Preparation And Application Of Specific Polyclonal Antibody Against Peanut Allergen Ara H2

Peanut was defined as one of the eight major allergenic foods by Food andAgriculture Organism of the United Nations (FAO) in 1995. Differing from otherfood allergies, peanut allergy usually doesn't outgrow in adults, and is becoming anissue in public health and food safety fields because of high prevalence and seriousatopic disorders. Thus, developing a quick, accurate and sensitive method to detectthe trace Ara h2 in foods is much more significant.In this research, three major parts are as follows: the purification of the peanutallergen Ara h2, the preparation of the specific polyclonal antibody against Ara h2,and the development of the indirect competitive ELISA for detecting Ara h2.Si Li Hong (Arachis hypogaea L.) was chosen as the material in this study.After peanuts were crushed, degreased, extracted, Ara h2 was purified by usingDEAE-Sepharose Fast Flow anion-exchange chromatography and protein recovery ofSDS- PAGE. The purity of purified Ara h2 was more than 95％by SDS-PAGE. Thetechnique using DEAE-Sepharose Fast Flow anion-exchange chromatography andprotein recovery of SDS- PAGE for purifying Ara h2 was developed.In the processing of preparation of specific antibody against peanut allergenAra h2, each rabbit was injected with 200ng Ara h2 in every two weeks for six times.The rabbits were immunized with complete Freund's adjuvant at first, and boostedwith incomplete Freund's adjuvant. Twelve weeks later, specific and high titer serumwas gained. The serum titer of the rabbits was 1:200,000 and 1:16 by using indirectELISA and agar double diffusion method respectively. Agar double diffusion methodwas also used to detect the specificity of the antibody, and results showed that therewere no cross reactions among Ara h2, bovine serum albumin, soybean proteins andegg white proteins. The indirect ELISA was developed to detect the titer of thespecific rabbit serum and a protocol to prepare polyclonal antibody against Ara h2was established.In the processing of developing indirect inhibition ELISA with out-platecompetitive reaction model, working ELISA conditions were as follows: coating for 2hours in 37℃with Ara h2 at 160ng/mL, blocking for 1 hour in 37℃with a 5％ (M/V) defatted milk powder; coating for 1 hour in 37℃with anti-Ara h2 antibody at1:16000 dilusion. In this method, the sensitive concentration, the detection limit, therecovery rate was 51ng/mL, 0.26ng/mL, 90％～100％respectively, and the standardcurve was linear within the range of 12.5ng/mL and 400ng/mL. The method wasapplied to detecting 6 commercial samples. The Ara h2 concentration of the peanutmilk sample was 812mg/kg, and other five samples showed no Ara h2 allergen.