The B. tropicalis mite has been the target of several studies highlighting its role as being one of the major asthma sensitizing agent in tropical areas of the world [15–17]. These studies have demonstrated that serum IgE antibodies in 42% to 98% of B. tropicalis-sensitized individuals react with Blo t 5 and Blo t 21 [2, 14]. In the present work it is shown that a large proportion of serum IgE antibodies from BtE-sensitized children and adults with asthma reacted with rBlo t 5 and rBlo t 21 obtained as described herein. The rBlo t 5 in acidic pH solutions, like under purification denaturing conditions, tends to form molecular aggregates . Likely, the rBlo t 21 behave like the rBlo t 5, forming aggregates under the same conditions, since both allergens share structural similarities . This could explain the nonspecific band recognition by antibodies from asthmatic patient sera that were used in immunoblotting assay of this work. B. tropicalis is found more frequently (71.8%) than D. pteronyssinus (39.9%) in bed dust in Salvador, a major city in Northeast of Brazil . In environments where B. tropicalis predominates in the mite fauna, it is common to find a high rate of co-sensitization to Blo t 5 and Blo t 21, as highlighted in our study (in 66.7% of 63 sera from B. tropicalis sensitized individuals) [4, 19]. Although several individuals had a higher IgE antibody reactivity to rBlo t 5 and rBlo t 21 than to BtE (i.e., their sera led to higher optical densities when tested in ELISA for IgE antibodies against these recombinant allergens than against the extract; Figure 2), there is a small percentage of sera (7.9%, corresponding to 5 out of 63 sera) that does not recognize any of the recombinant allergens, a fact that would limit the sensitivity of assays using these allergens. When the two recombinant allergens were used together in the same immunoassay, the IgE reactivity increased so that 96.4% of the sera from asthmatic patients had anti-BtE antibodies. Some studies indicate that mite allergy could be detected using a mixture of two or more major recombinant allergens (component-resolved diagnostics), replacing the natural extract . Furthermore, the use of multiple recombinant allergens to detect sensitization profiles could be very important to guide and improve immunotherapy for mite allergies [19–21].
This study, despite not comparing the reactivity of IgE antibodies to rBlo t 5 and rBlo t 21 with the reactivity to their native counterparts, suggests that recombinant allergens produced as described in this paper can replace natural allergen extracts in the diagnosis of allergies, confirming previously published data showing that rBlo t 5 expressed in E. coli and native rBlo t 5 have comparable IgE reactivity in terms of percentage of sera with antibodies . However, our data also indicate that it is desirable to introduce other recombinant antigen(s), in addition to rBlo t 5 and rBlo t 21, in an assay for IgE antibodies to diagnosis hypersensitivity to B. tropicalis in order to increase its sensitivity. Blo t 7, which has been shown to be as reactive as Blot t 5 with IgE antibodies in the sera from allergic children in a study also carried out in a tropical environment , may be a good candidate to be included to a pool of recombinant antigens to increase the sensitivity of an immunoassay. Meanwhile, Blo t 10 and Blo t 11, which shown to cross-react with A. lumbricoides, would not be good candidates to be included in an immunoassay with multiple recombinant antigens.
The relationship between house dust mite sensitization and triggering of asthma and other atopic diseases is well documented [24, 25]. Given the difficulty of completely eliminating the sensitizing agents of the affected individuals' homes, immunotherapy has been playing a key role in alleviating the clinical aspects of allergic diseases. Thus, a more specific mite allergy diagnosis is necessary and it is also a key point to develop appropriate, more specific and individualized therapies . The antigenic extracts used in the diagnosis and immunotherapy of allergic diseases are obtained from natural sources. This fact brings many disadvantages, such as the presence of large amounts of non-antigenic proteins; contamination by other potentially immunostimulating compounds, like endotoxin; allergen variability in the sample composition (depending on the season of the year, the mite life cycle and differences in protein extraction protocols). In addition, the more sensitizing allergenic proteins in the mite extract can be found in low concentrations, requiring the use of higher doses of total extract, which is not always convenient in clinical practice [20, 25, 27, 28].
In addition to the problems described above with the use of whole allergenic extracts in the immunodiagnosis of allergic diseases, there is another factor that should be considered by the companies that commercialize these products: the common association of B. tropicalis sensitization with helminth infection in tropical and undeveloped regions of the world. It is in fact believed that about 1.5 billion people worldwide are infected with A. lumbricoides[10, 29]. Salvador city, where the donors of the sera used in the present work live, is one helminth parasite endemic areas in Brazil, having high prevalence of A. lumbricoides, Trichuris trichiura and Toxocara spp (Toxocara canis and Toxocara cati) infections. In fact, most of the sera studied in the present work had anti-Ascaris IgG antibodies, indicating present and/or past A. lumbricoides infection. In this area, an overlap of parasitic endemicity and high prevalence of allergic diseases is observed [30–32]. There is often dissociation between positive skin prick test results and detectable serum IgE antibodies in allergic individuals who are also co-infected with A. lumbricoides. This finding could be explained by the existence of IgE antibodies, raised in response to the helminth infection, that cross-react with allergens but are unable to lead to degranulation of mast cells, consequently increasing anti-allergen IgE antibody levels but not SPT positivity [9, 33]. The increase of knowledge on cross-reactive IgE antibodies to common environmental respiratory allergens and helminth antigens will certainly improve allergy diagnosis and perhaps even immunotherapy. In the present study, pre-incubation of sera with A. lumbricoides extract led to varying degrees of inhibition of the binding of IgE to mite allergens. It would also be useful to further demonstrate this cross-reaction by inhibiting the binding of IgE antibodies to A. lumbricoides extract by incubation of the sera with B. tropicalis antigens. As the high levels of IgG antibodies raised in the immune response to this helminth hinders the detection of IgE anti-Ascaris antibodies, a possible way to carry out the experiment would be to use an assay to detect IgE antibodies more sensitive than the one used in the present work.
The inhibition by adsorption with A. lumbricoides extract was higher for the binding of antibodies to B. tropicalis crude extract than to rBlot 5 and rBlo t 21, although in a few sera there was also a high level of IgE cross-reaction between the helminth extract and the recombinant allergens. Cross-reactivity to both recombinant allergens was observed in the same sera (data not shown). This finding could be explained if the anti-Ascaris antibody response in these sera donors had been more polyclonal than the immune response of the other serum donors, so that antibodies against additional cross-reactive epitopes would be produced. Indeed, it is known that the repertoire of the antigens that are recognized in complex antigenic mixtures may vary greatly in different individuals of the same species .
The observed difference in the degree of absorption by A. lumbricoides extract of anti-rBlo t 5 and anti-rBlo t 21 in relation to anti-BtE antibodies cannot be explained by the presence of lower levels of anti-recombinant allergen antibodies, since the amounts of anti-rBlo t 5 IgE antibody activity, on the contrary, were found to be higher than those of anti-BtE IgE antibody activity. Due to this less intense cross-reactivity, assays using a mixture of rBlot 5 and rBlo t 21 as antigen should be more specific than assays using the crude extract.
IgE antibodies raised by helminths can cross-react with several epitopes of mite antigenic extracts often used in diagnosis of allergic diseases possibly affecting the correct diagnosis. It becomes increasingly relevant, therefore, to obtain more specific allergens to make an accurate diagnosis of allergy and, consequently, improving immunotherapy for mite allergy. However, the price of assays using bacteria-produced recombinant antigens, as compared to those of assays using crude extracts obtained from mite cultures, is a factor that should be taken into consideration.