- Top of page
- Material and methods
Background: Current allergy diagnosis is performed with allergen extracts which contain a variety of allergenic and nonallergenic components. The availability of highly purified and well-characterized allergen molecules seems to be an advantage of component-based diagnosis.
Methods: With the immunoenzymatic CAP FEIA System, we measured specific IgE levels to the recombinant allergens rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and native Phl p 4 in 77 sera of patients allergic to grass pollen, in order to evaluate the IgE-binding frequency to these purified grass-pollen allergens and their relationship to rBet v 4, rBet v 2, and other allergens.
Results: The frequency of sensitization was as follows: rPhl p 1=93.5%; rPhl p 2=67.5%; rPhl p 5=72.7%; rPhl p 6=68.8%; rPhl p 7=7.8%; rPhl p 11=53.2%; rPhl p 12=35.1%; and native Phl p 4=88.3%. As expected, rPhl p 7 and rPhl p 12 had a very good correlation (Spearman's r) with Bet v 4 (r=0.95%, P<0.05) and rBet v 2 (r=0.99, P<0.05), respectively. Good correlations of rPhl p 12 with papain (r=0.93, P<0.05), latex (r=0.92, P<0.05), and bromelain (r=0.86, P<0.05) were found. Highly variable individual sensitization patterns were observed.
Conclusions: A new clinical approach has allowed the determination of specific allergograms for the different patients and may therefore be of great importance for more specific diagnosis. The use of component-resolved diagnostics may be useful to evaluate the allergen content of an extract for immunotherapy by monitoring patient's IgE and IgG directed to relevant allergens.
Grass-pollen extracts used for diagnosis and therapy are mixtures of different grass species and contain a number of relevant and minor allergens (1, 2). The availability of highly purified and well-characterized allergen molecules seems to be promising since they can be easily quantified.
The techniques of genetic engineering applied to allergens have enabled the production of recombinant allergens (RAs) (3).
Since the work of Marsh and colleagues (4–6) with the perennial ryegrass groups 1, 2, and 3 allergens during the 1960s and 1970s, a number of new allergens have been identified, isolated, and characterized.
The grass group 1 allergens are acidic glycoproteins with molecular masses in the 27–35-kDa range. Histochemical examination has localized this glycoprotein in the exine and cytoplasm of the pollen grain, but its function is still unknown (7). Extensive immunologic cross-reactivity among the group 1 allergens from taxonomically related grasses was established (8–10), and over 95% of allergic subjects were highly reactive to the respective group 1 allergens. The grass group 2 allergens are acidic proteins to which 35–50% of grass-allergic patients are sensitive (10). The grass group 3 allergens are basic proteins (11–14 kDa) with a frequency of sensitization of 35–70% among grass-pollen-allergic subjects (11–13). The grass group 4 allergens are high-molecular-mass basic glycoproteins (50–60 kDa) of unknown biochemical function, and they may be responsible for sensitization rates of 50–75%. Studies with the group 4 allergens from Bermuda grass (11–17) suggest that the carbohydrate moiety, accounting for about 7.5% of the mass, may be an important allergenic determinant of the molecule. The group 5 allergens (includes group 9 allergens) are a heterogeneous group of proteins and have similar molecular mass to the group 1 allergens (27–35 kDa for group 1 and 27–38 kDa for group 5). The group 5 allergens, together with the group 1 allergens, account for most of the IgE binding of most grass-allergic sera (18, 19). The IgE-binding determinants are localized in the N-terminal and C-terminal ends of Phl p 5 (20), which has been shown to possess ribonuclease activity (21).
The group 6 allergens from timothy grass pollen, Phl p 6, are polypeptides of 13 kDa, to which a majority of timothy grass-pollen-sensitive subjects react (22). Both single and shared epitopes have been detected on Phl p 5 and Phl p 6 allergens by immunoadsorption studies with antibodies to Phl p 5 and Phl p 6 (23).
Approximately 35% of grass-pollen-allergic subjects show specific IgE against the group 7 allergen rCyn d 7 (24). The deduced amino-acid sequence of this protein showed sequence similarity with Bet v 4 from birch pollen and calmodulin from the fungus Fusarium oxysporum.
The group 10 antigens have been proposed as a model system for studying the molecular basis of the cross-reactivity of cytochrome c (25). The group 11 antigens are a group of glycoproteins of 16–18 kDa structurally similar to the Kunitz soybean trypsin inhibitor, the N-glycan structure of which appears to be involved in the IgE binding, as is bee venom phospholipase A2. Approximately 65% of sera from grass-allergic subjects possess IgE to Lol p 11 (26). Specific IgE against the group 12 antigen, profilin, purified from grass pollens, can be detected in 20–50% of grass-sensitive subjects (27). The recently characterized Phl p 13 (28) shows homology with polygalacturonases and is clearly different from the allergen previously designated as Phl p 4.
In this study, we measured specific IgE levels to the RAs rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, and native Phl p 4 (nPhl p 4) in patients with specific IgE to Phleum pratense, in order to evaluate the frequency of sensitization to purified grass-pollen allergens among grass-pollen-allergic subjects. Furthermore, a possible relationship of specific IgE against purified timothy allergens to rBet v 2, rBet v 4, papain, bromelain, and latex was investigated. A new clinical approach allowed the determination of specific allergograms for the different patients with the aim of identifying and monitoring the disease-eliciting molecules.
Material and methods
- Top of page
- Material and methods
All sera were obtained from patients allergic to grass pollen (n=77, 45 males, mean age 21.6 years, range 14–27; 32 females, mean age 29.4 years, range 13–46). Because all patients lived in the province of Cuneo (Italy), they were probably similarly exposed to grass pollen. They all had positive responses to the skin prick test (SPT) performed with timothy grass-pollen extract (Stallergenes Italia s.r.l., Saronno [VA], Italy). The results of the SPT were evaluated by the European Academy of Allergology and Clinical Immunology guidelines. None of the patients had previously undergone specific immunotherapy.
In vitro test
Sera were obtained from October 2000 to February 2001, and all contained timothy grass-pollen-specific IgE, as determined by the immunoenzymatic CAP method (Pharmacia Upjohn, Uppsala, Sweden). Moreover, the sera were characterized in detail by determination of IgE antibodies to rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and nPhl p 4. Total IgE and specific IgE to rBet v 1, rBet v 2, and rBet v 4 were also measured by the CAP System (Pharmacia Upjohn). Specific IgE to bromelain, papain, Hevea brasiliensis latex, olive tree, and birch were also evaluated.
Specific IgE levels were considered positive at the level of 0.35 kUA/l or higher (class 1).
The correlation index (r) was determined with the Spearman rank correlation coefficient matrix.
- Top of page
- Material and methods
The frequencies of sensitization to different timothy RAs and nPhl p 4 are indicated in Fig. 1. The mean levels of specific IgE against RAs and nPhl p 4 are summarized in Table 1.
Table 1. Levels of specific IgE to rPhl p 1, rPhl p 2, rPhl p 5, native Phl p 4, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, rBet v 1, rBet v 2, rBet v 4, and natural extracts in 77 sera from patients allergic to grass pollen. n=77 (mean age: 24.8 years)
| ||Geometric mean kUA/l||SD||Mean kUA/l||(25th–75th) percentile||Positive sera (%)|
|rPhl p 1||9.4||29.6||9.1||(3.4–34.6)||72 (93.5)|
|rPhl p 2||4.6||10.4||5.0||(1.9–11.3)||52 (67.5)|
|rPhl p 5||13.3||33.0||13.8||(5.9–31.4)||56 (72.7)|
|nPhl p 4||5.5||20.8||5.1||(2.2–14.8)||68 (88.3)|
|rPhl p 6||4.6||17.9||6.1||(1.7–12.0)||53 (68.8)|
|rPhl p 7||4.3||14.2||4.2||(1.9–11.6)||6 (7.8)|
|rPhl p 11||3.9||15.1||3.4||(1.4–11.5)||41 (53.2)|
|rPhl p 12||1.3||4.4||1.1||(0.5–3.0)||27 (35.1)|
|rBet v 1||4.9||28.6||4.1||(1.1–17.4)||15 (19.5)|
|rBet v 2||1.6||5.8||1.1||(0.7–3.6)||26 (33.8)|
|rBet v 4||2.2||13.8||2.4||(0.6–3.1)||5 (7.1)|
Quantitative IgE reactivity profiles were established for 77 grass-pollen-allergic subjects; some interesting individual allergograms are shown in Fig. 2. Patients 1, 4, 5, and 7 had IgE antibodies to a limited number of RAs. However, the sera of patients 2, 3, 6, and 8 display IgE antibodies to most RAs (Fig. 2).
Figure 2. Some quantitative IgE reactivity profiles of grass pollen-allergic patients (1–8) showing individual allergograms.
Download figure to PowerPoint
As expected, rPhl p 7 and rPhl p 12 were extremely positively correlated with rBet v 4 (r=0.95, P<0.05) and rBet v 2 (r=0.99, P<0.05), respectively. Other interesting correlations are summarized in Table 2.
Table 2. Correlation between specific IgE to recombinant allergens and some allergens in 77 sera of patients allergic to grass
|rPhl p 7 vs|
| rBet v 4||0.95||<0.05|
| Olive tree||0.33||<0.05|
|rPhl p 12 vs|
| rBet v 2||0.99||<0.05|
|rPhl p 1 vs|
|rPhl p 5 vs|
|rBet v 2 vs|
|rBet v 1 vs|
In this study, the equivalence of the natural counterparts was not observed in several cases.
- Top of page
- Material and methods
The results demonstrate that rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, rPhl p 11, rPhl p 12, and nPhl p 4 from P. pratense specifically bind IgE antibody in the sera of patients allergic to grass pollen. However, the reactivity to these purified allergens varied considerably with different patients. In our study, the IgE-binding frequency for rPhl p 1, rPhl p 2, rPhl p 5, rPhl p 6, rPhl p 7, and rPhl p 11, roughly corresponded to values reported in previous studies (9, 10, 16, 29–37). In contrast, Valenta et al. (38) reported that 20% of patients allergic to grass display IgE reactivity to profilins. Our findings showed that 35% of sera had specific IgE to Phl p 12. Moreover, previous studies reported that group 4 allergens may be responsible for sensitization rates of 50–75%. In our study, the IgE-binding frequency to nPhl p 4 was 88.3%. These last differences may be due to geographic differences in the study group. The quantitative IgE reactivity profile from patient 8 (Fig. 2) showed an individual sensitization pattern in which a simultaneous increase of specific IgE against rPhl p 7 and rBet v 4 was observed. In fact, a remarkable characteristic of the group 7 allergens is their cross-reactivity with the allergen Bet v 4 of birch pollen (24).
The class of thiol proteases includes proteolytic plant enzymes such as papain from papaya and bromelain from pineapple. Patients with allergic reactions to kiwi fruit also have IgE reactive to bromelain and papain (39).
Recently, Nettis et al. described a patient presenting an IgE-mediated reaction to bromelain and specific IgE to papain, grass pollen, cypress pollen, celery, fennel, and carrot (40). Papain appears to cross-react with latex, birch pollen, and kiwi (39). Moreover, in our previous study (41), we found that the higher prevalence of antilatex IgE was seen in sera containing specific IgE to rPhl p 1, rPhl p 5, and rBet v 2.
In the present study, we found that bromelain and papain were extremely well correlated with rPhl p 12 and rPhl p 7. This supports the concept that Phl p 7 may predict clinical sensitization to pollens of unrelated plant species.
There is evidence that certain RAs fail to behave comparably with their natural counterparts (42, 43), and many isoforms of allergens exist. However, for allergy to tree and grass pollen, it was previously demonstrated that mixtures of a few recombinant allergens can reproduce the allergenic complexity of natural extracts required for in vitro allergy diagnosis (44). Moreover, these allergens contained most of the IgE epitopes present in the corresponding natural pollen extract (29).
The individual patient allergograms that we have documented clearly demonstrate that grass-pollen-allergic patients differ widely with respect to their reactivity profiles. The main implication of previous studies (29, 42–45) and present work is that component-resolved diagnostics (46) allow us to document the changes in allergen-specific IgE levels after natural allergen exposure, and the possible appearance of IgE with specificities not detected before immunotherapy. Moreover, the current simple measurement of IgE against crude allergen extracts can mask serologically relevant allergens. Finally, the use of RAs may be useful to evaluate the allergen content of an extract for immunotherapy by monitoring patients' IgE and IgG against relevant allergens.