Screening Of Nanobody Specific For Peanut Major Allergen Ara H 3 By Phage Display And Fag T 6-a Novel Major Allergen In Buckwheat Seeds

Food allergies are widespread,affecting more than 10%of the population.In the past 20 to 30 years,the prevalence of food allergies has been on the rise.The development of specific nanobodies against food allergens and the discovery and characterization of new food allergens are very important in detecting food allergens.Peanut is one of the eight major allergic foods,and peanut allergy is also one of the most common fatal food allergies.The main allergen of peanuts,Ara h 3,was selected for specific nanobody research.Buckwheat is widely consumed in Asian countries.At Peking Union Medical College Hospital in China,it is observed that 3.6%of patients with allergic diseases have been diagnosed with buckwheat allergy.Accoding to the clinical data of Peking Union Medical College Hospital in China,we discovered and characterized a new buckwheat allergen,Fag t 6.Now peanut allergy has become one of the important health problems worldwide,especially in developed countries.In United States,peanut allergy is one of the most common fatal food allergies.Peanuts are cheap and often eaten without processing.Peanuts are also ingredients of many processed foods.Although a large number of clinical treatment studies have been conducted in the past decade,avoiding peanuts is still the only effective way to prevent peanut allergy.Therefore,it is very important to establish a reliable method for detecting peanut allergens.Because the enzyme-linked immunosorbent assay has high sensitivity,high specificity,easy operation and high sample throughput,enzyme-linked immunosorbent assay is currently the preferred method for detecting allergens in most food industries.Nanobodies are unique antibodies derived from camelid animals and sharks.Compared with conventional antibodies,there are antibodies with only two heavy chains in camelid animals and sharks.The variable region of the heavy chain is the main antigen-binding region.Because of its nanometer size,it is also called nanobody.Due to the high stability,small molecular weight and convenient expression of nanobodies,nanobodies show unique advantages in biological detection.However,no nanobody specific for peanut allergen has been developed yet.In this study,we screened a humanized camel nanobody phage display library with main peanut allergen Ara h 3,and obtained several candidate nanobodies,among which Nb16 is the best nanobody.Subsequent biochemical characterization methods such as gel filtration chromatography,isothermal titration calorimetry,co-crystallization and western blotting proved its interaction with Ara h 3.Nbl6 specifically binds to Ara h 3,which is the main peanut allergen,with a dissociation constant of 400 nM.In addition,we obtained Ara h 3-Nb16 complex crystals.According to structural analysis,we found that the stacking method of Ara h 3-Nb 16 complex crystals is completely different from that of natural Ara h 3 crystals.Combined with the results of western blot after crystal dissolution,it proves the existence of complexes in the crystals.Nb16 may have application value in the development of peanut allergen detection biosensor.Buckwheat is widely consumed in Asian countries and becoming more and more popular in the United States,Canada and Europe.However,buckwheat may be a strong allergen and may cause life-threatening allergic reactions.Buckwheat allergies are often reported in Asia.In Peking Union Medical College Hospital in China,it is found that 3.6%of patients with allergic diseases have been diagnosed with buckwheat allergy.Like peanuts,a small amount of buckwheat can also cause various allergic reactions,such as asthma and anaphylactic shock.Buckwheat cross-reacts with many foods or raw materials,so accurate diagnosis of buckwheat allergy is very important.However,the current understanding of its pathogenic allergens is still limited.So far,five important common buckwheat allergens and one tartary buckwheat allergen have been identified.These six allergens are all water-soluble proteins.Because our commonly used allergy diagnostic reagents are water-soluble,it is impossible to diagnose oleosin allergens,that is,allergy diagnostic reagents do not contain oleosin allergens,and false negative results may occur.In fact,oleosin allergens have been identified in beans,nuts and seeds.In this study,we firstly analyzed the fat-soluble crude extract of tartary buckwheat via western blotting,and found that between 15-20 kDa,a protein that obviously binds to IgE in the serum of patients with buckwheat allergy appeared.It is speculated that oleoprotein may also be presented in buckwheat.Then the natural tartary buckwheat oleosin was purified,and the mass spectrometry analysis showed that it was indeed oleosin.We extracted the tartary buckwheat genome,cloned the tartary buckwheat oleosin gene sequence,submitted it to the National Center for Biotechnology Information,and obtained the gene number MW567728.Apply for the name of tartary buckwheat oleoresin from the World Health Organization/International Union of Immunological Societies Allergen Nomenclature Database,and obtain the new name Fag t 6.Dot blot and enzyme-linked immunosorbent assay were used to detect the sensitization of the purified natural protein.It was found that half of the sera of 20 buckwheat allergic patients had strong reactivity with purified tartary buckwheat Fag t 6.We used a circular dichroism experiment to detect the change of Fag t 6 with temperature to evaluate its thermal stability.We found that the thermal stability of Fag t 6 protein is low,and the thermal denaturation is irreversible.Buckwheat has cross-reactions with many foods or raw materials.We conducted enzyme-linked immunosorbent assay analysis on buckwheat,wheat,peanuts,hazelnuts,quinoa,latex and rice,and found that the cross-reactions between buckwheat and wheat are the most obvious.It is hoped that the research on the new allergen Fag t 6 may be used to improve the diagnostic test of buckwheat allergy in the future.