A method for constructing an animal model of infant nutritional rice flour food allergy

Abstract

This invention relates to the field of prevention and treatment of food allergies in infants and young children, and a method for constructing an animal model of food allergy in infant nutritional rice cereal. The method includes: 1) Randomly dividing experimental animals into several groups according to body weight for adaptive feeding; 2) From day 1 to day 7, different test adjuvants are used to conduct experiments on each group of experimental animals, with one group receiving a negative control without adjuvants, and the remaining groups further divided into subgroups according to different test substances; the animals are fed at fixed times and in fixed quantities each day, with the negative control group fed a basic liquid feed without adjuvants, and the other groups fed the same amount of liquid feed containing 0.5 mL of immune adjuvant solution; 3) At 9:00 AM on day 8, allergic stimulation is performed, with the negative control group fed 5 g of purified water, and the other groups fed 5 g of infant nutritional rice cereal liquid feed, observing clinical allergic symptoms and collecting blood and tissue samples; then the animals are sacrificed, and blood samples are collected; the experimental animals are preferably 3-week-old female Wistar rats of SPF grade, weighing 45 g to 65 g.

Description

Technical Field

[0001] This invention relates to the field of prevention and treatment of food allergies in infants and young children, and in particular to a method for constructing an animal model of food allergy in infants and young children caused by rice cereal. Background Technology

[0002] Food allergies are a major public health issue, attracting global attention due to their high incidence. Approximately 5% of adults and 8% of children worldwide suffer from food allergies, and the prevalence has been steadily increasing over the past three decades.

[0003] According to epidemiological surveys, the incidence of food allergies among Chinese children rose from 3.5% in 1999 to 11.1% in 2019. Furthermore, food allergies affect 8% of children in the United States and 6.7% of the total population in Canada. The associated economic, emotional, and safety burdens of these diseases are substantial.

[0004] Based on their pathogenesis, allergic reactions are mainly classified into four types (I-IV). Type I is a reaction to sensitizing foods mediated by immunoglobulin E (IgE), types II and III involve IgG-mediated reactions, and type IV is attributed to the reaction of allergen-specific T cells. Common food allergies belong to type I allergic reactions. Over the past few decades, approximately 8% of children and 5% of adults worldwide have experienced food allergies, and the incidence of food allergies is on the rise. Most foods can trigger allergies, but the eight major categories of allergenic foods jointly identified by the Food and Agriculture Organization of the United Nations and the World Health Organization mainly include milk, peanuts, nuts, wheat, eggs, sesame seeds, fish, and shellfish.

[0005] Food allergies are related to multiple factors. In terms of age of onset, egg and milk allergies are most common in infants and young children, and the allergic reactions gradually disappear with age. Differences in the prevalence of food allergies are also closely related to local dietary habits. Studies have shown that among school children in southern my country, the prevalence of shellfish allergies diagnosed by doctors is significantly higher in urban areas than in rural areas. Geographical environment is another factor influencing food allergy rates. In Western countries, peanuts and nuts are the most common allergens, while in Eastern countries, fish and shellfish are more prevalent. Furthermore, the incidence of food allergies due to genetics is also a significant concern. Research has found that children of parents with a history of peanut allergy have a seven times higher risk of developing peanut allergy.

[0006] Food allergy is an adverse stress response to food in allergic individuals or those with sensitive constitutions, posing a serious threat to the health of many. Clinically, food allergies mainly manifest as adverse symptoms in the skin (allergic dermatitis and urticaria, etc.), the oral cavity and digestive tract (slurred speech, oral swelling, stomach pain, abdominal pain, and diarrhea, etc.), the respiratory tract (sneezing, runny nose, asthma, shortness of breath, and difficulty breathing, etc.), the cardiovascular system (palpitations and rapid heartbeat, etc.), and the nervous system (confusion and loss of consciousness, etc.). Severe food allergies can lead to shock or even death in allergy sufferers.

[0007] Most food allergens are highly resistant to heat, pH, and gastrointestinal enzymes. They are defined as specific components in food that can be recognized by allergen-specific immune cells, triggering a specific immune response and leading to allergic symptoms. To date, the World Health Organization (WHO) / International Union of Immunological Societies (IUIS) Allergen Nomenclature Group has named as many as 1062 allergens, of which 399 are food-derived. Further classification shows that approximately 60% are plant-derived allergens and 40% are animal-derived allergens.

[0008] Infant rice cereal is a complementary food suitable for infants and toddlers aged 6 months and older, made primarily from rice with added nutritional fortifiers and / or other ingredients. Rice accounts for approximately 95% of the raw materials, making it the main component. Infant rice cereal is a food specially designed for infants and toddlers, typically containing nutrients such as protein, carbohydrates, fat, vitamins, and minerals—all essential for their growth and development. Because it is easily digestible, it helps infants better absorb these nutrients, promoting healthy growth. More importantly, infant rice cereal does not contain any of the eight major allergens, meaning it is unlikely to cause allergic reactions, making it an excellent choice for infants and toddlers prone to allergies. Therefore, compared to other foods, infant rice cereal has a relatively low allergy rate, making it a good food source for reducing or alleviating allergy symptoms. However, in recent years, food allergies caused by rice have become increasingly common, and the incidence of allergies to infant formula rice cereal continues to rise, which has had many adverse effects on the development and health of infants. Therefore, conducting allergen assessments on infant formula rice cereal and reducing its allergenicity is of great practical significance in preventing allergic diseases in infant consumers and promoting their healthy growth.

[0009] The rising prevalence of food allergies and the challenges posed by various novel protein resources to food allergy risk management make food allergy assessment fundamental to the rational control of food allergy risks and the assurance of food safety. Currently, various methods exist for evaluating food allergenicity, but different methods have different advantages, disadvantages, and applicable scopes.

[0010] The application of bioinformatics in allergenicity assessment is mainly concentrated in the field of genetically engineered proteins. It cannot directly determine whether a target protein is an allergen; instead, it predicts allergenicity by analyzing the homology and cross-reactivity between the target protein and known allergens. Antibody-based immunological analysis methods rely on antibody preparation and antigen isolation and purification. Furthermore, complex food processing techniques can alter the structure of allergen proteins, causing them to lose their original antigenic activity, easily leading to false positive or false negative results. Nucleic acid-based PCR analysis targets DNA rather than proteins, and the results do not truly reflect the allergenicity of food; moreover, numerous factors influence amplification efficiency. The difference in amplification efficiency between actual samples and standard samples can affect the cycle threshold, making it difficult to meet high-precision quantification. Mass spectrometry detection technology has the characteristics of high sensitivity, strong specificity, good accuracy and high throughput, but the high cost of mass spectrometry equipment and the need for professional personnel make the detection cost and labor cost of this method high, which also limits its widespread application in food allergen detection. Assessing the resistance of target proteins to gastrointestinal digestion through in vitro simulated digestion experiments is also one of the commonly used methods for evaluating sensitization. However, a positive result in in vitro simulated digestion, i.e., the presence of anti-digestion characteristics, cannot directly determine that the target protein is sensitizing. Further verification through serological experiments or biological models is required. Although in vitro sensitization evaluation methods have the advantages of low cost and high efficiency in general, in vitro evaluation methods can only partially reflect the sensitization of target proteins, especially in accurately evaluating the sensitization of potential primary allergens, and cannot well simulate the complex environment inside the human body. Therefore, in vivo evaluation methods remain necessary in controlling the risk of allergic reactions to new foods in the future. Human trials can provide more reliable evaluations of food allergenicity, and commonly used evaluation methods include skin irritation tests and dietary challenge experiments. However, their development and application are greatly limited due to their high execution difficulty, high risk, high cost, and ethical issues. Currently, only a few hospitals have the conditions and qualifications to conduct human trials.

[0011] Compared to in vivo assessment in humans, animal model assessment has advantages such as abundant animal sources, simple methods, and no harm to humans. It is also valuable for understanding the complex immunological and pathophysiological mechanisms involved in the occurrence and development of food allergic reactions. Therefore, animal models are often used to evaluate the potential sensitization of allergens.

[0012] Animal models used for evaluation include rats, dogs, and pigs. The choice of animal model requires careful consideration of its operational characteristics, limitations, and reliability under different environments. Currently, rats are the most frequently studied because their immune system is well understood, they are a tool in many immune-related studies, and they are the most commonly used experimental animal in food safety evaluation studies. Results from other food safety studies can serve as the basis for sensitization studies of related substances. There are reports of using BN rats to evaluate food allergies, but there are no reports of using Wistar rats to establish animal models of food allergens. Furthermore, there are three commonly used sensitization routes in animal experiments for sensitization evaluation: oral gavage, intraperitoneal injection, and free-flowing feeding. Intraperitoneal injection can study the intrinsic sensitization of proteins, but this route is not a natural route for humans to ingest food allergens. Therefore, oral gavage and free-flowing feeding are often used because their exposure routes are most relevant to human exposure to food allergens.

[0013] Furthermore, existing food sensitization testing technologies and allergy potential assessment methods cannot accurately reflect the allergenicity or risk, and some methods are too costly and unsuitable for testing low-sensitivity products. Therefore, an assessment method that can accurately reflect the sensitization of allergens has broad application value in scientific research and production, and is of great significance for promoting basic research and technological applications related to allergies.

[0014] Currently, in studies on animal sensitization models, most researchers conduct animal experiments for 4-6 weeks or more after weaning. However, infant formula rice cereal is a supplementary food for infants, with a feeding period of 6 to 36 months. Therefore, existing animal models have drawbacks such as long duration and inability to fully simulate food allergic reactions in infants. Furthermore, infants' digestive systems are not yet fully developed, and their absorption and processing of food components differ from adult animals, which can affect the distribution of potential allergens and the manifestation of immune responses. In addition, infants' immune systems are not fully developed, and their responses to allergens may differ from those of adult animals or humans, exhibiting different allergic reaction mechanisms. Traditional animal models, mostly used for adult animals, may not accurately reflect allergic reactions in infants. Moreover, the mechanisms of allergies in infant populations are complex, and allergic reactions vary depending on the infant's living environment and age. This is because some allergens may not elicit a reaction in adult animal models, but in real life, they can cause severe allergic reactions in infants. For example, rice protein may not be identified as an allergen in some adult mouse models, but it can trigger allergic symptoms in infants and young children. Therefore, it is necessary to use animal experiments that are matched to the environment and age to ensure data accuracy. According to relevant reports, 2 to 3 weeks in rats is roughly equivalent to 1 year in humans. Therefore, the conventional animal testing period is not feasible. The animal testing period must be controlled within the required range, and the animal model must have the characteristics of accurately reflecting allergic symptoms in infants and young children to simulate the changes in food allergic reactions during infancy, thereby establishing an optimal animal model. In addition, BN rats have a high similarity to humans in terms of immune system and physiological characteristics, especially in terms of the mechanism of allergic reaction and immune pathways, but they are expensive, resulting in high animal testing costs. Wistar rats, on the other hand, are economical and can meet the requirements for food allergy sensitization experiments. Therefore, establishing a humanized animal model that simulates the allergic characteristics of infants and young children is of great significance for accurately studying the specific mechanisms of allergic reactions caused by infant rice cereal. This is beneficial for conducting in-depth research on the pathogenesis and treatment of the corresponding diseases without posing treatment risks to clinical subjects. Furthermore, the animal model is inexpensive and has excellent results, and it provides a theoretical basis for the accurate clinical diagnosis of infant rice cereal allergies. Summary of the Invention

[0015] The purpose of this invention application is to address the shortcomings of the prior art and solve the above-mentioned technical problems. The first objective is to provide a method for constructing an animal model of infant rice cereal food allergy and the application of the animal model.

[0016] The second objective of this invention is the application of the animal model constructed by the aforementioned construction method in the study of allergic diseases caused by allergens, wherein the study is for the purpose of non-disease diagnosis or treatment.

[0017] A third objective of this invention is to provide a method for screening potential substances that resist infant rice cereal allergies, the method comprising: applying relevant candidate substances to an animal model constructed by the above-described construction method, and detecting sensitization indicators of the animal model.

[0018] A fourth objective of this invention is to provide the application of the animal model constructed by the above-described method in screening drugs for allergies to infant rice cereal or in experimental studies of allergies to infant rice cereal and complementary foods.

[0019] To achieve the above objectives, the present invention adopts the following technical solution: a method for constructing an animal model of infantile rice cereal food allergy, comprising the following steps:

[0020] Step 1: After the experimental animals were acclimatized, the experiment was conducted. The animals were randomly divided into several groups according to their weight, and they had free access to water and food during the experiment.

[0021] Step 2: From day 1 to day 7, each group of experimental animals was tested with different test adjuvants. One group of experimental animals received a negative control without adjuvant, and the remaining groups were further divided into subgroups according to different test substances. During the experiment, each animal was fed 5g of food at 9:00 am and 4:00 pm each day. The negative control group was fed a basic liquid feed without adjuvant, which consisted of 2.4g + 2.6g water with a consistency of 48%. The other groups were fed the same amount of liquid feed containing 0.5mL of immune adjuvant solution.

[0022] Step 3: At 9:00 AM on the 8th day, allergy stimulation was performed. The control group was fed 5g of purified water, while the other groups were fed 5g of infant nutritional rice cereal liquid feed. Allergy stimulation was performed. The animals were fasted for 12 hours before feeding. Clinical allergic symptoms of the animals were observed within 30 minutes to 1 hour, and corresponding blood and tissue samples were collected.

[0023] Then, the animals were euthanized, and blood samples were collected.

[0024] The preferred experimental animals are 3-week-old female Wistar rats of SPF grade, weighing 45g to 65g.

[0025] Furthermore, the animals were randomly divided into 11 groups according to their body weight, with 6 experimental animals in each group. One group received lipopolysaccharide as the test substance, which was administered orally by gavage. The test substances in the other groups were given freely, and they included physiological saline, lipopolysaccharide, β-glucan, chitosan, wolfberry polysaccharide, astragalus polysaccharide, mannan oligosaccharide, mannose, ginseng stem and leaf saponins, and zinc hydroxide.

[0026] Furthermore, the basic liquid feed formula is as follows: casein, L-cysteine, cellulose, xanthan gum, maltodextrin, sucrose, soybean oil, mixed minerals, mixed vitamins, and choline tartrate.

[0027] Furthermore, the dosage of the test substance and the basic liquid feed preparation are as follows:

[0028] 1. Preparation of immune adjuvants

[0029] All immunoadjuvant solutions were prepared fresh for use, with the following specific concentrations: lipopolysaccharide 0.10 mg / mL; β-glucan, chitosan, wolfberry polysaccharide, and astragalus polysaccharide 0.244 g / mL; mannan oligosaccharide, mannose, and riboflavin 10.0 mg / mL; ginseng stem and leaf saponins 3.0 mg / mL; and zinc hydroxide 2.0 mg / mL.

[0030] 2. Basic liquid feed preparation

[0031] Each mouse was fed a fixed amount of 5g each time. The control group was fed 2.4g of basic liquid feed + 2.6g of water. The preparation method was as follows: accurately weigh 2.4g of liquid feed, mix it evenly with 2.6g of water, and stir to make a basic liquid feed with a consistency of 48%. The other groups were fed the same amount of liquid feed containing 0.5mL of immune adjuvant solution.

[0032] Further, the blood collection and testing steps in step 3 are as follows: Each rat is anesthetized by intraperitoneal injection of 10% (w / v) chloral hydrate solution at a dose of 0.3g / 100g (body weight). Then, the abdominal skin is disinfected with alcohol, and the abdominal skin and muscles are cut open with surgical scissors. Under sterile conditions, blood from the rat's abdominal aorta is collected into negative pressure blood collection tubes and EDTA anticoagulant tubes, respectively. After collection, the tubes are centrifuged at 3500 rpm / min for 15-20 min at 4℃. The supernatant from both tubes is collected and placed in cryopreservation tubes. After liquid nitrogen cold extraction, the tubes are stored at -80℃ for the determination of serum and plasma specific antibodies and histamine.

[0033] The specific antibodies mentioned above are specific IgG, specific IgE, mast cell protease mMCP-1, trypsin MCT, and histamine HIS.

[0034] Furthermore, the detection steps for specific antibodies are as follows:

[0035] The obtained serum and plasma were analyzed according to the same method described in step 3, following the instructions of the ELISA kit:

[0036] 1. Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Seal the remaining strips in a resealable bag and store them at 4°C. Set up standard wells and sample wells. Add 50 μL of different concentrations of standard to each standard well, and then add 50 μL of the sample to be tested to each sample well. Do not add any to the blank wells. If the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required.

[0037] 2. Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add to the standard wells or blank wells.

[0038] 3. Discard the liquid, pat dry on absorbent paper, and fill each well with washing solution (350μL). Let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times.

[0039] 4. Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min.

[0040] 5. Discard the liquid and pat dry on absorbent paper. Fill each well with washing solution (350 μL), let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times.

[0041] 6. Add 50 μL each of substrate A (chromogenic agent A: H2O2) and B (chromogenic agent B: TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Within 15 min, use a microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0042] Furthermore, the high-dose infant nutritional rice cereal liquid feed is prepared by mixing 5g of infant nutritional rice cereal with 10g of water.

[0043] The beneficial effects of this invention are as follows: Extensive and in-depth research has shown that infants and young children develop increased tolerance to food allergies as they grow and age. Therefore, this invention utilizes an animal model constructed using this method. The animal model obtained by this invention overcomes the limitations of in vitro evaluation methods, which cannot fully simulate the complex immune response to infant formula. Compared to existing food allergy animal models, it overcomes the drawback of long animal modeling periods that prevent the complete simulation of food allergy reactions during infancy. This model can be used to evaluate the allergenicity of infant formula and various complementary foods for infants, fundamentally reducing the risk of food allergies in infants and young children. It can simulate the characteristics of food allergies in infants and young children, has strong clinical relevance, and can be applied to experimental research on allergies to infant formula and complementary foods. Furthermore, it provides a research model for clinical research on infant food allergies or for drug screening against infant food allergies, offering important options and basis for the clinical application of basic research. Attached Figure Description

[0044] Appendix Figure 1 This is a grouped clinical symptom scoring chart for Embodiment 3 of the present invention.

[0045] Appendix Figure 2 This is a diagram showing the grouped body temperature in Embodiment 3 of the present invention.

[0046] Appendix Figure 3 This is a diagram showing the diarrhea situation in groups according to Embodiment 3 of the present invention.

[0047] Appendix Figure 4 This is a graph showing the IgG content in groups in Example 3 of the present invention.

[0048] Appendix Figure 5 This is a diagram showing the IgE content of groups in Example 3 of the present invention.

[0049] Appendix Figure 6 This is a graph showing the mMCP-1 content in group 3 of Example 3 of the present invention.

[0050] Appendix Figure 7 This is a graph showing the content of trypsin-like enzymes (MCT) in group 3 of the present invention.

[0051] Appendix Figure 8 This is a grouped HIS content diagram for Example 3 of the present invention. Detailed Implementation

[0052] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0053] All raw materials and equipment used in this invention are known products, obtained by purchasing commercially available products. The infant rice cereal used in this invention is a commercially available Stage 1 infant rice cereal from a certain brand, and all test substances were purchased from reagent suppliers.

[0054] Example 1:

[0055] This embodiment provides a method for constructing an animal model and sensitizing / stimulating it. The specific steps are as follows:

[0056] Construction of sensitized animal models:

[0057] (1) Animal requirements:

[0058] Wistar rats, SPF grade, 3 weeks old, female, 45g-65g, purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0059] (2) Test substance

[0060] Physiological saline, lipopolysaccharide, β-glucan, chitosan, wolfberry polysaccharide, astragalus polysaccharide, mannan oligosaccharide, mannose, ginseng stem and leaf saponins, zinc hydroxide.

[0061] (3) Animal grouping

[0062] The experimental animals were randomly divided into 11 groups (6 animals in each group) according to their body weight: negative control group, lipopolysaccharide group, β-glucan group, chitosan group, wolfberry polysaccharide group, astragalus polysaccharide group, mannan oligosaccharide group, mannose group, riboflavin group, ginseng stem and leaf saponins group, and zinc hydroxide group.

[0063] (4) Formulation of basic liquid feed

[0064] The basic liquid feed formula is: casein, L-cysteine, cellulose, xanthan gum, maltodextrin, sucrose, soybean oil, mixed minerals, mixed vitamins, and choline tartrate;

[0065] (5) Dosage of the test substance and requirements for liquid feed:

[0066] 1) Preparation of immune adjuvants

[0067] All immunoadjuvant solutions were prepared fresh for use, with the following specific concentrations: lipopolysaccharide 0.10 mg / mL; β-glucan, chitosan, wolfberry polysaccharide, and astragalus polysaccharide 0.244 g / mL; mannan oligosaccharide, mannose, and riboflavin 10.0 mg / mL; ginseng stem and leaf saponins 3.0 mg / mL; and zinc hydroxide 2.0 mg / mL.

[0068] 2) Liquid feed preparation

[0069] Each mouse was fed a fixed amount of 5g each time. The control group was fed 2.4g of basic liquid diet + 2.6g of water (accurately weigh 2.4g of basic liquid diet, mix it with 2.6g of water, and stir to make a basic liquid diet with a consistency of 48%). The other groups were fed the same amount of liquid diet containing 0.5mL of immune adjuvant solution.

[0070] (6) Sensitization and stimulation

[0071] The sensitization test for high-dose infant nutritional rice cereal liquid feed was prepared by mixing 5g of infant nutritional rice cereal with 10g of water.

[0072] (7) Establishment of the sensitization model

[0073] The purchased Wistar rats were housed in metabolic cages, one rat per cage, with an indoor temperature of 26±2℃, humidity of 50% to 60%, and a day-night cycle of 12:12h, for 7 days to allow them to adapt to the feeding.

[0074] From day 1 to day 7, each group of experimental animals was tested with different test adjuvants. One group of experimental animals received a negative control without adjuvants, and the remaining groups were further divided into subgroups according to different test substances. During the experiment, each animal was fed 5g of food at 9:00 am and 4:00 pm each day. The negative control group was fed a basic liquid feed (2.4g + 2.6g water, 48% dilution) without adjuvants, while the other groups were fed the same amount of liquid feed containing 0.5mL of immune adjuvant solution.

[0075] On the morning of the 8th day at 9:00, allergy stimulation was performed. The control group was fed 5g of purified water, while the other groups were fed 5g of infant nutritional rice cereal liquid feed to stimulate allergy (the animals were fasted for 12 hours before feeding). Clinical allergic symptoms of the animals were observed within 30 minutes to 1 hour, and corresponding blood and tissue samples were collected.

[0076] Example 2:

[0077] In the embodiments of this application, the sensitization indicators detected include allergy clinical symptom scores, body temperature, diarrhea status, and specific antibodies IgG, IgE, mast cell protease (mMCP-1), trypsin (MCT), and histamine (HIS) in serum.

[0078] (1) Allergy clinical symptom score

[0079] According to Table 1, the clinical allergic symptoms of rats were evaluated within 30 min to 1 h after allergic provocation. The allergic reaction symptoms were recorded and observed, and continuous observation and real-time monitoring and scoring were performed.

[0080] Table 1 Clinical Symptom Scores

[0081]

[0082]

[0083] (2)Body temperature

[0084] Within 30 minutes to 1 hour after allergic stimulation, the rectal temperature of rats was measured multiple times using a rectal thermometer to observe the changes in rat body temperature.

[0085] (3) Diarrhea

[0086] Within 30 minutes to 1 hour after allergic stimulation, the occurrence of diarrhea in rats was observed, and the proportion of diarrhea in each group of mice was calculated.

[0087] (4) Determination of specific antibodies (IgG, IgE) in serum

[0088] The obtained serum was analyzed according to the ELISA kit instructions, as follows:

[0089] 1) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Seal the remaining strips in a resealable bag and return them to 4°C. Set up standard and sample wells. Add 50 μL of different concentrations of standard to each standard well. Then add 50 μL of the test sample to each sample well; do not add any to the blank wells. If sample dilution is required, dilute the sample with the sample diluent provided with the kit as instructed.

[0090] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add to the standard wells or blank wells.

[0091] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). Let stand for 1 minute, then shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0092] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min.

[0093] 5) Discard the liquid and pat dry on absorbent paper. Fill each well with washing solution (350 μL), let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0094] 6) Add 50 μL of substrate A (chromogenic agent A: H2O2) and 50 μL of chromogenic agent B (TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Within 15 min, use a microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0095] (5) Mast cell protease (mMCP-1)

[0096] The obtained serum was analyzed according to the ELISA kit instructions, as follows:

[0097] 1) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Seal the remaining strips in a resealable bag and return them to 4°C. Set up standard and sample wells. Add 50 μL of different concentrations of standard to each standard well. Then add 50 μL of the test sample to each sample well; do not add any to the blank wells. If sample dilution is required, dilute the sample with the sample diluent provided with the kit as instructed.

[0098] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add to the standard wells or blank wells.

[0099] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). Let stand for 1 minute, then shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0100] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min.

[0101] 5) Discard the liquid and pat dry on absorbent paper. Fill each well with washing solution (350 μL), let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0102] 6) Add 50 μL of substrate A (chromogenic agent A: H2O2) and 50 μL of chromogenic agent B (TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Within 15 min, use a microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0103] (6) Trypsin-like enzymes (MCT)

[0104] The obtained serum was analyzed according to the ELISA kit instructions, as follows:

[0105] 1) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Seal the remaining strips in a resealable bag and return them to 4°C. Set up standard and sample wells. Add 50 μL of different concentrations of standard to each standard well. Then add 50 μL of the test sample to each sample well; do not add any to the blank wells. If sample dilution is required, dilute the sample with the sample diluent provided with the kit as instructed.

[0106] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add to the standard wells or blank wells.

[0107] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). Let stand for 1 minute, then shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0108] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min.

[0109] 5) Discard the liquid and pat dry on absorbent paper. Fill each well with washing solution (350 μL), let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0110] 6) Add 50 μL of substrate A (chromogenic agent A: H2O2) and 50 μL of chromogenic agent B (TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Within 15 min, use a microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0111] (7) Histamine (HIS)

[0112] The obtained plasma was analyzed according to the ELISA kit instructions, as follows:

[0113] 1) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 minutes. Seal the remaining strips in a resealable bag and return them to 4°C. Set up standard and sample wells. Add 50 μL of different concentrations of standard to each standard well. Then add 50 μL of the test sample to each sample well; do not add any to the blank wells. If sample dilution is required, dilute the sample with the sample diluent provided with the kit as instructed.

[0114] 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min. Do not add to the standard wells or blank wells.

[0115] 3) After discarding the liquid, pat dry with absorbent paper and fill each well with washing solution (350μL). Let stand for 1 minute, then shake off the washing solution and pat dry with absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0116] 4) Except for the blank wells, add 100 μL of horseradish peroxidase (HRP) labeled detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min.

[0117] 5) Discard the liquid and pat dry on absorbent paper. Fill each well with washing solution (350 μL), let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times (or use a plate washer).

[0118] 6) Add 50 μL of substrate A (chromogenic agent A: H2O2) and 50 μL of chromogenic agent B (TMB) to each well, incubate at 37°C in the dark for 15 min, then add 50 μL of stop solution to each well. Within 15 min, use a microplate reader to measure the OD value of each well at a wavelength of 450 nm.

[0119] (8) Data Processing

[0120] All experimental results are expressed as mean ± standard deviation. One-way ANOVA using SPSS was used for significance analysis, and a p < 0.05 was considered statistically significant.

[0121] Example 3:

[0122] Based on the animal model construction method in Example 1 and the experimental method in Example 2, the specific experimental results and analysis are as follows:

[0123] 1. Clinical symptom score

[0124] When the body is stimulated by a certain allergen, a series of clinical allergic reactions will occur. The experiment scored the clinical symptoms exhibited by Wistar rats after induced allergy, and the results are as follows: Figure 1 As shown.

[0125] After the rats were challenged, according to Figure 1 It was found that, except for the control group, rats in all other groups exhibited varying degrees of clinical symptoms. Figure 1 As shown, no signs of allergic symptoms were observed in the blank control group, while some rats in the lipopolysaccharide group showed more severe allergic symptoms, including one rat exhibiting respiratory distress. In addition, rats in the other groups scratched their noses, rubbed their heads, and even showed swelling around their eyes and mouths, as well as increased respiratory rate. Compared with the blank control group and the other groups, the allergic symptom scores of the rats in the lipopolysaccharide group were significantly higher.

[0126] The results showed that lipopolysaccharide (LPS) induced allergy in rats, resulting in the highest allergy symptom scores and triggering a significant allergic reaction. The rat sensitization model constructed by the method described in this paper was successful.

[0127] 2. Rat body temperature index

[0128] The experiment statistically analyzed the body temperature of Wistar rats after allergic stimulation, and the results are as follows: Figure 2 As shown.

[0129] Studies have shown that when rats exhibit clinical allergic symptoms, their body temperature drops rapidly and their activity level decreases significantly.

[0130] Depend on Figure 2 It can be seen that the body temperature of the control group was normal, with an average body temperature of 38.08℃. Except for the control group, the body temperatures of all other groups decreased, at 36.98℃, 37.40℃, 37.23℃, 37.37℃, 37.43℃, 37.50℃, 37.25℃, 37.47℃, 37.70℃, and 37.70℃, respectively. Compared with the control group and the other groups, the lipopolysaccharide group showed the most severe trend of body temperature decrease, and the difference was highly significant compared with the control group (P < 0.01).

[0131] The results showed that when lipopolysaccharide was used as an immune adjuvant to induce allergy in rats, the rats exhibited the most significant decrease in body temperature and the lowest average body temperature, which triggered a significant allergic reaction. The rat sensitization model constructed by the method described in this paper was successful.

[0132] 3. Diarrhea in rats

[0133] The experiment statistically analyzed the diarrhea of ​​Wistar rats after allergic provocation, and the results are as follows: Figure 3 As shown.

[0134] like Figure 3 As shown, no diarrhea symptoms were observed in the control group, while all other groups experienced diarrhea to varying degrees. Compared with the control group, the diarrhea rates in the other groups were 66.6%, 33.3%, 33.3%, 33.3%, 33.3%, 50.0%, 33.3% and 33.3%, 50%, and 33.3%, respectively. Compared with the control group and the other groups, the lipopolysaccharide group showed the most severe diarrhea, with a highly significant difference compared to the control group (P < 0.01). These results indicate that using lipopolysaccharide as an immune adjuvant to induce allergy in rats resulted in the highest rate of diarrhea, triggering a significant allergic reaction. The rat sensitization model constructed using the method described in this paper was successful.

[0135] 4. Analysis of specific antibodies in rat serum

[0136] The experiment analyzed the specific antibodies IgG and IgE in rat serum, and the results are as follows: Figure 4-5 As shown.

[0137] The levels of IgG and IgE in the body are important indicators for assessing food allergies. Allergies are mainly type I hypersensitivity reactions mediated by IgE, and IgE-mediated allergic reactions inevitably lead to an increase in the expression levels of IgE and IgG.

[0138] The experiment analyzed the specific antibodies IgG and IgE in the serum of Wistar rats, and the results are as follows: Figure 4-5 As shown in the figure. The results showed that the serum IgG and IgE levels in the blank group were very low, with almost no change, indicating that no allergic reaction occurred in the blank group mice. After feeding mice with different adjuvants, the serum IgG and IgE levels in rats increased to varying degrees. Compared with the blank group and the other groups, the serum IgG and IgE levels in the lipopolysaccharide group were significantly increased, showing a highly significant difference from the blank group (P < 0.01). This indicates that the rat's immune system played a role, leading to sensitization under the induction of allergens and the production of IgE and IgG antibodies that mediate the allergic reaction, resulting in elevated serum IgG and IgE levels. The results indicate that using lipopolysaccharide as an immune adjuvant to induce allergy in rats resulted in the highest serum IgG and IgE levels, triggering a significant allergic reaction. The rat sensitization model constructed by the method described in this paper was successful.

[0139] 5. Analysis of rat serum mast cell protease (mMCP-1)

[0140] The experiment analyzed rat serum mMCP-1, and the results were as follows: Figure 6 As shown.

[0141] mMCP-1 is an important inflammatory mediator released during the degranulation effect of activated basophils or mast cells, and it can serve as an important indicator of the strength of mast cell degranulation. Figure 6 It was found that the serum mMCP-1 level in the blank group was very low, showing almost no change, indicating that no allergic reaction occurred in the mice in the blank group. After feeding mice with different adjuvants, the serum mMCP-1 level in rats increased to varying degrees. Compared with the blank group and the other groups, the serum mMCP-1 level in the lipopolysaccharide group was significantly increased, and the difference was extremely significant compared with the blank group (P < 0.01). The results indicate that lipopolysaccharide, as an immune adjuvant, induces allergy in rats, resulting in the highest serum IgG and IgE levels, which triggered a significant allergic reaction. The rat sensitization model constructed by the method described in this paper was successful.

[0142] 6. Rat serum trypsin-like protein (MCT) analysis

[0143] The serum trypsin-like protein (MCT) of Wistar rats was analyzed in the experiment, and the results are shown in Table 7.

[0144] When the body is stimulated by a certain allergen, a series of clinical allergic reactions will occur. Figure 7 It was found that feeding rats with different adjuvants increased serum MCT levels to varying degrees. Compared with the control group and the other groups, the serum mMCP-1 level in the lipopolysaccharide group was significantly increased, showing a highly significant difference compared with the control group (P < 0.01). The results indicate that lipopolysaccharide, as an immune adjuvant, induces allergy in rats, resulting in the highest serum IgG and IgE levels, triggering a significant allergic reaction. The rat sensitization model constructed using the method described in this paper was successful.

[0145] 7. Histamine (HIS) analysis in rat plasma

[0146] The experiment analyzed HIS in the plasma of Wistar rats, and the results are as follows: Figure 8 As shown.

[0147] Histamine is widely present in body tissues. When body tissues are damaged or allergic reactions occur, histamine is released, which in turn leads to increased capillary permeability and even local edema.

[0148] Depend on Figure 8 It was found that feeding rats with different adjuvants increased the plasma HIS content to varying degrees. Compared with the control group and the other groups, the plasma HIS content of rats in the lipopolysaccharide group was significantly increased, and the difference from the control group was extremely significant (P < 0.01). The results indicate that lipopolysaccharide, as an immune adjuvant, induces allergy in rats, resulting in the highest plasma HIS content and triggering a significant allergic reaction. The rat sensitization model constructed by the method described in this paper was successful.

[0149] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for constructing an animal model of infantile rice cereal food allergy, characterized in that: It includes the following steps: Step 1: After the experimental animals were acclimatized, the experiment was conducted. The animals were randomly divided into several groups according to their weight, and they had free access to water and food during the experiment. Step 2: From day 1 to day 7, each group of experimental animals was tested with different test adjuvants. One group of experimental animals received a negative control without adjuvants, and the remaining groups were further divided into subgroups according to different test substances. During the experiment, each animal was fed a fixed amount of 5 g at 9:00 am and 4:00 pm each day. The negative control group was fed a basic liquid feed without adjuvants, which consisted of 2.4 g + 2.6 g of water with a consistency of 48%. The other groups were fed the same amount of liquid feed containing 0.5 mL of immune adjuvant solution. Step 3: At 9:00 AM on the 8th day, allergy stimulation was performed. The control group was fed 5 g of purified water, while the other groups were fed 5 g of infant nutritional rice cereal liquid feed. Allergy stimulation was performed. The animals were fasted for 12 hours before feeding. Clinical allergic symptoms of the animals were observed within 30 minutes to 1 hour, and corresponding blood and tissue samples were collected. Then, the animals were euthanized, and blood samples were collected. The experimental animals were SPF-grade 3-week-old female Wistar rats weighing 45 g to 65 g. The animals were randomly divided into 11 groups according to their body weight, with 6 animals in each group. One group received lipopolysaccharide as the test substance orally via gavage. The other groups received the test substances freely, which included physiological saline, lipopolysaccharide, β-glucan, chitosan, wolfberry polysaccharide, astragalus polysaccharide, mannan oligosaccharide, mannose, ginseng stem and leaf saponins, and zinc hydroxide. The basic liquid feed formula is as follows: casein, L-cysteine, cellulose, xanthan gum, maltodextrin, sucrose, soybean oil, mixed minerals, mixed vitamins, and choline tartrate. The dosage of the test substance and the preparation method of the basal liquid feed are as follows: 1) Preparation of immune adjuvants All immunoadjuvant solutions were prepared fresh for use, with the following specific concentrations: lipopolysaccharide 0.10 mg / mL; β-glucan, chitosan, wolfberry polysaccharide, and astragalus polysaccharide 0.244 g / mL; mannan oligosaccharide, mannose, and riboflavin 10.0 mg / mL; ginseng stem and leaf saponins 3.0 mg / mL; and zinc hydroxide 2.0 mg / mL. 2) Basic liquid feed preparation Each mouse was fed a fixed amount of 5 g of food each time. The control group was fed 2.4 g of basic liquid diet + 2.6 g of water. The preparation method was as follows: accurately weigh 2.4 g of liquid diet, mix it evenly with 2.6 g of water, and stir to make a basic liquid diet with a consistency of 48%. The other groups were fed the same amount of liquid diet containing 0.5 mL of immune adjuvant solution.

2. The method for constructing an animal model of infantile rice cereal food allergy according to claim 1, characterized in that: Step 3, the blood collection and testing procedure, is as follows: Each rat is anesthetized by intraperitoneal injection of 10% chloral hydrate solution at a dose of 0.3 g / 100 g of body weight. Then, the abdominal skin is disinfected with alcohol, and the abdominal skin and muscles are cut open with surgical scissors. Under sterile conditions, blood is collected from the rat's abdominal aorta into negative pressure blood collection tubes and EDTA anticoagulant tubes, respectively. After collection, the tubes are centrifuged at 3500 rpm / min for 15-20 min at 4 ℃. The supernatant from both tubes is placed in cryopreservation tubes, cold extracted with liquid nitrogen, and stored at -80℃ for the determination of serum and plasma specific antibodies and histamine.

3. The method for constructing an animal model of infantile rice cereal food allergy according to claim 2, characterized in that: The specific antibodies are specific IgG, specific IgE, mast cell protease mMCP-1, and trypsin MCT, and histamine is HIS.

4. A method for constructing an animal model of infantile rice cereal food allergy according to claim 2 or 3, characterized in that: The detection steps for the specific antibody are as follows: Collect the obtained serum and plasma, and perform the assay according to the method described in step 3, following the instructions of the ELISA kit: 1) Remove the required strips from the aluminum foil bag after equilibration at room temperature for 60 min. Seal the remaining strips in a self-sealing bag and store them at 4℃. Set up standard wells and sample wells. Add 50 μL of different concentrations of standard to each standard well, and then add 50 μL of the sample to be tested to the sample well. Do not add any to the blank wells. If the sample needs to be diluted, dilute the sample with the sample diluent provided with the kit as required. 2) Add 50 μL of biotin-labeled antibody to each well, seal the reaction wells with sealing film, and incubate in a 37°C water bath or incubator for 30 min. Do not add to the standard wells or blank wells. 3) Discard the liquid and pat dry on absorbent paper. Add 350 μL of washing solution to each well, let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times. 4) Except for the blank wells, add 100 μL of horseradish peroxidase-labeled detection antibody to each of the standard and sample wells, seal the reaction wells with sealing film, and incubate at 37°C in a water bath or incubator for 30 min. 5) Discard the liquid and pat dry on absorbent paper. Add 350 μL of washing solution to each well, let stand for 1 minute, then shake off the washing solution and pat dry on absorbent paper. Repeat this washing process 5 times. 6) Add 50 μL each of substrate chromogenic agent A (H2O2) and chromogenic agent B (TMB) to each well. Incubate at 37°C in the dark for 15 min. Then add 50 μL of stop solution to each well. Within 15 min, use an ELISA reader to measure the OD value of each well at a wavelength of 450 nm.

5. The method for constructing an animal model of infantile rice cereal food allergy according to claim 1, characterized in that: The preparation method of infant nutritional rice cereal liquid feed in step 3 is as follows: 5 g of infant nutritional rice cereal is mixed and stirred with 10 g of water.

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