya bean allergy in Japanese children.

BACKGROUND: Soya bean is a major food allergen in children. Component-resolved diagnostics has improved the accuracy of diagnosing immunoglobulin E (IgE)-mediated food allergies. OBJECTIVE: We aimed to develop a novel component for the diagnosis of soya bean allergy using recombinant technology. METHODS: Japanese paediatric patients with suspected soya bean allergy (n = 91) were included, and symptomatic (n = 40) and asymptomatic (n = 51) cases were divided through oral food challenge testing. Specific IgE (sIgE) antibodies to each recombinant allergen component were analysed by enzyme-linked immunosorbent assay, and the diagnostic performances of the components were assessed by area under the receiver operating characteristic curves (AUC). RESULTS: Among the recombinant components, sIgE antibody levels to Gly m 8 showed the highest AUC (0.706). A combination of Gly m 8 and α' subunit of Gly m 5, improved the diagnostic performance of the single components. Moreover, the N-terminal extension region of α' subunit of Gly m 5, which has low cross-reactivity among the vicilins, showed higher diagnostic performance (AUC 0.695) than the full-length α' subunit of Gly m 5 (AUC 0.613). Based on these findings, we developed a fusion protein of Gly m 8 plus the extension region of α' subunit of Gly m 5; this fusion protein was very efficient for diagnosing soya bean allergy (AUC 0.801). CONCLUSION: A fusion protein of Gly m 8 and the extension region of α' subunit of Gly m 5 could potentially diagnose soya bean allergy in paediatric patients. Fusion proteins may be useful for producing novel allergen components with improved diagnostic value.

Specific IgE for Fag e 3 Predicts Oral Buckwheat Food Challenge Test Results and Anaphylaxis: A Pilot Study.

BACKGROUND: Buckwheat (BW) is the source of a life-threatening allergen. Fag e 3-specific serum IgE (sIgE) is more useful than BW-sIgE for diagnosis; however, it is unknown whether Fag e 3-sIgE can predict oral food challenge (OFC) results and anaphylaxis. This study aimed to clarify the efficacy of Fag e 3-sIgE in predicting OFC results and anaphylaxis. METHODS: We conducted a retrospective review of BW- and Fag e 3-sIgE data obtained using the ImmunoCAP® assay system and fluorescent enzyme-linked immunosorbent assay from children who underwent OFC using 3,072 mg of BW protein between July 2006 and March 2014 at Sagamihara National Hospital, Kanagawa, Japan. RESULTS: We analyzed 60 patients aged 1.9-13.4 years (median 6.0 years); 20 (33%) showed objective symptoms upon BW OFC. The patients without symptoms had significantly lower Fag e 3-sIgE than those with non-anaphylactic (p < 0.001) and anaphylactic reactions to BW (p = 0.004). Fag e 3-sIgE was the only tested factor that significantly predicted positive OFC results (odds ratio 8.93, 95% confidence interval 3.10-25.73, p < 0.001) and OFC-induced anaphylaxis (2.67, 1.12-6.35, p = 0.027). We suggest that a threshold Fag e 3-sIgE level of 18.0 kUE/L has 95% probability of provoking a positive reaction to BW. CONCLUSIONS: Fag e 3-sIgE predicted OFC results and OFC-induced anaphylaxis. We further emphasize paying careful attention to the risk of BW OFC-induced anaphylaxis.

Deposition mode of transforming growth factor-ß expressed in transgenic rice seed.

KEY MESSAGE: Mouse TGF-ß highly accumulated by expressing as a secretory homodimeric protein in transgenic rice endosperm. It was tightly deposited in ER-derived PBs by interaction with cysteine-rich prolamins. TGF-ß is one of the key players involved in the induction and maintenance of mucosal immune tolerance to dietary proteins through the induction of regulatory T cells. In order to utilize rice-based TGF-ß as a tool to promote oral immune tolerance induction, high production of TGF-ß is essentially required. When the codon-optimized mTGF-ß was expressed as a secretory protein by ligating an N-terminal signal peptide and C-terminal KDEL ER retention signal under the control of the endosperm-specific rice storage protein glutelin GluB-1 promoter, accumulation level was low in stable transgenic rice seeds. Then, to increase the accumulation level of mTGF-ß, it was expressed as fusion proteins by inserting into the C terminus of acidic subunit of glutelin GluA and the variable region of 26 kDa globulin. When fused with the glutelin, it could accumulate well as visible bands by CBB staining gel, but not for the 26 kDa globulin. Unexpectedly, expression of homodimeric mTGF-ß linked by a 6×Gly1×Ser linker as secretory protein resulted in higher level of accumulation. This expression level was further enhanced by reduction of some endogenous prolamins by RNA interference. The monomeric and dimeric mTGF-ßs were deposited in ER-derived PBs containing prolamins. When highly produced in rice seed, it is notable that most of ER-derived PBs were distorted and granulated. Step-wise extraction of storage proteins from rice seeds suggested that the mTGF-ß strongly interacted with cysteine-rich prolamins via disulfide bonds. This result was also supported by the finding that reducing agent was absolutely required for mTGF-ß extraction.

Recent advances in component resolved diagnosis in food allergy.

Due to the high prevalence of food allergic diseases globally there are increasing demands in clinical practice for managing IgE-mediated conditions. During the last decade, component resolved diagnostics has been introduced into the field of clinical allergology, providing information that cannot be obtained from extract-based tests. Component resolved data facilitate more precise diagnosis of allergic diseases and identify sensitizations attributable to cross-reactivity. Furthermore it assists risk assessment in clinical practice as sensitization to some allergenic molecules is related to persistence of clinical symptoms and systemic rather than local reactions. The information may also aid the clinician in prescription of oral immunotherapy (OIT) in patients with severe symptoms, and in giving advice on food allergen avoidance or on the need to perform food challenges. The use of allergen components is rapidly evolving and increases our possibility to treat food allergic patients with a more individual approach. Using molecular allergology, we can already now better diagnose, prognose and grade the food allergy. In summary, daily routine molecular allergy diagnostics offers a number of benefits that give us a higher diagnostic precision and allow for better management of the patient.

Synchronous quadruple multiple primary cancers of the tongue, bilateral breasts, and kidney in a female patient with a disease-free survival time of more than 5 years: a case report.

BACKGROUND: Reports of synchronous multiple primary cancers in patients with oral cancer have recently been increasing because of progress in radiographic diagnostic techniques. Multiple primary cancers in patients with oral cavity cancer mainly occur in the head and neck region, lung, and esophagus. 2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography is usually used to identify synchronous multiple primary cancers. CASE PRESENTATION: We herein describe a 69-year-old woman diagnosed with synchronous quadruple multiple primary cancers, namely a squamous cell carcinoma of the mobile tongue, invasive ductal carcinoma of the right breast, intraductal carcinoma of the left breast, and chromophobe renal cell carcinoma of the right kidney. We removed the four tumors over three surgical procedures to reduce the surgical risk because the patient had diabetes mellitus. To the best of our knowledge, this combination of multiple primary cancers has not been reported to date. Importantly, we followed this case for 5 years after surgery. The patient was alive and well with no clinical or radiologic signs of recurrent or metastatic disease at the time of this writing. CONCLUSIONS: In the present case, the kidney cancer could not be detected by 2-[18F]-fluoro-2-deoxy-D-glucose positron emission tomography but could be detected by contrast-enhanced computed tomography. To avoid overlooking multiple primary cancers of the kidney, we suggest that contrast-enhanced computed tomography should cover a region extending to the inferior margin of the kidney, rather than only to the liver, in patients with oral cavity cancer.

Development of a novel transgenic rice with hypocholesterolemic activity via high-level accumulation of the α' subunit of soybean ß-conglycinin.

Soybean 7S globulin, known as ß-conglycinin, has been shown to regulate human plasma cholesterol and triglyceride levels. Furthermore, the α' subunit of ß-conglycinin has specifically been shown to possess low-density lipoprotein (LDL)-cholesterol-lowering activity. Therefore, accumulation of the α' subunit of ß-conglycinin in rice seeds could lead to the production of new functional rice that could promote human health. Herein, we used the low-glutelin rice mutant 'Koshihikari' (var. a123) and suppressed its glutelins and prolamins, the major seed storage proteins of rice, by RNA interference. The accumulation levels of the α' subunit in the lines with suppressed glutelin and prolamin levels were >20 mg in 1 g of rice seeds, which is considerably higher than those in previous studies. Oral administration of the transgenic rice containing the α' subunit exhibited a hypocholesterolemic activity in rats; the serum total cholesterol and LDL cholesterol levels were significantly reduced when compared to those of the control rice (var. a123). The cholesterol-lowering action by transgenic rice accumulating the α' subunit induces a significant increase in fecal bile acid excretion and a tendency to increase in fecal cholesterol excretion. This is the first report that transgenic rice exhibits a hypocholesterolemic activity in rats in vivo by using the ß-conglycinin α' subunit.

Knockdown of the 7S globulin subunits shifts distribution of nitrogen sources to the residual protein fraction in transgenic soybean seeds.

KEY MESSAGE: A platform of gene silencing by amiRNA had been established in fertile transgenic soybean. We demonstrated that knockdown of storage protein shifted the distribution of nitrogen sources in soybean seeds. Artificial microRNAs (amiRNAs) were designed using the precursor sequence of the endogenous soybean (Glycine max L. Merrill) miRNA gma-miR159a and expressed in transgenic soybean plants to suppress the biosynthesis of 7S globulin, which is one of the major storage proteins. Seed-specific expression of these amiRNAs (amiR-7S) resulted in a strong suppression of 7S globulin subunit genes and decreased accumulation of the 7S globulin subunits in seeds. Thus, the results demonstrate that a platform for gene silencing by amiRNA was first developed in fertile transgenic soybean plants. There was no difference in nitrogen, carbon, and lipid contents between amiR-7S and control seeds. Four protein fractions were collected from defatted mature seeds on the basis of solubility at different pH to examine the distribution of nitrogen sources and compensatory effects. In the whey and lipophilic fractions, nitrogen content was similar in amiR-7S and control seeds. Nitrogen content was significantly decreased in the major soluble protein fraction and increased in the residual fraction (okara) of the amiR-7S seeds. Amino acid analysis revealed that increased nitrogen compounds in okara were proteins or peptides rather than free amino acids. Our study indicates that the decrease in 7S globulin subunits shifts the distribution of nitrogen sources to okara in transgenic soybean seeds.

Metastatic gallbladder cancer presenting as numb chin syndrome: A case report and literature review.

Gallbladder cancer (GBC) is an uncommon malignancy that is highly aggressive in the advanced stages. However, it rarely metastasizes to the mandible. Numb chin syndrome (NCS) is a rare neurological manifestation associated with various underlying causes, including occult primary cancers and distant metastases. It is often considered to be a significant indicator of malignancy, and thorough investigation is essential in the presence of unclear etiology. The current study reported on the case of a 69-year-old Japanese woman who presented with numbness and mild pain in the lower lip and chin area for three months. No other systemic symptoms were observed. Immunocytochemical examination revealed the presence of an adenocarcinoma and TNM staging as per the Union for International Cancer Control and the American Joint Committee on Cancer guidelines confirmed stage IVb GBC. Comprehensive full-body positron emission tomography-computed tomography examination using 18F-fluoro-2-deoxy-D-glucose revealed additional bone and soft-tissue metastases. Palliative chemotherapy and radiation treatment were initiated based on the advanced stage of disease at the time of diagnosis. However, the patient succumbed to multiple organ failure six months later. The simultaneous occurrence of GBC, mandibular metastasis and NCS is rare and associated with poor prognosis. Despite the widespread nature of the disease, it can often manifest as non-specific oral symptoms without any systemic indications. The current study emphasizes the critical importance of timely confirmatory testing for accurate diagnosis and initiation of appropriate management for such complex conditions.

High-level production of lactostatin, a hypocholesterolemic peptide, in transgenic rice using soybean A1aB1b as carrier.

Hypercholesterolemia, a form of cardiovascular disease, is one of the leading causes of deaths worldwide. Lactostatin (Ile-Ile-Ala-Glu-Lys), derived from ß-lactoglobulin in cow's milk, is a bioactive peptide with hypocholesterolemic activity higher than sitosterol, a known anti-hypercholesterolemic drug. Here, we successfully developed a transgenic rice accumulating a much higher level of lactostatin by inserting 29 IIAEK sequences into the structurally flexible (nonconserved) regions of soybean seed storage protein, A1aB1b, and introducing it into LGC-1 (low glutelin content mutant 1) as host variety. A1aB1b containing 29 lactostatins was expressed in the endosperm of rice seed cells by using seed specific promoters and sorted into novel compartments distinct from normal PB-I (ER-derived protein body) and PB-II (protein storage vacuoles). Transgenic rice seeds accumulated approximately 2 mg of lactostatins/g of dry seeds, which is relatively high compared with previous reports. Our findings suggest that the introduction of a high copy number of bioactive peptide into seed storage proteins as carrier is one of the effective means in producing higher amounts of bioactive peptides in rice.

Purification, crystallization and preliminary crystallographic analysis of soybean mature glycinin A1bB2.

Glycinin is one of the most abundant storage-protein molecules in soybean seeds and is composed of five subunits (A1aB1b, A1bB2, A2B1a, A3B4 and A5A4B3). A1bB2 was purified from a mutant soybean cultivar containing glycinin composed of only A5A4B3 and A1bB2. At 281 K the protein formed hexagonal, rectangular and rod-shaped crystals in the first [0.1 M imidazole pH 8.0, 0.2 M MgCl2, 35%(v/v) MPD], second [0.1 M sodium citrate pH 5.6, 0.2 M ammonium acetate, 30%(v/v) MPD] and third (0.1 M phosphate-citrate pH 4.2, 2.0 M ammonium sulfate) crystallization conditions, respectively. X-ray diffraction data were collected to resolutions of 1.85, 1.85 and 2.5 Å from crystals of the three different shapes. The crystals belonged to space groups P6322, P21 and P1, with unit-cell parameters a = b = 143.60, c = 84.54 Å, a = 114.54, b = 105.82, c = 116.67 Å, ß = 94.99° and a = 94.45, b = 94.96, c = 100.66 Å, α = 107.02, ß = 108.44, γ = 110.71°, respectively. One, six and six subunits of A1bB2 were estimated to be present in the respective asymmetric units. The three-dimensional structure of the A1bB2 hexamer is currently being determined.

Degradation of ß-conglycinin ß-homotrimer by papain: independent occurrence of limited and extensive proteolysis.

Limited and extensive proteolysis occur when ß-conglycinin ß homo-trimer (ß(3)-conglycinin) from soybeans is attacked by papain. Slow limited proteolysis is restricted to cleavage of ß(3)-conglycinin polypeptides into subunit halves (N- and C-terminal domains) that are further slightly truncated. The kinetics of limited and extensive proteolyses analyzed separately indicates that the two processes occur independently from the very beginning of the reaction. In contrast, limited proteolysis of phaseolin from common beans has been found to be prerequisite for the onset of its extensive proteolysis. The dramatic distinction between the degradation patterns of ß(3)-conglycinin and phaseolin, homologous storage 7S globulins, suggests the existence of intrinsic differences in their structures. This hypothesis is supported by comparative analysis of the accessibilities to the solvent of amino acid residues in phaseolin and ß(3)-conglycinin structures, which indicated the relatively low packing density of the latter, resulting in enhanced susceptibility of it to extensive proteolysis.

Odontogenic keratocyst in the mandibular condyle base region: A case report.

Odontogenic keratocysts (OKCs) often occur in the molars in the mandibular ramus; they often progress asymptomatically and are discovered only after widespread development. Some cases of OKC progress to the mandibular condyle; however, very few cases exist only in the condyle. To the best of our knowledge, in all of the previously reported cases, OKCs occurred in the mandibular ramus, which underwent resection. The present study reports the case of a 31-year-old man in whom an OKC (13x12x6 mm) occurred discretely in the base of the condyle, in which the condylar head was successfully preserved. The tumor was removed under general anesthesia using the approach of shaving the anterior surface of the mandible. The extraction cavity was managed using the packed open technique and with an obturator. Approximately 20 months post-operation, the patient remained recurrence-free. This report presents a rare case of an OKC in the mandibular condyle base region. Resection was performed under general anesthesia and the condylar process was successfully preserved.

Measurement of Ara h 1-, 2-, and 3-specific IgE antibodies is useful in diagnosis of peanut allergy in Japanese children.

BACKGROUND: Food challenges are time-consuming, expensive, and not always possible to perform. Therefore, new tools to diagnose food allergy are desired. The aim was to evaluate IgE antibodies to peanut allergens in the diagnosis of peanut allergy in Japanese children using ImmunoCAP(®) and IgE immunoblotting. METHODS: The study included 2-13-yr-old consecutive patients (n = 57) referred to our specialist clinic for investigation of current peanut allergy using food challenge. All children had a previous doctor's diagnosis of peanut allergy and were on elimination diet. Serum samples were analyzed for IgE reactivity to peanut, recombinant (r) Ara h 1, 2, 3, 5, 8, and 9. IgE immunoblotting (n = 23) was performed using extracts from raw and roasted peanut. RESULTS: Twenty-six of the children failed (allergic group), and 31 passed the peanut challenge (tolerant group). The rAra h 2 ImmunoCAP test was superior in its ability to differentiate between children in the allergic and tolerant groups with a sensitivity and specificity of 88% and 84%, respectively (cutoff, 0.35 kU(A)/l). The combination of rAra h 1, 2, and 3 resulted in a higher specificity (94%) when IgE to all of them was the criteria for positivity. ImmunoCAP generally showed a good agreement with immunoblotting using both raw and roasted peanut for IgE reactivity to Ara h 1, 2, and 3. CONCLUSIONS: Measurement of IgE antibodies to rAra h 1, 2, and 3 is useful in the diagnosis of peanut allergy and in the investigation of reactions to raw and roasted peanut.