Assessment of grass pollen allergens and efficacy of ELISA Protocol towards the binding ability of IgE
Grass pollens are a major cause of high fever and allergies to asthma. They affect about 500 million people globally and is the cause of various disease, reduction of quality of life and declined productivity levels. Allergen-specific immunotherapy can diminish symptoms and reduce the overall levels of asthma and treatment of pollen allergy cased by temperate grass. The world’s highest population densities live in the subtropics where subtropical Bahia, Johnson and Bermuda grass pollens are key allergens. 1 The Davies Allergy Research laboratory has shown that the content of allergens and recognition of immune allergens of subtropical grass pollens appears to differ from those of temperate grasses. Research shows differences in the regions and allergic sensitivity to subtropical and temperate grass pollen. Furthermore, there were species-specific IgE binding to antigenic determinants of subtropical grass pollen allergens that were not represented in temperate grass pollens. 2
To enable understanding of the full diversity of subtropical grass pollen allergens, the QUT Allergy Research Laboratory has characterized all the clinically important Panicoideae allergen components. Group 1 allergens (beta-expansins), including Pas n 1 of Bahia and Sor h 1 of Johnson grass, are major allergens of all grass pollens. Group 13 (polygalacturonase) allergens e.g. Pas n 13 of Bahia grass pollen, constitute the second most important allergen of the subtropical Bahia and Johnson grass pollens, but this allergen family appears to be less important for temperate grass pollen. In contrast, group 5 (ribonuclear protein) allergens, e.g. Lol p 5 of Ryegrass, are restricted to temperate grasses and are reportedly absent from subtropical grass pollens. The absence of the group 5 allergens in subtropical grasses is significant because extracts for temperate grass pollens are standardized by group 5 allergen content and serum IgE reactivity with group 5 allergens are associated with severity and risk of asthma.
Measurement of IgE reactivity with allergen components can have the ability to be sensitivity to the temperate or subtropical pollen grass and lead to appropriate application of immunotherapy among patients with severe disease. . Tests for IgE to subtropical allergens for use as diagnostic and prognostic markers are needed. However, strong evidence of immune recognition among patients of various regions with local exposure patterns is needed to encourage the necessary investment in developing more specific tests and treatment for patients in subtropical regions. Specific diagnostic management of subtropical grass pollen allergy have the potential to reduce the social, medical and economic burden of allergic diseases, providing benefit to patients in Queensland as well as other subtropical regions globally.
In this view this study protocol aimed at undertaking a comparisons of the avidity, level, and specificity of IgE binding of grass pollen allergens of subtropical and temperate grass and efficacy determination of ELISA assessment for a prognosis for human allergic disease.
This study utilized randomized controlled design both having none allergic and allergic controls for the two regions. A total of 50 patients were selected per group from the two regions of subtropical and temperate regions were selected to participate in the study. Patients diagnosed with allergic conditions based on positive prick tests results were involved in the study. Patients were offered consent forms and assented. The study was conducted after approval by the Ethics Committee of Metro South Human Research. Guidelines adherence was followed while undertaking the skin prick assessment according to the Australian Society for Clinical Immunology and Allergy. 3 Panels of common allergens were utilized based on the ryegrass, timothy, Bahia, Johnson and Bermuda grass allergens. No history of allergens was reported from nonatopic donors and no positive skin prick tests assessment from the allergens extracts utilized in the study. Random selection was done on the allergic patients. Prick test was done among the allergens presented for assessment and serum IgE reactivity was performed on the allergens response. Serum samples were further assessed for ImmunoCap assays for specific IgE activity.
ELISA serum reactivity for the allergens was performed. Proteins were obtained from the pollen grains allergens. Extraction was done using ammonium bicarbonate and complete inhibitor of protease cocktail and left for 3 hours at 4 degrees Celsius. The allergens were extracted in the ammonium bicarbonate solution; the extracts were filtered using phosphate-buffered saline and coating undertaken using microtiter filters. Confirmation of the allergens was done using the immune bloating process. The serum samples underwent dilution and tested for IgE reactivity for both the extracts.
Statistical analysis
Data were analyzed using unpaired T-tests and one way ANOVA analysis for the multiple comparisons of the various allergens from the two regions being assessed. Significance results was assessed based on P<0.05, ** p<0.01, ***p<0.001 and ****p<0.0001. the results were illustrated using bar graphs both for the specificity of IgE binding ability and the ELISA development for the prognostic assessment for the human allergens.
Graph overview of grass allergens and specificity levels
Figure 1 Grass Allergens from the two regions and their endpoint specificity levels
Key ; *P<0.05, ** p<0.01
ELISA efficacy assessment of the allergic and nonallergic cohorts
Figure 2 IgE allergen reaction for both the groups
The results signify that grass allergens from ryegrass, Bahia, and Bermuda showed significant results. Ryegrass was obtained from the Victoria region while the rest of the two; Bahia and Bermuda showed high specificity and avidity levels. The serum extracts from the Bermuda and Bahia regions of subtropical among the patients showed high specificity and avidity levels. This study demonstrates similarities with other observations made which showed that patients within Queensland have high rates of allergen specificity and sensitivity to the subtropical exposure of allergens than those from the Victoria areas which are regarded as temperate grass pollens. Cross inhibition of studies indicates that there is an existence of Vitro IgE reactivity response to the subtropical allergens and occurrence of a specific immune response.
IgE allergen reaction on both the groups as observed in figure 2 illustrated a significant level with p-value 0.001 among the control group signifying reaction to IgE reaction response compared to the nonallergic control groups. Thus the developed ELISA essay is a crucial aspect in the determination of human prognostic allergic disease which is key in clinical Responses aspects.
Grass pollens are an essential aspect in clinical practice, they are essential in triggering allergic responses and assessing diseases occurrence. 2 Grass pollen allergens arise from various subfamilies species such as the temperate Pooideae and subtropical Chloridoideae; they include Cynodon dactylon; Bermuda grass and Panicoideae such as Paspalum notatum; Bahia grass. The allergic sensitivity and reaction of these grass species and its sensitivity have been focussed previously for both the temperate and subtropical grasses. The subtropical grasses have demonstrated higher IgE reaction activity and allergen sensitivity levels. Although studies have shown that all grass pollens have a certain degree of allergen reactivity levels, they have demonstrated clinical significance in the reception of immune responses from the various subfamilies of the individual grasses.4
Subtropical grasses have a phylogenetic distinction from temperate grasses. These distributions have been affected by climate change. These changes have affected human bodies respond to various production of allergens. This is shaping the process of the general diagnosis and treatment of patients in subtropical regions. Increased knowledge on the sensitivity of the subtropical grass pollens and immunological globulin reaction is essential in the clinical practices and its application ion clinical management. 5
Assessment with patients from the temperate regions reveals they have developed grass pollen allergy and are thus show sensitivity to the temperate grass pollens and sensitivity to allergens and serum immunoglobulin IgE reactivity towards the subtropical grass pollens. Studies undertaken in Europe among 143 subjects showed that subtropical specific serum IgE related to grass species of C. dactylon, P. notatum, and S. Halepense were not directly related to the IgE reactivity to the P. Pratense grass pollens compared to other temperate grass pollens. 6 Among 5000 subjects from Northern Europe and North America showed a weak IgE reactivity correlation between grass pollens of P. pratense and C. dactylon grass pollens across the temperate grass regions. 7
A report by Rossi et al 8 observed an IgE reactivity with allergens aspects from P. Pratense and C. Dactylon grass pollens. Studies have demonstrated that C. Dactylon grass pollens have been observed to have significant levels of peak airborne pollen during months of June-July. In this study, there was an observation of six times more specificity of IgE to P. Pratense with a median 48.9 kUA/L) than C. Dactylon with a median of 8.0 kUA/L of grass pollens. This signifying a higher sensitivity towards temperate grass pollens 8 similar to IgE reactivity patterns of P. Pratense grass pollens.9 Furthermore, in the temperate region of Australia, studies have shown that about 85% of grass pollen allergic patients from the Victoria region had sputum responses to the P. Notatum grass pollens.10 There was observed higher serum IgE reactivity with L. perenneGP compared to P. Notatum or C. Dactylon but consistent with L.perenne grass pollens being dominant grass fodder in Victoria.
Patients originating from subtropical climates characterized in this study from Queensland climate have shown evidence of grass pollen sensitivity from various species and various families of subtropical grasses as observed in figures 1 and 2 above. Grass pollens of Rhodes grass and the Eragrostis sp have been key allergens in Queensland Australia as demonstrated by studies undertaken. 12 Further Dichanthium sericeum 13 and Sorghum verticilliformum 14 and Stenotaphrum secundatum 15 are key allergens in Queensland, Australia, India, and S. Africa. P. Notatum has been identified as a fundamental clinically significant allergens source in parts of the USA and Australia. 16,17,18
Subtropical grass pollens play an essential role in allergic disease causation in the subtropics compared to the tropical regions as observed by its high frequency of allergic sensitivity. Allergic sensitivity detection has often been limited to presence of the grass pollens for diagnostic assessment while other subtropical grasses have shown an effect on the contribution of allergic sensitivity to grass pollen allergy. 19 Studies undertaken in Thailand and Taiwan have shown that allergic sensitivity of pollen found in temperate grasses such as the P. Pratense and Dactylis glomerata and Orchard grass. The overall ability of temperate grasses to be able to respond in temperate grass pollen has reflected the overall sensitivity of pollens to local grass.
A study conducted in South Africa in a mixture of grass pollen in a subset of 25 children who presented with an allergy to pollen, about 92% demonstrated serum IgE reactivity towards P. Pratens. 20 Assesment by Westritching et al 9, demonstrated allergic reactivity among patients with exposure of C. Dactylon at 38% compared to P. Pratense at 26%. The allergen patterns of the component recognition reveal that the subtropical grass pollens are key sensitizing aspects of grass pollens allergens for patients.
In Australia, the sensitivity of allergens to P. notatum and C. Dactylon among patients in NSW have been linked to asthma and allergic rhinitis. Among patients from the subtropical regions of Queensland, P. notatum and C. Dactylon they exhibit a higher allergic sensitivity and increased IgE reactivity levels with P. Notatum.21 These studies are in line with results obtained in this study which have shown higher sensitivity reactions to grass pollen species. Subjects with the origin of birth in Queensland, greater sensitivity and avidity have been observed on the serum IgE with the subtropical allergens of P. notatum, S. Halepense and C. Dactylon 22, findings similar to results of this study.
This study has demonstrated key findings with regards to the testing of various grass pollen allergens. The assessment reviewed their reactivity, avidity, and specificity of their IgE binding ability. The findings have demonstrated that P. Notatum and C. dactylon have affinity levels and respond to high specificity levels and avidity among patients of Queensland. The ELISA developed was effective in identifying allergic cohort among the participants in the study, thus an essential tool to be employed in medical diagnostic approaches.
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