Produksi dan Aplikasi Ekstrak Protein Kacang- Kacangan dan Produk Laut sebagai Reagen Uji Alergi Makanan dengan Metode Skin Prick Test (SPT)

Abstract

Food allergy is an abnormal immune response of a sensitive person to food proteins that was known as allergen. Most of them have a protein or glycoproteins structure. The reaction of food allergies are mediated by immunoglobulin E (IgE) antibodies. The allergen may stimulate the immune system to produce a large amounts of immunoglobulin E antibodies of allergic individuals, then bind to their receptor on the surface of mast cells. These IgE could cross-linked with the same allergens on subsequent exposure. The cross-linked of IgE and allergen could be activated mast cells and the subsequent release rapidly pathological mediators such as histamine, proteases and leukotrienes. These mediators are causing itching, bumps, swelling, shortness of breath, cough, and anaphylactic shock. To date, there is no cure for food allergies except avoiding allergenic foods. Avoiding food should be established according to the result of allergy tests, such as skin prick test and double-blind placebo-controlled food challenges (DBPCFC). Skin prick tests (SPTs) are widely used for detecting allergen that caused IgEmediated food hypersensitivity. The main component on skin prick test was known as SPT reagent which were extracted from food. A miniscule amount of SPT reagent was pricked into the skin by gently. The aim of study are extraction protein of soybean, peanut, bambara nut, shrimp, tuna and asian green mussel Indonesian local then making them as SPT reagents. The protein extract of nuts were prepared by isoelectric precipitation method and phosphat buffer extraction for seafood product. Autoclaved glycerol and phenol were mixed with the protein extracts then packed on 5 ml sterile vial bottles. The specification of food allergen extracts (SPT reagents) referred to the European Pharmacopoeia Monograph on Allergen Products 2010:1063. Characterization of protein extract was performed using electrophoresis, indirect ELISA method was done to detect specific IgE. Immunoblotting method was done to identify allergen proteins. The study was approved by Ethics Committee of Faculty of Public Health, University of Diponegoro No. 190/EC/FKM/2014. SPT reagent have been tested on forty subjects allergic and non-allergic food. They gave written informed consent before joint with the study as participants. Sensitivity and specificity of SPT reagent were calculated based on SPT and specific IgE results. The yield of soybean extract was 53.10% and 23.50g /100g sample as recovery value. The results of SDS-PAGE showed that numerous molecular weight were detected: 147 kDa, 102 kDa, 91 kDa, 82 kDa, 76 kDa, 66 kDa, 56 kDa, 30 kDa, 28 kDa, 23 kDa, 22 kDa, 20 kDa, 18 kDa, and 17 kDa. Sensitivity and specificity of soybean protein extract were 100%. Immunoblotting test shown that extract protein of soybean contain protein allergenic, they have molecular weight between 17 kDa and 61 kDa. The yield and recovery of peanut extract were 74.98% and 51.31g/100g sample respectively. Ten bands were detected on protein peanut extract by SDSPAGE method with molecular weight are: 122 kDa, 70 kDa, 57 kDa, 41 kDa, 37 kDa, 31 kDa, 20 kDa, 19 kDa, 18 kDa and 17 kDa. Potential allergenic protein were shown by molecular weight between 10 kDa and 49 kDa. The sensitivity and specificity of SPT extract were 90.9 % and 100% respectively. The negative error rate was 9.1%. The yield and recovery of bambara nut extract were 53.58% and 12.53g/100 g sample. Protein profile shown that it was consists of fourteen bands of protein: 159 kDa, 115 kDa, 60 kDa, 51 kDa, 42 kDa, 36 kDa, 30 kDa, 27 kDa, 26 kDa, 22 kDa, 20 kDa, 19 kDa, 18 kDa and 17 kDa. Just four bands of protein as allergens, their molecular weight are: 26 kDa, 38 kDa, 41 kDa and 48 kDa. Sensitivity and specificity of bambara nut reagent were 90.9% and 100% respectively. The negative error rate was 9.1%. Shrimp extract of jerbung was very sensitive. From the forty subjects were tested, twenty-four was stated positive. The yield of jerbung shrimp extract was 57.91% whereas recovery value was 31.27g /100 g sample.SDS-PAGE analysis could be identified twelve protein bands with molecular weight are: 185 kDa,125 kDa, 103 kDa, 76 kDa, 72 kDa, 66 kDa, 51 kDa, 49 kDa, 43 kDa, 30 kDa, 23 kDa dan 18 kDa. Potential allergenic protein were shown by molecular weight between 31 and 65 kDa. Sensitivity and specificity of shrimp extract were obtained 96% and 93.3% . Negative and positive error rate were 4% and 6.7%. Protein content of tongkol was 29.58% and the yield and recovery of tongkol extract were 60.09% and 27.7g/100g sample respectively. SDS-PAGE analysis showed that the tongkol extract contains fifteen protein bands with their molecular weight were: 152 kDa, 135 kDa, 97 kDa, 78 kDa,59 kDa, 49 kDa, 45 kDa, 34 kDa, 27 kDa, 24 kDa, 22 kDa, 21 kDa, 19 kDa, 18 kDa and 17 kDa. Potential allergenic proteins were presented by the molecular weight between 12 kDa and 50 kDa. Sensitivity of SPT reagent of tongkol extract was 78.6% and 21.4% as a negative error value while the specificity values obtained was100%. The yield protein extract of asian green mussel was 58.16% and recovery value was 25.29g/100g sample. Twelve protein bands were detected by SDSPAGE method, their’s molecular weight are: 117 kDa, 103 kDa, 70 kDa, 54 kDa, 46 kDa, 36 kDa, 29 kDa, 27 kDa, 22 kDa, 21 kDa, 19 kDa and 18 kDa. Sensitivity of asian green mussel extract was obtained 86% and 14% as a negative error value. The specityvity was 100%. Allergen protein are: 55; 56; 57; 58; 59; 68 and 79 kDa. SPTs extract of Indonesian local could be used to determine the allergen and concurrently were able for the diagnosis of food allergies. Their sensitivity and specificity were high. Each of extract protein was composed of several protein allergens and the molecular weight are different for each subjects sera, so that SPT extract which will be used for diagnosis allergies by skin prick test should be in the form of whole protein or the crude extract.