Jinjin Hung,Chngjun Liu,Ynbo Wng,b,Chong Wng,Menghu Xie,Yi Qin,Linglin Fu,b,*

a School of Food Science and Biotechnology,Zhejiang Gongshang University,Hangzhou,China

b Zhejiang Engineering Institute of Food Quality and Safety,Zhejiang Gongshang University,Hangzhou,China

c Xiangshan County Aquatic Technology Promotion Station,Xiangshan,315700,China

Keywords:

ABSTRACT

1.Introduction

In recent years,food allergy and other related diseases have increased rapidly worldwide,with 1%–2% adults and 4%–8% children have high reactivity mediated by Immunoglobulin E(IgE)[1,2].Food allergy is a kind of inflammatory disease,which is known to be the cause of some serious complications that affect people’s quality of life,arousing widespread concerns.

In a broad sense,food allergy can be classified into two categories:IgE-and non-IgE-mediated hypersensitivities.The former includes the sensitization stage,the excitation stage,and the effect stage,while the mechanism of the latter is not very clear at present.Food allergy is mostly IgE-mediated type I hypersensitivity,when food allergens first enter the body,the cells were activated and secreted interleukin(IL)-4,IL-5,IL-13 and other cytokines that induce IgE antibody production in B cells,the interaction of IgE and the target cells(mast cells,basophil granulocyte cells)makes the body hypersensitive.When the same food allergen enters the body again,it interacts with the IgE molecules on the target cells to mediate the bridging reaction,activating the downstream signal pathway,causing the target cells to degranulate and release the mediators such as histamine,5-hydroxytryptamine,and leukotriene,resulting in an allergic reaction[3],at present,the mechanism of food allergy is very clear,we show the mechanism of food allergy,as shown in Fig.1,it increases the permeability of intestinal epithelial cells to the antigen,thereby affecting the pathway of certain cytokines,and producing a Th2-type inflammatory response.The food allergy by IgE stress is the most serious type of food immune response as well as a clinically important allergic disease,which has two characteristics:food specific IgE antibody and food related allergic symptoms.Notably,some allergic patients have high level of allergen-specific IgE in their bodies,but do not develop an allergic reaction to the allergen and thus cannot be diagnosed as IgE-mediated food allergy[4].

National Allergens and Institute of Medical Science(NAIMS)has defined food allergens as foods or ingredients that can be identified by specific immune cells and elicit hapten responses that produce specific symptoms,food allergens are generally protein compounds that may bind to proteins(called semi-antigens)[5].The Food and Agriculture Organization of the United Nations(FAO)has published a list of common allergenic foods,including peanuts,soy,eggs,milk,crustaceans,nuts,wheat,fish etc.,more than 90%of the allergic reactions were caused by these 8 common allergens[6],the detailed information of which,including the specific allergen protein molecules and allergen family,is list in Table 1.Food allergies often cause severe allergic reactions including anaphylaxis and can be life threatening,and are usually lifelong.

Fig.1.Diagram of Food Allergy Mechanism.

Table 1 The major food allergens.

In recent years,researches assessed food allergens mainly through bioinformatics analysis,simulated gastric digestion,serological analysis,cell model animal tests and model tests.Animal model test is the most direct method to evaluate the potential allergenicity of food while cell model is a more convenient and flexible one.In this paper,the current status of food allergenicity evaluation is briefly introduced,and the advanced researches of the allergen evaluation methods(based on animal,cell and clinical approaches)are reviewed.Specifically,because zebrafish is a typical model organism and is widely used in the biomedical field,we introduced some related research on zebrafish and hope to have some inspiration in the field of food allergy.

2.Cell models of food allergy

The occurrence and severity of food-induced allergies depend on the releasement of allergenic mediums such as histamine and amino glycosidase and the subsequent responsiveness of the target organs,which are produced from the degranulation of basophil granulocyte or mast cell triggered by the interaction between surface-bound IgE and the corresponding allergen.Therefore,immunological methods based on in vitro cell degranulation models can directly reflect the allergenicity of food allergens.In general,the ideal cell model should meet the following aspects:the allergen can trigger the releasement of active medium with good repeatability;the specificity of different allergens can be distinguished;and a small amount of allergen can induce significant degranulation.A number of cell models have been used to study allergenicity and sensitization mechanisms,including mast cell models,human basophil granulocyte models,etc.

2.1.Mast cell models

In the 1877,Paul Ehrlich firstly discovered mast cell,an important immune cell type involved in allergic reactions.Originating from bone marrow stem cells,mast cells are mainly distributed in mucous membrane and subcutaneous connective tissue,and play a central role in inflammatory and allergic reactions.When the high affinity IgE receptor(FcεRI)expressed on the surface is stimulated by specific allergen-IgE interaction,mast cell can release inflammatory mediators such as histamine and newly synthesized media such as leukotriene and prostaglandins,causing contraction of smooth muscles,dilatation of blood vessels,leakage of small blood vessels,and secretion of mucus.Additionally,mast cells do not release histamines only when they are activated by an allergenic substance,but also when they are damaged.

Mast cells play a central role in the pathogenesis of food allergy.It is the key link between induction stage and effect stage of allergic reaction,and amplifies the signal of allergen.Histamine is one of the most frequently studied and important agents of allergy,mainly used to mark the degree of degranulation of mast cells.However,Schwartz et al.have shown that histamine has a short half-life of only a few minutes under physiological conditions,and the release is not stable enough,which leads to instability and poor repeatability of detection results[7].On the other hand,trypsin is a pre-formed protein enzyme in mast cells,which is released into the medium and involved in an allergic reaction together with other mediators.Results showed that the content of the trypsin in the cell was high and easy to detect.In addition,the detection method of trypsin is simple and does not require the formation of fluorescence to participate in the reaction as histamine do,so it can be more accurate and stable to reflect the degree of granulation of mast cells,and therefore is a more suitable marker for allergic reactions.

Since mast cells play an important role in food allergies,high concentrations of mast cell produces such as histamine,IL-5 and IL-13 can be detected in the serum of food allergic patients[8].Therefore,detecting the releasement of the granule contained in the mast cell can be used as a quick method to screen food allergens.Based on this,Yang et al.stimulated OVA allergen in vitro from rat peritoneal rat(rat peritoneal mast cell)and found that the mast cells became larger and the cell membrane shrank after stimulation,the cytoplasm is filled with bubbles and the degranulation surrounds the cell[9].Through the observation of mast cells by transmission microscope,it was found that the cell membrane was damaged,the contents of the cell were reduced and the distribution was not uniform.Thus,the morphology of mast cells and the release of particulate matters can serve as indicators for the detection of allergens.By inducing mast cells in bone marrow in vitro,Toyoshima et al.revealed the role of splenomegaly in food allergic diseases,significant expansion of MC was also observed in the spleen in the Th2 response mediated food allergy model.The incidence of allergic diarrhea was significantly reduced in splenectomy mice,the in vitro induced transfer of spleen MCs to these mice was restored to allergic symptoms,indicating that spleen MCs play a role in pathogenic cells in the development of food allergy[10].But this assay takes about five weeks for the mast cells,which are derived from bone marrow,to mature.Consequently,time consuming and high cost limit the assay for food allergen detection,and therefore commercial cell lines such as human mast cell line 1(HMC-1)and laboratory of allergic diseases 2(LAD2)are the most commonly used cell models for the study of food allergenicity assessment.Nagai et al.used HMC-1 and LAD2 cells to reveal the relationship between hyperglycemia and hypersensitivity[11].In addition,Zhang et al.used HMC-1 cell to screen anti-allergenic components and identified the critical role of JAK2/STAT5 signal pathway,which further elucidates the intracellular pathway involved in anti-allergy[12].Similarly,Han et al.used HMC-1 cells and rat peritoneal mast cells to screen for anti-allergenic components in vitro,through the combination of mouse model in vivo,it is proved that mast cell model has great potential in the field of allergy research[13].Overall,these studies all show the important role of mast cells in the evaluation of food allergenicity.

2.2.Human basophil models

The basophil granulocytes are originated from CD34+bone marrow precursor cells and developed in the bone marrow.Mature basophil granulocytes are presented in blood and only moved out by the induction of chemokines in the event of inflammation.It has been reported that the activity of basophils and the degree of degranulation were closely related to allergen allergenicity.The basophil granulocyte is found in peripheral blood,bone marrow and spleen and accounts for less than 1% of the peripheral white blood cells,despite the low percentage,basophils play an important role in allergic reactions.Basophil not only participates in the chronic allergic reaction mediated by IgE,but also plays an important role in modulating immune response,enhancing immune response memory and inducing Th2-type reaction[14–16].Like mast cells,human basophil express high affinity IgE receptors on the surface and are the main effector cells involved in the type I hypersensitivity.Its cytoplasm contains basophilic particles which release histamine and other bioactive substances after being stimulated by antigens.As a result,basophil histamine release test is commonly used in studies of allergenicity[17–19].

2.3.RBL-2H3 cell models

RBL-2H3 cell line is a subset of the rat alkaline leukemia granulocyte,which is similar to mast cells on many properties and functions.The high affinity receptor of IgE on the surface of RBL-2H3 cells can be activated by the IgE-antigen complex and release the inflammatory mediators such as histamine,5-hydroxytryptamine(5-HT)and hexokinase.Compared with primary basophils,RBL-2H3 has the advantages of good homogeneity,small variation,unlimited generation and strong operability[20].In recent years,the role of RBL-2H3 cells in the detecting IgE has aroused wide interest of researchers worldwide.Compared with the results of classical method such as IgE binding test,the results of RBL-2H3 cells showed good consistency[21].

RBL-2H3 cells are often used as complementary models of mast cells or human basophils to evaluate food allergenicity and identify allergen-specific IgE,furthermore,the humanized RBL-2H3 cell line plays a role in the diagnosis of food allergies and the identification of food allergens.Many studies used transfected RBL-2H3 cell models to assess allergies to peanuts,wheat,eggs and milk,and the results were consistent with the results of traditional detection methods(scratch tests)[5,22–25].By detecting the fluorescence signal of RBL-2H3 cells,Jing et al.identified the primary fish allergen albumin.In addition,Vogel et al.identified the allergenicity of complex food matrix by analyzing the humanized RBL-2H3 cells,a stable’humanized’cell strain was established,which can be a useful tool for the standardization of allergy extract,and it can also be used for detecting trace of potentially sensitive proteins[26].Porterfield et al.identified Ara as the main allergen causing peanut allergy by using the RBL-SX38 cell model(a humanized RBL-2H3 cell line)[27].The study of Kaul et al.showed that the medium release test of RBL-2H3 cells could be used to detect the allergenic potential of allergen and the titer of IgE antibody in serum.However,there are some controversies about the use of RBL-2H3 cell model.Dearman et al.showed that the sensitivity of RBL-2H3 cell model was lower than that of passive cutaneous anaphylaxis(PCA)and was not suitable for the analysis and detection of specific IgE antibody[22].Although RBL-2H3 cells have the characteristics of mast cells and basophils,there are many differences between RBL-2H3 cell and mucosal mast cell(MMC)and connective tissue mast cell(CTMC),for example,RBL-2H3 cells does not express TLR2 or the TLR4 signal pathway,and some non-immune stimuli can also cause RBL-2H3 cell degranulation[5,20].In addition,the phenotype and function of RBL-2H3 cells are also susceptible to culture and test conditions.Therefore,RBL-2H3 cells can not completely take the place of mast cells or basophils,and research on food allergies with RBL-2H3 needs to be treated with caution,as further validation is needed for the cell model to be used in food allergenicity assessment[20].

3.Murine models of food allergy

Animal models are used in the studies of food allergies to overcome the possibility of life-threatening,difficult operations and ethical limitations in the clinical studies.The ideal animal model must have a similar gastrointestinal system to humans,can develop allergic reactions with allergens that are only exposed to humans,and can produce human-like IgE antibodies and similar antigenic reactions.Taking into account the species,strains,ages,differences in immune responses to proteins,exposure pathways,times,doses of allergens,and the use of adjuvants,the mouse strains BALB/c,C57BL/6 and A/J are most common used to establish the allergy models[28].On the other hand,the use of rat strains included SD,Wistar and BN,and other animals such as pigs and dogs were also reported.

3.1.BALB/c mice models

Mouse models have been widely used in studies of cellular and molecular mechanisms of various IgE-mediated allergic diseases[29].BALB/c mouse is a near-crossing high-IgE response strain and is widely used in the study of hypersensitive diseases[30].

Due to the high-reactivity and readily available,BALB/c mice are widely used in food allergy studies[31–33].For example,Fotschki,et al.sensitized the BALB/c mice by intraperitoneal injection of lactoglobulin and casein showed that the milk allergen could significantly increase the level of IgE antibody in serum to eight folds[34].Similarly,Wavrin,et al.exposed BALB/c mouse to peanut allergen Ara h 1 by means of contact,inhalation and oral administration[35].It was found that the contact and inhalation pathways tended to produce specific IgG1 antibodies,and that the allergic patterns were significantly dose dependent.Gizzarelli,et al.used cholera toxin(CT)as adjuvant to establish the IgE-mediated BALB/c mouse soy protein-sensitized model by oral gavage,in addition to soy protein specific IgE and IgG1 antibodies,the levels of IL-4 and IL-5 also increased[36].In addition,they also detected changes in cytokines and IgE in mice by oral administration of genetically modified soy extracts,demonstrating that BALB/c mice can provide some valuable information in the assessment of food allergens.Gomes-Santos and colleagues also selected the BALB/c mice to establish a sensitive model[37].The results showed that the content of IgE,Siga,and IL-5 in the intact whey-sensitized mice were increased and their body weight was reduced,the contents of IL-4 and IL-10 in intestinal mucosa were decreased,showing that the cells were biased toward TH2 levels,and the mice had an allergenic effect.On the other hand,the contents of these indexes in the mice of the sensitive group of whey hydrolysate were kept at normal level without enteritis.Orafo et al.performed food allergy studies on BALB/c mice by intragastric administration with the adjuvant CT[38].The study found that BALB/c mice had elevated levels of IFN-γ,IL-4,and IL-10.However,no allergic reactions were observed to milk after avage in BALB/c mice.

However,the sensitivity of BALB/c mice was easily tolerated by the administration of allergens.Dearman et al.compared the effects of the two allergenic pathways of intraperitoneal injection and gastrectomy on OVA specific IgG and IgE antibodies and found that only intraperitoneal injection of OVA resulted in the production of more potent IgE antibodies.Consequently,the BALB/c mouse model is more suitable for intraperitoneal injection to determine the intrinsic allergenicity of proteins[39].

3.2.C3H/HeJ mice models

In recent years,some researchers used C3H/HeJ mice to investigate the mechanism of food allergy[40,41].C3H/HeJ mice are Toll-like receptor 4(TLR4)mutant and high-IgE response strains.The mutation of TLR4 gene may induce immune system disorders and affect the composition of intestinal flora,making mice more susceptible to anaphylaxis[42–44].Berin et al.studied the hypersensitivity of C3H/HeJ mice induced by milk and peanut allergens[45].The results showed that the differentiation of T cells in C3H/HeJ mice with TLR4 defect was biased toward allergenic TH2 cells.

Similar with the case in BALB/c mice,milk can also cause anaphylaxis in C3H/HeJ mice through oral,stomach,and skin contact,but skin contact can maximize the level of IgE,and adjuvant will also make the allergic reaction more pronounced[46].A threeweek-old C3H/HeJ mouse cow-milk protein allergy model induced by IgE was established by using a variety of techniques[40].Morafo et al.found that the level of milk-specific IgE in C3H/HeJ mice was significantly increased in the sixth week after sensitizing,and the levels of Il-4,IL-10,and IFN increased significantly[38].In addition,the increase in plasma histamine levels associated with allergic reactions was also found in the C3H/HeJ mouse model.

CT is protein complex secreted by the bacterium vibrio cholerae.The most classic of these was the ingestion of mice with CT combined with milk.After 6 weeks,the level of milk-specific IgE antibody was significantly increased,the model is valuable for the study of the mechanism of IgE-mediated food allergy and the cellular regulation reaction.Van Esch et al.used whey protein as an antigenic agent and combined with CT to study the recognition ability of allergen in C3H/HeJ mice with milk allergy[47].The results showed that the concentration of serum specific IgE and IgG1 and the concentration of mouse mast cell protease-1(mMCP-1)were increased,and the model was able to distinguish between whey and whey hydrolysates,it proved that the C3H/HeJ mouse model can be used as a new screening model for low-sensitive milk formula in vivo.However,the reliability of C3H/HeJ mouse as an allergenicity model remains to be verified.Andreassen et al.showed that cry 1 ab was used as an allergen-sensitized female C3H/HeJ mice[48].Results showed that neither the addition of adjuvant nor the use of the same dosage of C3H/HeJ mice could observe the hapten of cry 1 ab protein.

In addition to the milk,other researchers used other allergens to stimulate mice.Li et al.established anoral model of peanut induced food allergy in C3H/HeJ mice[49].They used low dose(5 mg/mouse)and high dose(25 mg/mouse)peanut protein with adjuvant CT to irrigate the stomach.It was found that the specific IgE antibody could be produced in different dosage groups,but the sensitization of low dose group was more efficient.After oral stimulation,the mice in the low-dose group showed severe and even fatal allergic reactions,plasma histamine level increment and mast cell degranulation in ear tissue,however,the high dose group did not see the occurrence of extreme allergic symptoms.In addition,the study of T and B cells found that they were similar to those in humans when they had an allergic reaction.Additionally,C3H/HeJ mice may be a useful animal model for the study of genetically modified food sensitivity[38].This model provides more opportunities for further study of food allergy mechanisms as well as the prevention and treatment of allergic diseases.

3.3.BN rat models

Compared with mice,the size of BN rats was moderate,and the dynamic analysis of serum specific antibody IgE and IgG was easily observed on a single animal.Moreover,rats could be sensitive to oral treatment without adjuvant,and the produced allergic symptoms are more similar to those in humans.BN rats are high immunoglobulin(especially IgE)response strains,and thus play an important role in the selection of milk-sensitive investigations.Knippels et al.established the food allergy BN rat model with eggsensitized ovaline(OVA)was used as a pattern allergen in the absence of adjuvant[50].The results showed that over 80% of BN rats produced OVA-specific IgE,moreover,the intestinal permeability of the sensitized rats increased after stimulation.Likewise,Akiyama et al.studied the oral sensitization model of BN rats and found that BN rats produce OVA-specific IgE and IgG1 antibodies and exhibit strong allergic symptoms[51].

By using intraperitoneal injection followed by oral administration of OVA as the antigen with alum as the adjuvant in BN rats.Abril-Gil et al.found that the increased concentration of OVA-IgG1,OVA-IgG2a and OVA-IgG2b in the serum by intraperitoneal injection were further increased after oral administration,especially the anti-OVA-IgE concentration in the first week after oral lavage[52].The treated mice also showed decrement in body temperature and activity,as well as the overexpression of mast cell protease II(RMCP II).Therefore,the authors concluded that BN rat is an effective animal model for the evaluation of food allergy and that the best sensitizing method is intraperitoneal injection followed by oral administration.

Rucheng Chen et al.found that parental exposure to OVA had an effect on the filial generations,and that BN rats had to reproduce at least one more generation without allergen in order to be used in food allergy models[53].In addition,Bogh et al.found that the hydrolysis products of β-lactoglobulin(BLG)and whey in BN rats were not sensitizing and lacking the ability of sensitivity and excitation,demonstrating that the hydrolysis product can be evaluated by intraperitoneal injection animal models,but the selection of animal strains and adjuvants may have a great influence on the results.Interestingly,it was also found that the IgE epitopes of Ara h 1,the main allergen of peanut,was identical between rats and humans[54].Taking into consideration that different allergens are diverse in the incidence and sensitivity in human and the ideal animal model should reflect these allergen-specific differences,BN rat is an ideal animal model for allergic researches.

4.Zebrafish model potentially used in the study of food allergy

In spite of the advantages above,the murine models have some disadvantages such as strong subjectivity,large variability,high cost and so on.Therefore,it attracts increasing researchers to develop other appropriate animal models for allergen detection.Zebrafish is an important vertebrate model widely used in research fields such as developmental biology,genetics,toxicology,and oncology.It is one of the four model organisms in biological research together with mouse,fruit fly,and nematode worm.In recent years,the application of zebrafish in the field of immunology has gradually been valued.The zebrafish’s immune system has both commonalities and specificities compared with higher vertebrate animals’.The study of the hematopoietic mechanism of zebrafish showed that human and zebrafish have very similar cellular composition in the immune system[55].Notably,the adaptive immune system of zebrafish does not begin to appear until 4–6 weeks after fertilization,and zebrafish which only have innate immune function during the first week of life exists in embryonic development[56].Because zebrafish embryos are developed in vitro and fully transparent,it is a good model for studying the innate immunity of vertebrates,but the direct study on food allergy is absent and needs far more investigations.

4.1.Zebrafish as an inflammation and infection model

Traumatic inflammation studies are an important aspect of zebrafish researches.The establishment of immune cell transgenic lines such as neutrophils(mpo:GFP),early macrophages(fli-1:EGFP)and neutrophil/macrophage double transgenic lines(Lyz:EGFP/DsRED2)enables the observation of inflammatory and immune responses in vivo.The main method is to trace the inflammatory behavior of immune cells by fluorescence microscopy after cutting off the tail,acupuncture or laser stimulation.Results have shown that in the early stage of zebrafish embryo development,macrophages and neutrophils participate in an acute inflammatory reaction.Neutrophils and macrophages respond almost simultaneously to traumatic inflammation but neutrophils migrate faster and reach the site of wound infection before macrophages arrive.The macrophages at the wound originate from the tissue,while the neutrophils are derived from blood vessels.

On the other hand,zebrafish infection models are commonly used for the investigation of bacterial and viral infections.In these models,the infection site,time,and amount of infected bacteria and viruses are all important influencing factors.Li et al.injected Staphylococcus aureus(S.aureus)into the eye,pericardial cavity,tail vein,fourth ventricle,yolk sac,and urothecium of zebrafish to establish multiple sites infection models[57].Xu et al.established a zebrafish infection model by intraperitoneal injection of infectious spleen and kidney necrosis virus(ISKNV)into adult fish,with systemic bleeding,vertical scales,and splenomegaly as the main symptoms[58].Black worm baculovirus(SHRV)can also infected zebrafish embryos and adult fish,resulting in a large number of mononuclear cells infiltrating in the embryonic,congested fish gills,subcutaneous edema and hemorrhage.During this process,the expression of interferon and Mx were upregulated in both embryonic and adult fish[58].Studies also showed that adult zebrafish is susceptible to infectious hematopoietic necrosis virus(IHNV),infectious pancreatic necrosis virus(IPNV),and spring viremia virus(SVCV),particularly,the SVCV model has been used to study the genetic basis of the disease[59,60].Additionally,Lu et al.demonstrated that neural necrosis virus(NNV)can cause zebrafish infection and tissue damage[61].

4.2.Zebrafish as a potential food allergy model

The largest and most complex immune site in the body is the intestinal mucosal immune system.Because the internal environment of the intestine is very complex and the gut is often stimulated by signals from pathogens,commensal bacteria and food proteins,the intestinal immune system relies on a strict regulation mechanism to distinguish these signal stimuli.Most systems of zebrafish have many common characteristics in phylogeny,structure and function compared with the corresponding systems of human,and the zebrafish also has the advantages of embryonic transparency,rapid development,easy sampling,etc.,so it has become an ideal model for studying intestinal immunity.

Gut microbe is the main source of lipopolysaccharide(LPS).In recent years,studies have found that LPS-induced inflammatory signals are associated with Toll-like receptors(TLRs),and LPS act as toxins by over-stimulation of TLRs innate immune signals and induce pathogenic inflammatory responses[61].In collaboration with LPS-binding protein(LBP),LPS binds to CD14 on the surface of monocytes/macrophages and interacts with the TLR4/Mengline Differentiation Protein-2(MD-2)complex to induce the nuclear factor-kappaB(NF-κB)activation and the up-regulation of proinflammatory cytokines[62].

Zebrafish only have innate immune function during the first week of life,and the specific humoral and cellular immune responses do not develop until 4–6 weeks after fertilization.Therefore,zebrafish is an ideal animal model for elucidating both innate immunity and adaptive immunity.In recent years,compared with systemic immunity,mucosal immunity has become a hotter spot in fish immunology researches,and new technologies such as gene chip technology which detects fish gene expression profile are widely applied.Several studies used the gene chip method to study the zebrafish infected by bacteria,and obtained genes related to immune response,inflammatory response,complement activation and defense response[63–65].Currently,there are many such studies,which are not discussed in detail here.As a powerful model organism,zebrafish has been widely used in the fields of growth and development,genetics,immunity and physiological pathology.However,the molecular mechanism of mucosal immunity in zebrafish is still unclear and further researches are needed.

5.Clinical studies for food allergy assessment

The diagnosis of food allergy is mainly based on clinical symptoms.It is important to identify allergens that trigger food allergies through in-depth inquiry of clinical history[62],and detailed dietary records are an important part of further diagnosis[63].However,the clinical diagnosis of food allergies is difficult.On the one hand,the reliability of the medical history provided by the patient is poor.On the other hand,the commonly used detection methods such as skin prick test and food-specific IgE have lower positive predictive value,and the correlation between test results and clinical findings is difficult to assess.Current testing methods include skin prick test(SPT),Serum-Specific IgE(sIgE),Component-Resolved Diagnosis(CRD)and Oral Food Challenge(OFC),etc.

5.1.Skin prick test

The SPT is a test for screening the presence of food-specific IgE in the body.Because it is easy to handle,non-invasive to infants and children,and cheaper than serum-specific IgE antibody detection,fast to show results,and high in negative predictive value,the method is widely adopted as the first step to check IgE-mediated food allergy.It can basically exclude IgE-mediated food allergy if the prick test results are negative[64].

The sensitivity of food allergen SPT is 30%–90%,and the specificity is 20%–60%[65].SPT in patients to foods such as eggs,peanuts,and fish allergens,has better sensitivity and negative predictive value(NPV,＞90%),but the specificity and positive predictive value(PPV)are poor(50%–85%),that is,negative result of SPT can basically exclude IgE-mediated food allergy,but the positive result can not accurately predict the clinical response.

5.2.Serum-specific IgE level

sIgE is an in vitro direct detection of the interaction of IgE antibodies with suspected food antigens.It is not affected by skin conditions and antihistamines and requires no special technicians to operate.It is currently believed that the higher value of sIgE test result,the higher positive predictive value(PPV)[66].But sometimes the sIgE test results are not consistent with the clinical manifestations,for example,some patients are negative for sIgE,but there may be an allergic reaction to certain foods,which reduces the negative predictive value and the relative specificity.On the other hand,many specific individuals are positive to sIgE but tolerant to the corresponding foods and have no clinical allergic reactions.This may be due to a cross-allergic reaction between food allergens and other allergens.Moreover,it may also be due to the lack of cofactors,low sIgE levels,low sIgE affinity or high binding threshold.

5.3.Component-resolved diagnosis

CRD,also known as molecular diagnosis(MA),is the use of extracting natural allergen components and recombinant techniques to identify allergen components at the molecular level.This diagnostic technology is a new type of allergen detection method that has emerged in the past 20 years[67].

CRD can effectively monitor the tolerance process of milk sensitization.Ford et al.found that casein is the best biomarker for identifying persistent allergies and transient allergies[68].The lower level of casein IgE in serum,the greater likelihood that patients will tolerate milk.Some other studies have also clarified the importance of casein[69].Elevated serum casein sIgE levels are associated with milk sensitization in children and may be associated with prolonged allergic disease.Furthermore,CRD has many applications in the detection of allergens such as eggs,wheat,and peanuts.

CRD diagnosis helps to identify sensitizing components that are truly allergic to patients at the molecular level,it improves the specificity of diagnosis and the effect of treatment,and optimizes the management of allergic patients as soon as possible,so as to further improve the quality of life of patients.

5.4.Oral food challenge

The gold standard for food allergy diagnosis is the oral food challenge(OFC),which provides direct evidence for the diagnosis of allergens in clinic,and is important for the risk stratification of related allergic reactions[70].

Oral challenge tests are simpler and more convenient to operate,so they are more commonly used clinically,and objective and clear allergic reactions are sufficient to diagnose food allergies[71].In the oral challenge test,the patient should stop using the drug,stop eating the suspected allergic food for 2–4 weeks,and then give the subject a suspected allergic food in a dose-increasing manner,which increase the dose every 15–20 minutes.The clinical manifestations of the patient’s skin,respiratory,digestive and circulatory systems should be closely observed during the procedure.When the objective symptoms or signs are positive,the test should be discontinued.If there is no response to the maximum dose,continue to observe for 2 h.During the oral challenge test,medical staff with formal training must be present to detect early adverse reactions and treat them promptly[72].At present,many scholars believe that OFC has a higher risk,but some other data show that the majority of patients during the OFC test only have mild allergic symptoms,saying the incidence of severe allergic reactions is about 2.2%–28%,and therefore it is safe to carry out OFC under the guidance of a specialist[73].

To assess the respective biomarkers of food allergy at the genetic level,the genome-wide association study(GWAS)in the current research method is a commonly used analytical tool.Hong et al.identified peanut allergy(PA)-specific loci in the HLA-DR and-DQ gene region at 6p21.32,tagged by rs7192 in 2,197 US participants.The result suggests that the mutation in HLA-DR and-DQ gene region probably result in a significant genetic risk to PA[74].In addition,Liu et al.examined maternal genotypic and parent-oforigin(PO)effects in 588 complete food allergy trios,and three suggestive loci with PO effect were observed.Findings from this family-based GWAS of food allergy provided some preliminary evidence on maternal genotypic or PO effects on food allergy[75].By better explored the effects of food allergies on gene levels,we are able to make allergens better circumvented by undiagnosed allergic patients.

6.Summary and future directions in different models of food allergy

The potential lethality of anaphylaxis is a major limiting factor in immune therapy for human food allergies,although there are many clinical tests for food allergies,the accuracy of these methods needs to be studied,on the other hand,for clinical studies of food allergies,oral stimulation and other methods have certain insecurity and should be used with caution.In which case the animal model provides an important platform for researches.Different animal models have their own characteristics:BALB/c mice are more selective in food allergens and may be better suited to determine the intrinsic allergenicity of proteins,C3H/HeJ mice can be used to study the mechanism of food allergy,and BN rats are easier to be sensitized by means of both oral and intraperitoneal injections.The systemic responses induced by sensitization in mice or rats are similar to those in humans,but the allergenic repeatability of BN rats is still to be verified.But animal models are flawed,so researchers are paying increasing attention to in vitro cell models,and the use of cell models has greatly contributed to the study of food allergies.However,we need further validation and standardization studies of animal models and cell models so as to better use them in food allergy assessments.Finally,as a convenient model organism,zebrafish should be applied to the mechanism study of food allergy as soon as possible.However,there is not enough research in this field to date,in addition,the rodent models and cell models still have some contradictions,so it is necessary to further improve and develop the present and novel food allergy models.

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgment

This work was financially supported by the State key research and development plan(2017YFD0400203).