An Evaluation of Immune-Profile
An Evaluation of Immune-Profile and IgE Binding Proteins in the Sera of Respiratory Allergy Patients of North India to Commonly Consumed Foods
Allergy is defined as an exaggerated response of the immune system typified by immunoglobulin E (IgE) levels against the offending substance called ‘allergen’. Allergic diseases include asthma, rhinitis, conjunctivitis, angioedema, food allergies, urticaria, eczema, and even life-threatening anaphylaxis. As per the World Health Organization (WHO) reports, the number of patients having asthma is 300 million and is expected to increase to 400 million by 2025. The total immunoglobulin E (IgE) level comprising of the cell-bound as well as circulating IgE antibodies form a clinical marker for allergy diagnosis, while allergen-specific IgE (sIgE) is a positive indicator of sensitization 13,41. Elevated total IgE levels are generally indicative of atopy but many clinically proven allergic individuals may have normal total serum Ie. levels. A number of epidemiological studies have shown a strong association between total serum IgE levels, skin prick test or specific IgE positivity to aeroallergens and asthma phenotype. Food allergy, one of the important allergic diseases is on a rise worldwide, affecting nearly 10% of the population. More than 170 foods have been reported to cause IgE-mediated reactions, with big eight foods- peanut, tree nuts, egg, milk, fish, crustacean shellfish, wheat, and soy being attributed as “major food allergens”.
Questionnaire-based estimates tend to overestimate the real rate of food allergy, whereas the “gold standard,” fond challenge, is logistically difficult and sometimes unethical and may lead to anaphylaxis. Determination of the true prevalence of food sensitization/allergy is still challenging and difficult to correlate with other factors as total serum IgE, food-specific IgE (sIgE), Skin Prick Test (SPT) and a good clinical history. Sporadic studies focused on Indian patients have highlighted for self-history based food sensitization patterns to be as high as 58- 70%. The present study is aimed at to find food hypersensitivity patterns due to five commonly consumed foods by screening for food-specific IgE and IgE binding protein profiles in the sera of respiratory allergy patients from North India besides total serum IgE.
Materials and Methods
Selection of Foods
The Indian food diet comprises a variety of cereals and legumes. Knowledge about the prevalence of food sensitization among commonly consumed foods in Indians is still limited. Keeping this in mind, five different foods comprising cereals like maize and rice, and legumes namely peanut, chickpea and soybean, is commonly consumed in India were selected for the present study.
Preparation of Food Antigen Extracts
Healthy seeds of selected five foods procured from the market were crushed to a fine powder and defatted in diethyl ether at 4 °C. The antigens were extracted from defatted seed powder in 1:20 (w/v) ammonium bicarbonate buffer (50 mM, pH 8.0) with 5mM ethylene diamine tetraacetate and 1mM phenyl methyl sulfonyl fluoride by continuous stirring for 6-8hrs at 4 °C [13]. The suspension was centrifuged at 12,000 rpm for 20 min at 4 °C. The filtrate obtained was passed through a 0.45 pm nitrocellulose filter membrane, aliquoted in small quantities, lyophilized and stored at -70 °C for further experimentation and assays.
Immunoblot
The SDS resolved proteins of food extracts were transferred on to nitrocellulose membrane (NCM) as described. The unbound sites were blocked by 3% Nonfat dry milk for 3hr at 37 °C. The NCM strips were washed and incubated with 1:10 v/v food-allergic sera at 4 °C, overnight. A healthy serum pool was taken as control. The strips were washed with PBST and incubated with 1:1000 diluted antihuman IgE-peroxidase for lhr. The IgE binding was detected by diaminobenzidine (DAB) with hydrogen peroxide in sodium acetate buffer (pH 5.0). The detection was finally stopped using 0.2N HCI.
Statistical Analysis
The statistical analysis was carried out using the MS EXCEL, Prism V software (Graph Pad Prism, San Diego, California, USA. Values of IgE measurements are represented as mean ± standard error mean. The variation of total serum IgE based on gender amongst the three disease groups was analyzed by the Student t-test (two-tailed). The extent of association of age with IgE was analyzed as a correlation function and the variation was estimated by ANOVA. A comparison of mean values involving more than two groups was conducted by ANOVA. The significance level as observed by student t-test or ANOVA was considered to be at p-value 5. 0.05. The cutoff point to define ELISA positive cases was absorbance / Optical Density (00492) Z3 times the value of healthy volunteers in each of the respective cases.