Development Of ELISA For Egg Immunocompetence And Extraction Of Peanut Allergy Proteins Ara H1by Immunoaffinity Chromatography

Allergy reaction is regarded as global hygienic and food safety problem, and is becoming seriously from year to year. According to epidemiological investigation,Food allergy affects up to8%of enfants and infants and2%of adults,and exceeding20%of total population in developed countries. Peanuts and eggs are respectively enrolled in8main foods allergy,and they are also favorite foods because they are rich in nutrients, but some people are affected by their allergenicity.so it is necessary to research the rapid testing and purification of allergic proteins.The main content and result were as follows:(1) Indirect ELISA kit for detecting egg allergy proteins had been developed. Firstly, egg allergy proteins were extracted by ammonium sulfate decantation,and verified by Western Blotting,and then got polyclonal antibody was collected by getting New Zealand white rabbit a shot with extracted proteins. The best condition for ELISA was optimized influencing factors, which shown as:oating with CBS(0.01mol/L,pH9.6) at4℃for12h and then37℃for1h, blocking with5%skim milk powder, the best react content for antigen1μg.mL-1,for antibody50000times’dilution and for HRP-IgG2000times’dilution, Based on those conditions, the kit was with lowest detectable limit at1.1ng.mL-1.Secondly,kit was evaluated:the sample recovery was at90%-105%, specificity was good, the average coefficient of variation of infra and inner plate were less than5%and10%, repeatability discrepancy was Non-significant. The ELISA kit was stable at37℃for at least4days,which could be accumulate more than6months at4℃. Kit could tolerate three times frozen.(2) The method of Immunoaffinityfor peanut allergy proteins Ara h1purification was developed.Firstly, Ara h1allergy protein were gained by ammonium sulfate decantation and molecular sieve chromatography,and the purity of Ara h1was above90%,then we got polyclonal antibody by getting New Zealand white rabbit a shot with Ara hl,whose titer was5×105.The next step was to couple the CBr-activated Sepharose4B with antibody(the coupling rate was up to95.9%), then chromatographic column was prepared.The best condition for chromatographic column was measured by optimizing influencing factors, which showed as, elution with glycine-HCI (pH2.4), elution velocity at0.5-2mL/min, the best incubation time was5min, loading capacity was11mg. The sample recovery was at73.6%-89.2%.Above results showed that the kit’s parameter,for example detectable limit stability and so on,fulfilled with technical specification, which supported help for egg allergic proteins detecting. From this study,we found that Immunoaffinity for proteins purification timesaving,highly efficient and strong specificit, which was a good support for proteins deep-going study.