In a previous study, we successfully produced rDer p 2 and found that rDer p 2 had a good solubility and high IgE-reactivity ( Kim et al., 1999). More characterizations of the group 2 allergens are needed, especially on the functions and distributions in the mites. However, anti-group 2 antibodies have been reported to show reactivity associated with the gut ( van Hage-Hamsten et al., 1995). They are similar in amino acid sequences, size, and the distribution of cysteine residues to a family of epididymal proteins ( Thomas and Smith, 1998). In the past, there has been a speculation that the group 2 allergens are lysozymes, but it is now clear that they are not ( Hakkaart et al., 1997). Although the group 1 allergens have been well known to be cysteine proteases ( Chua et al., 1988), the group 2 allergens have not been clearly characterized as yet. The level of group 2 allergens in house dust mite has been reported to be as high as that of group 1 allergens ( Yasueda et al., 1990). However, most recent researches have been focusing on the two major allergens (groups 1 and 2). Immunoblot analysis using patients' sera selected on the basis of high anti-mite IgE has revealed 32 different IgE-binding bands with molecular weights from 11 to over 100 kDa ( Tovey and Baldo, 1987 Thomas and Smith, 1998). pteronyssinus are the major source of HDM allergens. Sensitization to allergens produced by HDMs is commonly associated with symptoms of asthma and rhinitis around the world, and D. Consequently, this approach using monoclonal antibodies allowed a two-site capture ELISA to be developed. In this study, we describe the production of monoclonal antibodies and the analysis of Der p 2 epitopes. Allergen-specific monoclonal antibodies have many advantages for allergen characterization, identification, and quantitation ( Chapman, 1988 Ovsyannikova et al., 1994), since they possess a unique specificity and a high level of selectivity for a single epitope and can be produced in unlimited amount in vitro. One way to characterize allergen extracts is to measure their major allergen levels with monoclonal antibodies. Due to the lack of available methods in measuring allergenic components in the extracts, the potency of these extracts might vary from batch to batch ( Platts-Mills and Chapman, 1991). The crude allergen extracts were prepared from cultured mites, and they probably contained a complex mixture of proteins of which only a few was allergenic. These two allergens are the most significant causative agents for the sensi-tization, invoking IgE and IgG antibody responses in 80% to 95% of patients allergic to HDMs, and each has been cloned and sequenced ( Chapman and Platts-Mills, 1980 Krilis et al., 1984 Platts-Mills et al., 1992 Hakkaart et al., 1998a). Recently, several research groups have described the characterization of the allergens from two species of mites, especially the group 1 and 2 allergens. However, it is generally believed that the predominant species are only D. pteronyssinus, and Tyrophagus putrescentiae, a storage mite, were reported to be the three most predominant species. ( 1997) identified 23 species of mites from house dust mites of which Dermatophagoides farinae, D. House dust mites (HDMs) have been well established as the most significant indoor sensitizing agents that are important in the induction of asthma and allergic rhinitis. The result suggested that the assay using monoclonal antibodies against rDer p 2 could be useful for the environmental studies and for the standardization of mite allergen extracts. The sensitivity limit was 4 ng/ml with rDer p 2 and 8 µg/ml with the D. Two-site capture ELISA was developed using two different monoclonal antibodies for quantitating Der p 2 in house dust.
Three monoclonal antibodies showed reactivities to the recombinant B fragment and to the full-length rDer p 2, but one monoclonal antibody reacted only with the full-length rDer p 2. For the analysis of epitopes, a Der p 2 cDNA encoding 126 amino acids (aa) was dissected into three fragments with several overlapping peptides, A (aa residues 1-49), B (44-93), and C fragment (84-126). Two of the monoclonal antibodies were found to be IgG1 and the others were IgM. Four monoclonal antibodies produced were species-specific and did not cross-react to the D. In order to analyze epitopes of the allergen and to develop a quantitative method of the allergen exposure, monoclonal antibodies against a recombinant Der p 2 (rDer p 2), one of the major allergens of Dermatophagoides pteronyssinus, were produced. House dust mite allergens have been well established as sensitizing agents that are important in the induction of allergic diseases.
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