The Standard Peanut Allergens Protein Arah2of Preparation Techniques

Food allergy refers to an allergic reaction in which certain compoundsin food, usually proteins, produce an immune response as an antigen to the body. Ithas become a major concern in the food industry to detect allergic proteins in foodquickly and accurately. Peanut is an important food allergen and allergy to peanut isbe coming a serious health problem because of its increasing prevalence, clinicalseverity, and chronically of the reaction which may occur at any age.Within theallergenic food, protein components are usually the trigger of allergenic reactions,therefore it is of utmost importance to obtain highly purifed proteins and begininvestigation on desensitization.The aim of this work is to propose application of high-throughputapproaches,peanut proteins are purifed in active form on the basis of theircharacteristics, such as solubility, size,and charge, by using centrifugation, ammoniumsulfate stepwise precipitation, DEAE ion-exchange chromatography andimmunoaffinity chromatography. Based on this theory and technology, we hope todevelop and prepare hypoallergenic or desensitized food for patient. It is vital and hasa good application in the future.The main contents and results are as follows:(1)According to the dissolution properties of peanut protein, peanut proteins areisolated into three soluble proteins.And then,author go a step further on those threetypes of proteins, centrifugation, membrane dialysis,and last it was purified by DEAEion-exchange chromatography, The results showed that Tris-HCl solution solubleallergic protein, eluting peak by0.3mol/LNaCl elution time can get high purity ofArah2.By SDS-PAGE and immunoblot analysis, results showed that it contains the tar get protein in19kD, but there are till mixed strips of other protein around7-15kD, need further purification experiment.(2) The spleen cells of BALB/c mice immunized by the allergic proteinArah2wasevaluated by western blotting,and Recovery with trace protein PAGE gel extractionkit.Then hybridomas secreting antibodies against Arah2were selected andclonedand.Two stable hybridoma cell lines E12H1and D1A1that MAb determinedsubclasses IgM, kappa light chain were established. The titer of the MAb determinedby indirect ELISA was1:80000in ascites.,lays a foundation for future researches ofsimilar kind.(3) Purify the antibody the allergic proteinArah2by immunoaffinity chromatography.The harvested MAb was purified and coupled with CBr–activated SepHarose4B toprepare immunoaffinity chromatograpHic column by which the allergic proteinArah2was purified from soluble proteins after DEAE ion-exchangechromatography,determined for purity and activity by SDS-PAGE and ELISArespectively．The coupling rate was up to95.9%,The best condition forchromatographic colum was optimizing influencing factors,which shows that elutionglycine-HCl(pH2.4) was best and the loading capacity was6.83mg.The samplerecovery was at89.5%-93.4%.After grayscale scanning, allergenic proteinsArah2purity is up to99%, the resulting of purification protein sequence a high degree ofmatching with the knownArah2immune site sequence.The above results meettechnical specification.