Immunological Characterization of Polyclonal Antisera Prepared Against Recombinant Rice RAG2 and Its Application in Detection of 14-16kDa .ALPHA.-amylase/trypsin Inhibitors from Processed Foods

Abstract

RAG2 is a member of the rice 14-16 kDa α-amylase/trypsin inhibitors, which are the main allergens in rice grains. Recombinant RAG2 protein was expressed in Escherichia coli, and polyclonal antisera were prepared against the protein. The polyclonal antisera specifically bound with 14-16 kDa α-amylase/ trypsin inhibitors from husked brown, red and black rice. No cross-reaction was observed between the polyclonal antisera and saline-soluble proteins from other tested cereal grains. By immunoblotting with the polyclonal antisera, 14-16 kDa α-amylase/trypsin inhibitors were found to be distributed throughout the endosperm of brown rice and their contents were estimated. Moreover, 14-16 kDa α-amylase/trypsin inhibitors were also detected in processed foods. Thus, the polyclonal antisera prepared against recombinant RAG2 protein could be used to compare the content of 14-16 kDa α-amylase/trypsin inhibitors in rice samples and probe the presence of the allergen in processed foods.Keywords: rice (Oryza sativa), RAG2, α-amylase/trypsin inhibitor, recombinant protein, polyclonal antisera, processed food *To whom correspondence should be addressed. E-mail: kaz@affrc.go.jp IntroductionCereal allergy is of great concern in Japan, as it is in other countries where cereals are a staple food and the major nutrition source. Rice is the second most important cereal crop in the world, after wheat, and is consumed by more than half of the world's population. The association between rice and allergic reactions was first reported in Japanese patients with a history of asthma induced by exposure to rice flour and eczema exacerbated by rice ingestion (Shibasaki et al., 1979). Rice-induced allergy is IgE-mediated in the case of bronchial asthma (Arai et al., 1998;González-Mendiola et al., 2003;Lezaun et al., 1994) or atopic dermatitis (Di Lernia et al., 1992;Ikezawa et al., 1992) associated with the ingestion of cooked rice or inhalation of vapor during its cooking. Although rice is commonly thought of as a "hypoallergenic" food, the clinical importance of rice allergy has been demonstrated by a report in which a high radioallergosorbent test (RAST) score for rice allergens had a close relationship to the development of severe atopic dermatitis (Ikezawa et al., 1992). Moreover, Yamada et al. (1985) reported that rice has apparently exceeded other cereals in allergenicity, and ranked second after egg whites as the most common potential allergy-causing food, as assessed by an IgE RAST. A recent report (Mehr et al., 2009) noted that rice was a common trigger of a severe type of allergic reaction called food proteininduced enterocolitis syndrome (FPIES) in children.Several rice proteins with molecular masses of 14-16, 26, 33 and 60 kDa were demonstrated to be recognized by serum IgE of patients showing hypersensitive reactions to rice ingestion (Urisu et al., 1991;Usui et al., 2001). The first reported allergen in rice was a 16 kDa protein with reactivity for IgE of rice-hypersensitive patients (Matsuda et al., 1991). The 16 kDa protein is ...

Evaluation of Extraction Solutions for Biochemical Analyses of the Proteins in Rice Grains

“…The rabbit antisera were prepared by immunizing rabbits with the respective recombinant proteins as previously described. 28,32) Immunoblotting and signal detection were also carried out as previously described. 28) The chemiluminescent signal was detected by the Pharos FX molecular imager system (Bio-Rad Laboratories), the signal intensity being quantified by using Quantity One 1-D Analysis software (Bio-Rad Laboratories).…”

“…18) Analyses of rice total proteins have also been widely used in rice composition studies; 16,[19][20][21][22][23] however, possibly because most reported rice allergens have been included in salt-soluble proteins, rice salt-soluble proteins have always been used for allergen analyses. [24][25][26][27][28] Even so, among the available reports, the composition of solutions for extracting both salt-soluble and total proteins varies from laboratory to laboratory (Table 1). To evaluate the possible influence of the extraction solution on the amount and composition of extracted rice proteins, we extracted the salt-soluble, salt-insoluble and total proteins from grains of Koshihikari (Oryza sativa cv.…”

Immunoblotting Analysis of nsLTP1 in Cereal Grains with Antiserum Raised against Recombinant Rice nsLTP1

“…Fractionation of brown rice grains by grain milling Husked grains of three kinds of brown rice cultivars (rice A, rice B and rice C) were fractioned by grain milling with a Satake Grain Testing Mill TM-05 (Satake Engineering, Tokyo, Japan) as described previously (Lang et al, 2010).…”

“…Otherwise, for immunoblotting analysis, proteins were transferred to polyvinylidene difluoride (PVDF) membranes by electroblotting in Towbin buffer (Towbin et al, 1979) with a Trans-Blot Semi-Dry Transfer Cell. Immunoblotting and signal detection was carried out as described previously (Lang et al, 2010) except that the primary antiserum was diluted 6,000-fold. The signal intensity was quantified using Quantity One 1-D Analysis Software (Bio-Rad Laboratories).…”

Differences and similarities of albumins from japonica rice bran, japonica, indica, and javanica rice endosperm in the suppressive effect of postprandial blood glucose elevation

“…9 in Jp‐RMBA. Although some spots were identified as proteins that differed between Jp‐REA and Jp‐RMBA, the major protein was Os07g0214300, which is a seed storage protein located in protein body II (PB‐II) in the endosperm cells (Kurokawa et al, 2014; Lang et al, 2010; Zhou et al, 2017). Os07g0214300 is also present in various species, including indica rice (Teshima et al, 2010), which is consistent with the result that In‐REA contains an indigestible 16‐kDa protein.…”