Food-specific igg antibody detection kit and application thereof

Abstract

The application provides a food-specific IgG antibody detection kit and application thereof, 57 kinds of foods are grouped, any one of four groups of food antigens will produce cross reaction with the antibody corresponding to any other group of food antigens, any one kind of antigen in any one group of food antigens does not produce cross reaction with the antibody corresponding to any other kind of antigen, and thus cross reaction possibly existing in the case of large flux optimization experiment is avoided, so that the detection result is accurate; a blocking agent is added to completely eliminate cross interference; microspheres coupled with anti-human IgG2 antibody are added in the mixed solution of capture microspheres coupled with food antigens in each group for quality control, and the detection result of the quality control microspheres can be used to judge the effectiveness of the kit and the correctness of the experimental operation in real time. A blocking agent suitable for the kit system of the application is found, no false positive appears in 100 parallel tests; the calibration curve is obtained by testing the four combinations only once, and the corresponding content of 57 kinds of food-specific IgG antibodies is obtained; and the application has good application prospect.

Description

[0001] This application claims priority to the earlier Chinese application, application number 202310100961.9, filed on February 13, 2023; all its contents are part of this invention. Technical Field

[0002] This invention relates to the field of biological detection technology, specifically to a food-specific IgG antibody detection kit and its application. Background Technology

[0003] Abnormal physiological manifestations caused by the ingestion of certain foods and / or food additives are often referred to as adverse reactions to food. Food intolerance is an adverse reaction to food mediated by immunoglobulin G (IgG). Its pathogenesis is that when a patient ingests a certain type of food, the body's immune system recognizes small molecule food antigens that have not been completely broken down due to a lack of relevant digestive enzymes as harmful antigens, thereby producing corresponding food-specific IgG antibodies, triggering an excessive protective immune response in the body.

[0004] If a patient consumes intolerant foods for an extended period, the specific IgG antibodies against those foods will accumulate in the body. These antibodies can then form immune complexes with the antigens of the intolerant foods, becoming free or deposited in the corresponding organs and tissues, causing type III hypersensitivity reactions. This can lead to abnormal chronic symptoms in various systems throughout the body, clinically manifesting as abnormal symptoms in the gastrointestinal tract, skin, nervous system, respiratory system, and musculoskeletal system. Examples include irritable bowel syndrome, skin diseases, headaches, anxiety, depression, fatigue, eczema, asthma, chronic rhinitis, joint pain, allergic purpura, and autoimmune diseases. Simultaneously, the body's immune system, which defends against invading pathogens and maintains overall health, will also become abnormal.

[0005] Many common clinical conditions are closely related to food intolerance. Therefore, food intolerance symptoms lack specificity, making self-diagnosis difficult for patients. Recent clinical studies have further demonstrated that if a patient's digestive enzymes are not improved or their diet remains unchanged, the immune response associated with food intolerance will persist. By detecting the presence and level of specific IgG antibodies in the serum after a food enters the digestive system, we can not only determine the type and severity of the intolerant food but also provide new insights for clinical diagnosis and valuable clinical evidence for dietary adjustments.

[0006] Currently, commercially available methods for detecting food-specific IgG antibodies include enzyme-linked immunosorbent assay (ELISA), protein microarray, band ELISA, Western blotting, immunofluorescence microarray, and chemiluminescence immunoassay. Among these methods, ELISA is the most commonly used for detecting food-specific IgG antibodies, followed by band ELISA and protein microarray. Western blotting, immunofluorescence microarray, and chemiluminescence immunoassay are less common. ELISA and band ELISA require multiple manual steps, are cumbersome, and have low repeatability. Protein microarray is prone to non-specific binding during detection, resulting in low sensitivity.

[0007] Current detection technologies for food-specific IgG antibodies suffer from cumbersome manual procedures and poor repeatability, severely impacting sensitivity, positive / negative concordance rate, and precision. Most commercially available food-specific IgG antibody detection technologies are single-indicator assays, resulting in high costs, large sample consumption, and an inability to achieve high-throughput testing. Furthermore, existing methods can only diagnose immediate allergic reactions, offering poor accuracy for delayed-type allergic reactions; current technologies do not effectively differentiate between cross-reactivity with different food antigens, leading to frequent false positives or false negatives; and the results of existing methods may be affected by individual differences, food processing methods, and food formulations, lacking accuracy and reliability.

[0008] Therefore, it is necessary to provide a novel food-specific IgG antibody detection kit and its usage method to solve the above-mentioned problems existing in the prior art. Summary of the Invention

[0009] To address the problems of existing technologies, this invention provides a food-specific IgG antibody detection kit and its application. By identifying different signals from solid-phase carrier microspheres, it distinguishes different food antigens. The concentration of the sample is calculated using the detection signal of the fluorescent antibody. It can simultaneously detect different types of food-specific IgG antibodies, exhibiting high sensitivity and specificity. It eliminates the need to detect each food-specific IgG antibody individually, solving the problem of cumbersome operations and shortening the detection time. Furthermore, it avoids the situation where multiple food-specific IgG antibodies exist in the sample, making accurate identification impossible, thus saving costs and showing promising application prospects. During sample detection, the detection results of the quality control microspheres in the captured microsphere mixture can simultaneously and in real-time determine the effectiveness of the kit and the correctness of the experimental operation. Calibration curves only require one calibration, making it more convenient for customers.

[0010] Compared with the prior art, this technical solution mainly solves the following technical problems: (1) It provides a food-specific IgG antibody detection kit and its application, which can simultaneously detect 57 food antigens in groups; (2) By designing the cross-reaction and non-cross-reaction of four groups of food antigens and blocking the microspheres of specific food antigens, the problem of misjudgment caused by cross-reaction between food antigens is solved; (3) A quality control microsphere coupled with a specific IgG antibody subtype is used for quality control, and the correctness of the current detection operation is judged in real time, thereby improving the accuracy of the detection results; (4) When the number of food-specific IgG antibody items to be tested is large, a method that only requires calibration once is realized, which greatly improves the convenience of detection and avoids human error; (5) It provides a more comprehensive and accurate food-specific IgG antibody detection method, which can help people discover food allergy problems in a timely manner.

[0011] On one hand, the present invention provides a food-specific IgG antibody detection kit, comprising a microsphere capture mixture and a fluorescent detection antibody reagent. The microsphere capture mixture comprises microspheres conjugated with food antigens and control microspheres. The food antigens consist of a first group of food antigens, a second group of food antigens, a third group of food antigens, and a fourth group of food antigens, each group of food antigens containing one type of control microsphere. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens, and there is no cross-reaction between any antigen in any group of food antigens and the antibody corresponding to any other antigen. The first group of food antigens includes eggs, milk, soybeans, wheat, rice, cod, and shrimp. The food antigens in the first group include at least three antigens from the following: crab, tomato, mushroom, corn, chicken, pork, potato, and beef; the food antigens in the second group include at least three antigens from the following: sesame, peanut, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, lamb, cashew, sweet potato, chocolate, black tea, and honey; the food antigens in the third group include at least three antigens from the following: scallop, clam, oyster, lobster, salmon, sardine, ribbonfish, grass carp, tuna, trout, sole, and chum salmon; and the food antigens in the fourth group include at least three antigens from the following: mango, orange, pineapple, watermelon, olive, peach, durian, banana, cantaloupe, grape, grapefruit, strawberry, blueberry, apple, and lemon.

[0012] During the research process of this invention, it was discovered that a large number of food antigens exhibit cross-reactivity. Different food antigens may cross-react due to having the same or similar antigenic epitopes or due to the low specificity of antigen-antibody binding. Moreover, cross-reactivity may exist within the same food category, making simultaneous detection impossible. To solve this problem, this invention screened for cross-immunological reactions among 57 foods and divided them into 4 groups. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens. However, there is no cross-reaction between any antigen in any food antigen group and the antibody corresponding to any other antigen. This achieves the avoidance of cross-reactivity in high-throughput screening, resulting in accurate detection results.

[0013] Furthermore, a blocking agent, including PEG6000, is added to the mixture of beef, green beans, scallops, and peach antigen-conjugated microspheres.

[0014] The PEG6000 mentioned is polyethylene glycol 6000, and the chemical formula of polyethylene glycol is HO(CH2CH2O). n H and n are called the degree of polymerization. Polyethylene glycol is a mixture of molecules with different degrees of polymerization. 6000 refers to the average molecular weight.

[0015] When any one or more of the beef, green bean, scallop, and peach antigens are selected and added to the corresponding food group for testing, the blocking agent PEG6000 is added to the microsphere mixture of the corresponding group to eliminate cross-reaction. It is worth noting that, unlike the above four food antigen groups, even with the addition of the blocking agent PEG6000, the cross-reaction between the above food antigen groups cannot be completely eliminated. Therefore, it is necessary to divide them into four groups for testing, and the weak cross-reaction within the group can be eliminated by using the blocking agent PEG6000.

[0016] Furthermore, the quality control microspheres for the first group of food antigens, the second group of food antigens, the third group of food antigens, and the fourth group of food antigens are microspheres conjugated with anti-human IgG2 antibodies.

[0017] In the testing process, this invention discovered the use of anti-human IgG antibody microspheres (containing four subtypes) as quality control microspheres. IgG antibodies are classified into four subtypes: IgG1, IgG2, IgG3, and IgG4, with the content of these four subtypes decreasing sequentially in the human body. The four sets of quality control microspheres were added to four different food combinations, and the most suitable anti-human IgG antibody subtype microspheres were obtained by testing the samples from each of the four food combinations. This invention experimentally determined that the quality control microspheres for the first, second, third, and fourth food antigen groups were conjugated with anti-human IgG2 antibodies.

[0018] Many kits include separate quality control components, which increases costs and extends testing time. This invention mixes quality control microspheres directly with food antigen-conjugated microspheres as a capture microsphere solution. On the one hand, quality control is tested simultaneously with the sample, simplifying the operation. On the other hand, simultaneous testing reduces errors, ensuring that the quality control accurately reflects the test results, further improving accuracy and reliability.

[0019] Furthermore, the microsphere capture mixture also includes an inhibitor, which includes inactivated rabbit IgG and HAMA inhibitors.

[0020] A large number of non-specific bindings still exist in the detection. This invention screened a variety of blocking agents and finally found a specific blocking agent suitable for the reagent kit system of this invention, including rabbit IgG and HAMA blocking agents. The two can play a synergistic role to enhance the blocking effect. No false positives occurred in 100 parallel tests. Other blocking agents can not achieve this effect except for this combination.

[0021] Furthermore, the kit also includes a washing buffer, a sample dilution buffer, a calibration microsphere mixture, a calibration-specific fluorescent detection antibody, and a calibrator. The calibration microsphere mixture is a mixture of anti-human IgG3 antibody microspheres at different levels, the calibration-specific fluorescent detection antibody is an anti-human IgG antibody labeled with phycoerythrin, and the calibrator is a human IgG antibody.

[0022] On the other hand, the present invention provides a quantitative method for the food-specific IgG antibody detection kit, which involves calibration once, obtaining the MFI value of each food-specific IgG antibody by testing serum, and then obtaining the concentration of each food-specific IgG antibody by regression through calibration curve.

[0023] The MFI value is the average fluorescence intensity value automatically read by the instrument. Substituting this value into the calibration curve yields the corresponding food-specific IgG antibody concentration. This invention reduces the number of calibration cycles and calibration points. When plotting the calibration curve, different concentrations of calibrators (human IgG antibodies) are sequentially subjected to immune reactions with the calibration microsphere mixture and calibration-specific fluorescent detection antibody in the food-specific IgG antibody detection kit. Fluorescence type and intensity are detected using flow cytometry. The calibration curve, plotted based on the fluorescence signal values ​​corresponding to a series of calibrator concentrations, requires only one calibration.

[0024] In conventional methods, calibration curves are often plotted separately for 57 different foods, requiring 57 calibrations. Alternatively, simultaneous calibration within the same group without overlap requires 4 calibrations. However, the quantitative method provided by this invention requires only 1 calibration, thus greatly reducing the workload in the testing process while still obtaining accurate results.

[0025] The reason why quantification can be achieved with only one calibration is that the grouping and blocking methods avoid cross-interference between food-specific IgG antibody detections, the quality control microspheres improve the accuracy and reliability of the results, and the blocking agent eliminates false positives. As a result, the MFI readings of the 57 food-specific IgG antibody detections show similar linearity and similar ranges. Without any of the above technical means, the MFI reading ranges of the 57 food-specific IgG antibody detections would not be uniform and would have poor linearity. Each detection would require individual calibration, the plotting of calibration curves, and quantification, making it impossible to perform quantification with only one calibration.

[0026] Among these factors, the choice of blocking agent has the greatest impact on the plotting of calibration curves.

[0027] In principle, although rabbit IgG and HAMA blocking agents are called blocking agents, their actual role here is to eliminate errors between calibration curves. Calibration curves are affected by various factors such as antigen and antibody types and microsphere levels. Rabbit IgG and HAMA blocking agents can eliminate microscopic adsorption and other interactions between microspheres, antigens, and antibodies. The possible mechanism is preferential binding to interfering antibodies and antigens unrelated to detection, as well as eliminating electrostatic interactions. The combination of rabbit IgG and HAMA blocking agents can have a synergistic effect, allowing different types of antigens, antibodies, and microspheres to be calibrated simultaneously without interference.

[0028] The aforementioned blocking agents (rabbit IgG and HAMA blocking agents) enable the present invention to draw calibration curves for each microsphere level with only one calibration, while reducing human intervention while ensuring accuracy, facilitating detection and reducing human error.

[0029] On the other hand, the present invention provides a method for grouping food antigens to avoid cross-reactions in the detection of food-specific IgG. The food antigens consist of a first group of food antigens, a second group of food antigens, a third group of food antigens, and a fourth group of food antigens. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens, but there is no cross-reaction between any antigen within any group of food antigens and the antibody corresponding to any other antigen. The first group of food antigens includes eggs, milk, soybeans, wheat, rice, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, pork, and potatoes. The first group of food antigens includes at least three antigens from beef; the second group of food antigens includes at least three antigens from sesame, peanut, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, mutton, cashews, sweet potatoes, chocolate, black tea, and honey; the third group of food antigens includes at least three antigens from scallop, clam, oyster, lobster, salmon, sardine, ribbonfish, grass carp, tuna, trout, sole, and salmon; and the fourth group of food antigens includes at least three antigens from mango, orange, pineapple, watermelon, olive, peach, durian, banana, cantaloupe, grape, grapefruit, strawberry, blueberry, apple, and lemon.

[0030] Furthermore, a blocking agent, including PEG6000, is added to the mixture of beef, green beans, scallops, and peach antigen-conjugated microspheres.

[0031] On the other hand, the present invention provides an application of anti-human IgG antibody subtype quality control microspheres to improve the accuracy and reliability of test results. The anti-human IgG antibody subtype quality control microspheres are microspheres conjugated with anti-human IgG2 antibodies. When the reagents are used correctly, the test results of the quality control microspheres will definitely show positive.

[0032] On the other hand, the present invention provides the use of an inhibitor for preparing a reagent to reduce the false positive rate of food-specific IgG detection, said reagent comprising inactivated rabbit IgG and HAMA inhibitor.

[0033] On the other hand, the present invention provides the use of an inhibitor for reducing the number of calibrations required to plot a calibration curve for the detection of food-specific IgG, the reagent comprising inactivated rabbit IgG and HAMA inhibitor.

[0034] The beneficial effects achieved by this invention are as follows:

[0035] 1. This invention provides a food-specific IgG antibody detection kit and its application, which can simultaneously detect different kinds of food-specific IgG antibodies, and has high detection sensitivity and strong specificity;

[0036] 2. This invention groups 57 food IgG antigens. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens. However, there is no cross-reaction between any antigen in any group of food antigens and the antibody corresponding to any other antigen. This achieves the avoidance of cross-reaction in high-throughput screening and makes the test results accurate.

[0037] 3. The PEG6000 blocking agent used in this invention can further block non-specific sites that cannot be completely blocked by protein blocking agents, thereby improving the blocking effect of microspheres, reducing intra-group cross-reactions, and improving the positive and negative consistency rate of reagents;

[0038] 4. The quality control microspheres are the microspheres conjugated with the anti-human IgG2 antibody. Quality control is tested simultaneously with the sample testing, simplifying the operation.

[0039] 5. This invention screened a variety of blocking agents before finding a specific blocking agent suitable for the reagent kit system of this invention, including rabbit IgG and HAMA blocking agents. The two can play a synergistic role in enhancing the blocking effect. No false positives were found in 100 parallel experiments.

[0040] 6. The rabbit IgG and HAMA blocking agents of the present invention can work synergistically to reduce the absolute deviation of calibration curve measurements between different foods, so that calibration curves can be plotted with only one calibration, reducing the number of calibrations while ensuring accuracy, facilitating detection and reducing human error.

[0041] 7. The present invention only requires one calibration to plot the calibration curve, and can calculate the concentration of food-specific IgG antibodies in serum corresponding to 57 kinds of food combinations in 4 combinations. The calibration curves for different food combinations are plotted with one calibration, which means that the calibration steps are simplified and the reagent kit components are reduced, and the probability of error will be further reduced.

[0042] 8. The kit provided by this invention involves only one incubation and one washing operation, and the samples can be used directly without dilution. It is simple to operate, quick, and produces good repeatability with minimal batch-to-batch variation. It achieves accurate detection of both venous blood and finger-prick blood samples, and has promising application prospects. Attached Figure Description

[0043] Figure 1 This is a schematic diagram illustrating the detection principle of the microspheres conjugated with food antigens in the kit of this invention.

[0044] Figure 2 This is a schematic diagram illustrating the detection principle of the quality control microspheres in the kit of this invention.

[0045] Figure 3 The distribution of fluorescent microspheres in an embodiment of the present invention;

[0046] Figure 4 This is a calibration curve of food-specific IgG antibodies in an embodiment of the present invention. Detailed Implementation

[0047] To describe the present invention more specifically, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are merely illustrative of how the present invention is implemented and do not limit the specific scope of the present invention. The scope of the present invention is defined in the claims. The described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention. Unless otherwise defined, the technical or scientific terms used herein should have the ordinary meaning understood by those skilled in the art to which this invention pertains. The terms "comprising" and similar expressions used herein mean that the element or object preceding the word covers the element or object listed after the word and its equivalents, but does not exclude other elements or objects.

[0048] Figure 1 This is a schematic diagram illustrating the detection principle of the food antigen-conjugated microspheres in the kit of this invention. (Refer to...) Figure 1 Microspheres coated with food antigen 2, specific IgG antibodies from the sample, phycoerythrin 5, and anti-human IgG antibodies 4 form an immune complex. Two beams of excitation light of different wavelengths emitted by the flow cytometer irradiate the immune complex. The fluorescence intensity of the microspheres coupled with different antigens determines the type of detection index, and the fluorescence intensity of phycoerythrin determines the content of each detection index.

[0049] Figure 2 This is a schematic diagram illustrating the detection principle of the quality control microspheres in the kit of this invention. (Refer to...) Figure 2 Anti-human IgG2 antibody 6 is immobilized on microspheres 1 to form quality control microspheres. The conjugated phycoerythrin 5 and anti-human IgG antibody 4 form an immune complex. During the reaction, the anti-human IgG2 antibody 6 immobilized on the microspheres 1 first specifically binds to IgG2 antibody 7 in the sample to be tested, and then specifically binds to the complex formed by the conjugated phycoerythrin 5 and anti-human IgG antibody 4. Under the illumination of two beams of excitation light of different wavelengths emitted by the flow cytometer, the fluorescence intensity of the antibody-conjugated microspheres determines the type of detection index, and the fluorescence intensity of the phycoerythrin determines the content of the detection index.

[0050] Example 1: Reagent kit and detection method

[0051] The specific components of the food-specific IgG antibody detection kit are shown in Table 1. The kit contains 100 samples per kit. The kit may or may not include calibration microsphere mixture, calibration-specific fluorescent detection antibody, calibrator, and sample diluent.

[0052] Table 1: Specific components of the food-specific IgG antibody detection kit

[0053]

[0054]

[0055] The manufacturers and product numbers of the raw materials used in the reagent kit of this invention are shown in Table 2.

[0056] Table 2: Raw material manufacturers and product numbers

[0057]

[0058]

[0059] (1) Preparation of microsphere-capturing mixture

[0060] The microsphere capture mixture includes microspheres conjugated with food antigens and microspheres conjugated with anti-human IgG2 antibodies.

[0061] Table 1 shows that the food-specific IgG antibody detection kit contains different polystyrene microspheres in the microsphere capture mixture labeled with anti-human IgG2 antibodies and 57 food antigens, including eggs, milk, soybeans, wheat, rice, potatoes, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, beef, pork, lamb, cashews, sesame, black tea, peanuts, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, chocolate, sweet potatoes, honey, scallops, clams, oysters, lobsters, salmon, sardines, ribbonfish, grass carp, tuna, trout, sole, salmon, mango, oranges, pineapples, watermelons, olives, peaches, durians, bananas, cantaloupes, grapes, grapefruits, strawberries, blueberries, apples, and lemons. The 57 food antigens mentioned in this invention, including eggs, milk, soybeans, wheat, rice, potatoes, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, beef, pork, mutton, cashews, sesame seeds, black tea, peanuts, tobacco, coffee, cottage cheese, cane sugar, green beans, spinach, oats, chocolate, sweet potatoes, honey, scallops, clams, oysters, lobsters, salmon, sardines, ribbonfish, grass carp, tuna, trout, sole, salmon, mangoes, oranges, pineapples, watermelons, olives, peaches, durians, bananas, cantaloupes, grapes, grapefruits, strawberries, blueberries, apples, and lemons, are all natural extracts and are sources of food intolerances.

[0062] To avoid cross-infection of immune antigens, this invention grouped 57 food antigens into four groups: Group 1 includes at least three antigens from the following: eggs, milk, soybeans, wheat, rice, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, pork, potatoes, and beef; Group 2 includes at least three antigens from the following: sesame, peanuts, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, mutton, cashews, sweet potatoes, chocolate, black tea, and honey; Group 3 includes at least three antigens from the following: scallops, clams, oysters, lobsters, salmon, sardines, ribbonfish, grass carp, tuna, trout, sole, and chum salmon; and Group 4 includes at least three antigens from the following: mango, oranges, pineapples, watermelons, olives, peaches, durians, bananas, cantaloupes, grapes, grapefruits, strawberries, blueberries, apples, and lemons.

[0063] 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxythiosuccinimide (NHS) were used as activators to activate the carboxyl groups on the surface of fluorescent microspheres, which then covalently bonded to the amino groups on the food antigen and the anti-human IgG2 antibody, thus forming a food antigen-conjugated microsphere and anti-human IgG2 antibody microsphere complex. The food antigen-conjugated microsphere and anti-human IgG2 antibody microsphere complex were mixed to obtain a capture microsphere mixture with a microsphere concentration of 0.34 × 10⁻⁶. 6 per mL.

[0064] Table 3 shows the labeling ratios of 57 food antigens on different microsphere surfaces.

[0065] Table 3: Labeling ratios of 57 food antigens on different microsphere surfaces

[0066]

[0067]

[0068] In this embodiment, the microspheres are carboxyl-modified polystyrene microspheres of different sizes, and each polystyrene microsphere contains a different amount of allophycocyanin (APC). Figure 3 This shows the distribution of fluorescent microspheres in an embodiment of the present invention. (Refer to...) Figure 3Different types of polystyrene microspheres can be distinguished by the fluorescence signal values ​​of the allophycocyanin channel (APC-A) and the allophycocyanin-conjugated A750 dye channel (APC-A750-A) on a Beckman DxFLEX flow cytometer. Here, A refers to the area under the curve of the wavy signal generated when the laser passes through it, representing the signal intensity; A750 refers to the dye name. In the food antigen-conjugated microsphere and anti-human IgG2 antibody microsphere complex described in this embodiment, the polystyrene microspheres can be divided into 16 different parameter microspheres, corresponding to the detection results of the APC-A and APC-A750-A channels of the fluorescent microspheres at levels L1 to L16. The fluorescence intensity of different levels of microspheres, i.e., the relationship between microsphere level, APC-A, and APC-A750-A, is shown in Table 4.

[0069] Table 4: Fluorescence intensity of microspheres at different levels

[0070] Microsphere level APC-A strength APC-A750-A Strength L1 13566.40 453.00 L2 37080.00 1167.80 L3 78013.50 2472.70 L4 163501.09 5351.90 L5 733456.31 25522.50 L6 1221935.00 43964.30 L7 2740964.50 117061.60 L8 4258213.50 236981.20 L9 314528.09 691.30 L10 1414405.88 3017.30 L11 4240300.50 9352.10 L12 102172.50 1057.60 L13 241791.09 2438.30 L14 591517.38 6179.60 L15 1308926.75 13942.80 L16 12229655.00 812205.38

[0071] Beef, green beans, scallops, and peach antigens were grouped into different groups, but within each group, there was a weak cross-reaction with other foods, affecting the accuracy of the detection. This invention solves this problem by adding a blocking agent: after microsphere labeling, blocking is required. Under normal circumstances, the main active ingredient in the blocking solution is bovine serum albumin (BSA). This invention adds the macromolecular substance PEG6000 as a blocking agent to the captured microsphere solution, thus solving the cross-reaction phenomenon.

[0072] The capture microsphere solution also includes blocking agents to block non-specific binding during the detection process. The blocking agents include 50 μg / mL inactivated rabbit IgG (Sigma) and 50 μg / mL HAMA blocking agent (Roche).

[0073] (2) Preparation of fluorescent detection antibody reagent

[0074] The fluorescent detection antibody reagent includes anti-human IgG antibodies conjugated with phycoerythrin.

[0075] Preparation of the fluorescent detection antibody reagent: Anti-human IgG antibody was conjugated with phycoerythrin to obtain phycoerythrin-conjugated antibody. The ratio of 1 mg of anti-human IgG antibody to 5 mg of phycoerythrin was controlled. The phycoerythrin-conjugated antibody was then diluted 50-fold with sample diluent to obtain the fluorescent detection antibody reagent. The concentration of the fluorescent detection antibody reagent (the concentration of anti-human IgG antibody conjugated with phycoerythrin) was controlled at 4 μg / mL. The anti-human IgG antibody was purchased from Medion Biotechnology Co., Ltd.

[0076] (3) Preparation of washing buffer (1×)

[0077] Dissolve 2.4 g potassium dihydrogen phosphate (KH₂PO₄), 36.32 g disodium hydrogen phosphate dodecahydrate (Na₂HPO₄·12H₂O), 8 g NaCl, and 2 g potassium chloride (KCl) in 1000 mL of pure water. Add 25 g BSA, 1% ProClin 300 preservative, and 0.5% Tween-20 to obtain a washing buffer (10×). Alternatively, stabilize the washing buffer (10×) to room temperature until all salts dissolve, then add 10 mL of the washing buffer (10×) to 90 mL of pure water to obtain a washing buffer (1×).

[0078] (4) Preparation of sample dilution solution

[0079] Dissolve 3.0275g of tris(hydroxymethyl)aminomethane (Tris) and 9.0g of sodium chloride (NaCl) in 800mL of pure water, add 1mL of Krovin 100 preservative, adjust the pH to 7.4, and after complete dissolution, add 20g of bovine serum albumin (BSA). After complete dissolution, bring the volume to 1000mL and adjust the pH to 7.4 for later use.

[0080] (5) Preparation of calibration microsphere mixture, calibration-specific fluorescent detection antibody, and calibrators.

[0081] The suspension of the calibration microsphere mixture contains 16 identifiable polystyrene microspheres, each containing a different amount of allophycocyanin (APC). By analyzing APC-A and APC-A750-A, the polystyrene microspheres can be classified into 16 microspheres with different parameters, corresponding to L1 to L16 levels.

[0082] The calibrator has 5 concentration points. The human IgG3 antibody is diluted to different concentrations (0U / mL, 50U / mL, 100U / mL, 200U / mL, 400U / mL) using sample dilution buffer to establish a calibration curve for detection.

[0083] The preparation of the calibration microsphere mixture and the calibration-specific fluorescent detection antibody is the same as the preparation of the capture microsphere mixture and the fluorescent detection antibody reagent.

[0084] (6) How to use the kit

[0085] The specific usage method of the food-specific IgG antibody detection kit is as follows:

[0086] Add 40 μL of the captured microsphere mixture to the sample tube and 40 μL of the calibration microsphere mixture to the calibration tube;

[0087] Add 10 μL of the sample to be tested to the sample tube, and add 10 μL of calibrator to the calibration tube;

[0088] Add 40 μL of the fluorescent detection antibody reagent to the sample tube, mix well, and incubate at room temperature in the dark for 0.5 h;

[0089] Add 40 μL of the calibration-specific fluorescent detection antibody to each calibration tube, mix well, and incubate at room temperature in the dark for 0.5 h.

[0090] Add 1000 μL of washing buffer (1×) to the sample tube and calibration tube respectively, centrifuge at 400 g for 4 min, and discard the supernatant;

[0091] Add 150μL-300μL of washing buffer (1×) to the sample tube and calibration tube respectively, mix well, and detect the fluorescence type and fluorescence signal intensity on a flow cytometer. The specific detection process is a routine technique for those skilled in the art and will not be described in detail here.

[0092] The washing buffer is prepared by diluting the washing buffer (10×) with pure water at a ratio of 1:9 (1 part washing buffer (10×) + 9 parts pure water) to prepare the washing buffer (1×) for later use.

[0093] The food-specific IgG antibody detection kit of the present invention requires a small sample volume, with only 10 μL of serum sample needed, and no sample dilution is required, making the operation simple.

[0094] (7) Calibration curve

[0095] The principle of calibration curve plotting: Within the same kit combination, calibration microsphere mixtures are mixed with a series of calibrators at different concentrations and incubated at room temperature in the dark to form a "calibration microsphere-calibrator" complex. Then, an optimized dose of calibration-specific fluorescent detection antibody reagent is added, and the mixture is incubated at room temperature in the dark to form a "calibration microsphere-calibrator-fluorescent detection antibody" complex. Finally, flow cytometry is used to detect the fluorescence type and intensity of this complex, and calibration curves are plotted based on the fluorescence signals corresponding to a series of calibrator concentrations. In this embodiment, the relative concentrations of food-specific IgG antibodies in serum corresponding to different food antigens are calculated using regression analysis of the calibration curves.

[0096] Plotting calibration curves: Add 40 μL of the calibration microsphere mixture to each of the five calibration microsphere tubes, then add 10 μL of calibrator solutions with concentrations of 0 U / mL, 50 U / mL, 100 U / mL, 200 U / mL, and 400 U / mL respectively (0 U / mL is the sample dilution), and mix well; add 40 μL of the calibration-specific fluorescent detection antibody, mix well, and incubate at room temperature in the dark for 0.5 h; add 1000 μL of washing buffer (1×), centrifuge at 400 g for 4 min, and discard the supernatant; add 150 μL-300 μL of washing buffer (1×), mix well, and detect the fluorescence type and fluorescence signal intensity on a flow cytometer. Plot a calibration curve with concentration on the x-axis and detection signal (MFI) on the y-axis.

[0097] Figure 4 This is a calibration curve of food-specific IgG antibodies in this embodiment of the invention. The calibration curve was directly generated by a flow cytometer and computer software. The detection results of the concentrations of 57 food-specific IgG antibodies were automatically calculated by the computer software. Specifically, L1 to L15 represent the microsphere levels of different food antigens. According to the microsphere level, the MFI values ​​were substituted into the calibration curve to obtain the detection results of 57 food-specific IgG antibodies.

[0098] (8) Quality control working principle

[0099] The microsphere capture mixture includes microspheres conjugated with food antigens and microspheres conjugated with anti-human IgG2 antibodies. Microspheres conjugated with anti-human IgG2 antibodies, used as quality control microspheres, are mixed with serum samples to form a complex of microspheres, anti-human IgG2 antibodies, and human IgG2 antibodies. Then, an optimized dose of fluorescent detection antibody reagent is added, and the mixture is incubated at room temperature in the dark to form a complex of microspheres, anti-human IgG2 antibodies, human IgG2 antibodies, anti-human IgG antibodies, and fluorescent proteins. Finally, flow cytometry is used to detect the fluorescence type and intensity of this complex. A positive test result for the quality control microspheres indicates that the kit is effective, the experimental procedure is correct, and the test result for the corresponding food-specific IgG antibody combination is valid.

[0100] (9) Evaluation of negative and positive concordance rates

[0101] Add 40 μL of the described capture microsphere mixture to the flow cytometry tube. For each project, select 50 negative and 50 positive samples, adding 10 μL to each sample. Add 40 μL of the described fluorescence detection antibody reagent, mix well, and incubate at room temperature in the dark for 0.5 h. Add 1000 μL of washing buffer (1×), centrifuge at 400 g for 4 min, and discard the supernatant. Add 150 μL-300 μL of washing buffer (1×), mix well, and detect the fluorescence type and fluorescence signal intensity on a Beckman DxFLEX flow cytometer. All test samples were tested using reagents produced by companies such as Jiangsu Haooubo Biopharmaceutical Co., Ltd., Hangzhou Zhejiang University Disun Biotechnology Co., Ltd., BioBio (Europe) Co., Ltd., and Beijing Xinhua Lianxiehe Pharmaceutical Co., Ltd. as control reagents. The detection process and interpretation of the results of the control reagents were performed in accordance with the instructions of the respective kits. In this kit, a positive result is indicated when the detection signal is greater than the positive cutoff signal; a negative result is indicated when the detection signal is less than the positive cutoff signal. Tables 5-1 to 5-4 show the positive and negative concordance rates for combinations 1 to 4, respectively. Referring to Tables 5-1 to 5-4, the kit's positive concordance rate is ≥95%, and its negative concordance rate is ≥95%.

[0102] Table 5-1: Evaluation results of negative and positive concordance rates for combination 1

[0103]

[0104] Table 5-2: Evaluation results of negative and positive concordance rates for combination 2

[0105]

[0106] Table 5-3: Evaluation results of negative and positive concordance rates for combination 3

[0107]

[0108]

[0109] Table 5-4: Evaluation results of negative and positive concordance rates for combination 4

[0110]

[0111]

[0112] (10) Repeatability evaluation

[0113] Two calibrators at different concentration levels were tested, and 10 parallel tests were performed according to the above operating requirements. The coefficient of variation (CV) was calculated. Tables 6-1 to 6-16 show the results of the repeatability test. Referring to Tables 6-1 to 6-16, the coefficients of variation (CV) of the low- and medium-value calibrators of this kit are both within 7%, indicating good repeatability.

[0114] Table 6-1

[0115]

[0116] Table 6-2

[0117]

[0118]

[0119] Table 6-3

[0120]

[0121] Table 6-4

[0122]

[0123] Table 6-5

[0124]

[0125]

[0126] Table 6-6

[0127]

[0128] Table 6-7

[0129]

[0130] Table 6-8

[0131]

[0132]

[0133] Table 6-9

[0134]

[0135] Table 6-10

[0136]

[0137]

[0138] Table 6-11

[0139]

[0140] Table 6-12

[0141]

[0142] Table 6-13

[0143]

[0144]

[0145] Table 6-14

[0146]

[0147] Table 6-15

[0148]

[0149] Table 6-16

[0150]

[0151]

[0152] (11) Test Results

[0153] The above-mentioned food-specific IgG antibody detection kit was used to detect 57 food-specific IgG antibodies in a serum sample of a test subject. The positive cutoff value was 50 U / mL. Tables 7-1 to 7-4 show the detection results of this serum sample. The levels of scallop, cantaloupe, pineapple, and mango-specific IgG antibodies in this sample were greater than 50 U / mL, indicating that the test subject was intolerant to these foods. Simultaneous detection of 57 food-specific IgG antibodies requires only 4 tubes, significantly reducing the testing time.

[0154] Table 7-1: Combination 1 Test

[0155]

[0156] Table 7-2: Combination 2 Test

[0157]

[0158] Table 7-3: Combination 3 Test

[0159]

[0160]

[0161] Table 7-4: Combination 4 Test

[0162]

[0163] Example 2: Cross-exclusion test for food-specific IgG antibody detection

[0164] Different food allergens may cross-react due to having the same or similar antigenic epitopes or due to low specificity of antigen-antibody binding. The food-specific IgG antibody detection kit given in Example 1 divides 57 food antigens into 4 groups (Group 1 includes antigens for eggs, milk, soybeans, wheat, rice, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, pork, potatoes, and beef; Group 2 includes antigens for sesame, peanuts, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, mutton, cashews, sweet potatoes, chocolate, black tea, and honey; Group 3 includes antigens for scallops, clams, oysters, lobsters, salmon, sardines, ribbonfish, grass carp, tuna, trout, sole, and salmon; Group 4 includes antigens for mangoes, oranges, pineapples, watermelons, olives, peaches, durians, bananas, cantaloupes, grapes, grapefruits, strawberries, blueberries, apples, and lemons). This grouping was obtained through experiments in this invention. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens, and there is no cross-reaction between any antigen in any group of food antigens and the antibody corresponding to any other antigen.

[0165] In conventional methods, similar foods in food taxonomy are considered to be able to be detected simultaneously. However, this invention has demonstrated through experiments that a considerable number of similar foods exhibit cross-interference when performing specific IgG detection. For example, multiple types of meat, multiple types of fish, and multiple types of fruits exhibit cross-interference. Therefore, it is inappropriate to test foods with cross-interference in the same group.

[0166] In this embodiment, the antigen combinations of the aforementioned similar foods were subjected to immunoassay. A single food-specific IgG antibody detection reagent was used as a control reagent, and a mixed food-specific IgG antibody was used as the test reagent, with the same sample tested simultaneously. The test value of the control reagent is the test value of a single food detection reagent, while the test value of this kit is the test value of a single food in the mixed reagent. If the test value of a certain food in the kit is significantly higher than the test value of the control reagent, it indicates that this food in the kit has cross-reactivity with one or more other foods. The test results are shown in Tables 8-1, 8-2, and 8-3, where N represents negative and P represents positive.

[0167] Table 8-1

[0168] Foods that humans are intolerant to chicken beef pork mutton Control reagent test value (U / mL) 25 25 27 23 Determination of positive or negative results from control reagents N N N N Test values ​​for this kit (U / mL) 23 22 29 90 This kit's results are determined as positive or negative. N N N P

[0169] Table 8-2

[0170] Foods that humans are intolerant to sardine salmon trout tuna sole salmon grass carp ribbonfish cod Control reagent test value (U / mL) 16 15 14 21 18 20 15 16 20 Determination of positive or negative results from control reagents N N N N N N N N N Test values ​​for this kit (U / mL) 18 16 16 20 15 18 17 13 83 This kit's results are determined as positive or negative. N N N N N N N N P

[0171] Table 8-3

[0172]

[0173] Based on the above test results, the test value of mutton in the kit was higher than that in the control kit, indicating that mutton has cross-interference with at least one of chicken, mutton, and pork. Similarly, cod has cross-interference with at least one of salmon, sardines, ribbonfish, grass carp, tuna, trout, sole, and chum salmon. Tomatoes have cross-interference with at least one of oranges, cantaloupe, bananas, grapes, grapefruits, lemons, peaches, pineapples, watermelons, durians, strawberries, mangoes, and blueberries.

[0174] To address the aforementioned overlap issues, mutton, cod, and tomatoes were further grouped, and combined with market demand, resulting in a total of four groups. Subsequently, the first, second, third, and fourth groups, along with the control group, were simultaneously tested on the same sample. The test results are shown in Tables 8-4, 8-5, 8-6, and 8-7.

[0175] Table 8-4

[0176]

[0177]

[0178] Table 8-5

[0179]

[0180] Table 8-6

[0181]

[0182] Table 8-7

[0183]

[0184] Based on the above experiment, after grouping, there was no longer any cross-interference between mutton, cod, and tomatoes, thus eliminating cross-interference between similar foods. However, it was found that there was cross-interference in beef in the first group, green beans in the second group, scallops in the third group, and peaches in the fourth group.

[0185] If measurements are required for beef, green beans, scallops, and peaches, cross-interference needs to be eliminated. This invention addresses this cross-reactivity by adjusting the blocking solution. After microsphere labeling, blocking is necessary. Normally, the main active ingredient in the blocking solution is bovine serum albumin (BSA). This invention adds the macromolecular substance PEG6000 to the blocking solution, thus resolving the cross-reactivity issues with beef, green beans, scallops, and peaches.

[0186] A single food-specific IgG antibody detection reagent was used as a control reagent, while a mixture of food-specific IgG antibodies was used as the test reagent, with the same sample tested simultaneously. After adding the blocking agent PEG6000, the test results for the four groups are shown in Tables 8-8 to 8-11.

[0187] Table 8-8

[0188]

[0189]

[0190] Table 8-9

[0191]

[0192] Table 8-10

[0193]

[0194] Table 8-11

[0195]

[0196] According to the experimental results, the test values ​​of the kit and the control reagent were similar in the four groups of tests, and the specificity of food IgG antibody detection was high. This proves that after adding the blocking agent PEG6000 to the microsphere mixture, no cross-reaction occurred in the detection of food IgG antibodies in the four groups.

[0197] During the research of this invention, various blocking agents were tested. Most blocking agents improved the cross-interference of food-specific IgG antibody detection in this invention. All food antigens were tested in the same way as described above, except that the blocking agents were changed. A blank control group and an experimental group with different blocking agents were set up. The cross-interference that still existed under different blocking agents was recorded. Some experimental results are shown in Tables 8-12.

[0198] Table 8-12

[0199]

[0200]

[0201] According to the experimental results, different blocking agents have different blocking effects on the surface sites of antigen-coupled microspheres, which leads to different blocking agents being able to avoid cross-interference of different antigens. The blank control group actually contains BSA. Compared with protein-type blocking agents (BSA, casein), the steric hindrance and surface properties of inorganic polymer blocking agents will affect the binding of small molecules. Therefore, polyvinylpyrrolidone and polyethylene glycol have certain blocking effects, but their blocking effects are different. Considering the influence of polymer structure, polyethylene glycol has a better blocking effect on the food antigen-coupled microspheres of the present invention, and the average molecular weight of polyethylene glycol will also affect the blocking effect. The experimental results show that, except for PEG6000, no blocking agent can completely eliminate all the above-mentioned cross-interference. Therefore, PEG6000 is preferred as the blocking agent.

[0202] Example 3: Selection of Quality Control IgG Antibody Types and Evaluation of Effect

[0203] Anti-human IgG antibodies include four types: IgG1, IgG2, IgG3, and IgG4. This invention has experimentally studied the types of food IgG quality control microsphere antibodies, selecting suitable quality control antibodies for 57 food antigens to achieve optimal detection results and avoid false positives or false negatives. According to Example 1: milk, wheat, rice, cod, tomatoes, corn, chicken, pork, sesame, peanuts, sucrose, green beans, spinach, oats, honey, scallops, clams, lobster, tuna, watermelon, olives, cantaloupe, and grapes. The quality control microspheres corresponding to the antigens of grapefruit, strawberry, and blueberry are anti-human IgG2 antibody subtypes; the quality control microspheres corresponding to the antigens of soybean, mushroom, sweet potato, salmon, sardine, and apple are anti-human IgG2 antibody subtypes; the quality control microspheres corresponding to the antigens of egg, tobacco, coffee, soft cheese, lamb, cashew, black tea, trout, sole, peach, and durian are anti-human IgG2 antibody subtypes; the quality control microspheres corresponding to the antigens of shrimp, crab, potato, beef, chocolate, ribbonfish, grass carp, salmon, mango, pineapple, and lemon are anti-human IgG2 antibody subtypes.

[0204] This embodiment tested the specific selections mentioned above. The quality control microspheres were conjugated with anti-human IgG1 antibody, anti-human IgG2 antibody, anti-human IgG3 antibody, and anti-human IgG4 antibody, respectively. The kit was prepared and tested according to the method of Example 1, and tested according to the evaluation method of Example 1. The total number of samples tested was 200. The test results are shown in Table 9.

[0205] Table 9: Selection of IgG antibody subtypes for quality control microspheres

[0206] Quality control microsphere antibody selection Number of positive quality control tests Positive rate of quality control microspheres Combined effect Anti-human IgG1 antibody 176 88％ better Anti-human IgG2 antibody 200 100％ good Anti-human IgG3 antibody 8 4％ Difference Anti-human IgG4 antibody 32 16％ Difference

[0207] According to the experimental results, the selection of the four IgG antibody subtypes affects the positive rate of the quality control microspheres. This is determined by the nature of the IgG antibody subtypes. Overall, the IgG antibody subtype conjugated to the quality control microspheres should be anti-human IgG2 antibody. It showed positive results in all 200 tests. Therefore, the operation can be judged by interpreting the positive result of the quality control microspheres. The results are accurate and reliable. If a negative result is found, it indicates that the operation is incorrect or the reagent has deteriorated, and the test needs to be repeated.

[0208] Example 4: Test for eliminating false positives with blocking agents

[0209] During the testing process, we found that even when the four food combinations optimized in Example 2 were selected, some samples still showed false positives. For example, when milk IgG antibody was found to be positive, beef IgG antibody in the same sample sometimes showed positive (and different test values) and sometimes negative in multiple tests. After confirmation, it was found that the sample did not contain beef IgG antibody. It was initially speculated that non-specific binding occurred between the capture microspheres and the sample, resulting in a higher test value. Moreover, the occurrence of non-specific binding was unstable, which led to the test result of a negative sample being positive.

[0210] To address this, this embodiment attempts to incorporate reagents that block non-specific binding. The experiment follows the method of Example 1, with the difference being the inclusion of a control group. The control group's microsphere capture solution did not contain any blocking agents. Multiple experimental groups were established, with various blocking agents added to the microsphere capture solution based on the control group. Since the probability of false positives in beef-specific IgG antibody detection is particularly high if milk-specific IgG antibodies are detected in actual testing, samples that have been verified as positive for milk IgG antibodies and negative for beef IgG antibodies were selected for immunoassay. A positive result for beef IgG antibody was considered a false positive. Each group underwent 20 parallel tests, and the number of false positives was recorded. The results are shown in Table 10.

[0211] Table 10: Selection of blocking agents and false positives of beef IgG antibodies

[0212]

[0213] According to the experimental results, when inactivated rabbit IgG and HAMA blocking agent are used in combination, they can almost completely block the non-specific binding in the detection of beef IgG antibodies and almost completely eliminate false positives; while the combination of rabbit IgG with any other blocking agent cannot achieve this effect, nor can the combination of HAMA blocking agent with other animal IgG.

[0214] Experiments showed that other food-specific IgG antibody tests that frequently produce false positives exhibited the same non-specific binding trend as beef IgG antibody tests. Adding rabbit IgG+HAMA blocking agent almost completely eliminated false positives.

[0215] Example 5: Regression calculation of calibration curves for serum concentrations of food-specific IgG antibodies corresponding to different food antigens

[0216] Plotting calibration curves: Add 40 μL of the calibration microsphere mixture to each of the five calibration microsphere tubes, then add 10 μL of calibrator solutions with assigned concentrations of 0 U / mL, 50 U / mL, 100 U / mL, 200 U / mL, and 400 U / mL respectively. The calibrator solution with an assigned concentration of 0 U / mL is a sample dilution. Add 40 μL of the calibration-specific fluorescent detection antibody, mix well, and incubate at room temperature in the dark for 0.5 h. Add 1000 μL of 1× wash buffer, centrifuge at 400 g for 4 min, and discard the supernatant. Add 150 μL-300 μL of 1× wash buffer, mix well, and detect the fluorescence type and fluorescence signal intensity on a flow cytometer. Plot a calibration curve with concentration on the x-axis and detection signal (MFI) on the y-axis. This example provides three forms of calibration curves, and the comparison is shown in Tables 11 and 12.

[0217] Table 11: Number of calibrations required for different calibration curve settings

[0218]

[0219] Table 12: Number of calibration points required for different calibration curve settings

[0220]

[0221] Based on the comparison of the two tables above, this reagent allows for a single test of four food combinations. This enables testing of 57 food items across four combinations with just one calibration, significantly simplifying reagent components and experimental procedures, reducing the probability of errors, and ultimately lowering both reagent and time costs.

[0222] Example 6: Selection of the blocking agent and test of the calibration curve

[0223] In the process of testing the calibration curves, this invention found that the blocking agent affects the plotting of the calibration curves. When the blocking agent is not added, there is a large error when microspheres with different food antigens coupled at the same level are calibrated at the same time. Therefore, it is necessary to measure the calibration curve for each food separately.

[0224] The method for determining the number of calibration curves is as follows: Calibration curves are plotted for each food individually, in groups, and with only one calibration. Three sample test values ​​are substituted into each curve, and the sample test results are calculated. If the error in the interpretation results of different calibration curves for the same sample is greater than 3%, then each food uses an individual calibration curve. If the error between the individual calibration curve and the grouped calibration curve is less than 3%, four combinations can be used for four tests. If the error in the interpretation of the individual calibration curve, the grouped calibration curve, and the calibration curve with only one calibration is less than 3%, then four combinations can be used for one test. The effect of adding different blocking agents on the plotting of calibration curves is shown in Table 13.

[0225] Table 13: Selection of Blocking Agents and Plotting of Calibration Curves

[0226]

[0227] According to the experimental results, the choice of blocking agent affects the plotting of calibration curves. Adding an appropriate blocking agent can reduce the number of calibrations. For example, adding anti-RF factor antibody can enable four combinations to be used for four tests, that is, each group can be calibrated once. The combined blocking agents are more effective. For example, mouse IgG+HAMA blocking agent can also enable four combinations to be used for four tests. However, only rabbit IgG+HAMA blocking agent can enable four combinations to be used for one test.

[0228] In principle, although rabbit IgG and HAMA blocking agents are called blocking agents, their actual function here is to eliminate the influence of microspheres coated with different levels of anti-human IgG3 antibodies on human IgG3 antibodies. Calibration curves are easily interfered with by different microsphere levels. Rabbit IgG and HAMA blocking agents can eliminate microscopic adsorption and other interactions between microspheres and antibodies. The possible mechanism is preferential binding to interfering antibodies unrelated to detection and elimination of electrostatic interactions. The combination of rabbit IgG and HAMA blocking agents can have a synergistic effect, allowing different types of antigens, antibodies, and microspheres to be calibrated simultaneously without interference.

[0229] The aforementioned blocking agent allows the present invention to draw calibration curves for each microsphere level with only one calibration, while reducing human intervention while ensuring accuracy, facilitating detection and reducing human error.

[0230] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. The use of a combination of PEG6000 and rabbit IgG and HAMA blocking agents to prepare a food-specific IgG antibody detection kit that divides 57 food antigens into 4 groups to eliminate cross-interference and false positives, characterized in that... The kit includes a microsphere capture mixture and a fluorescent detection antibody reagent. The microsphere capture mixture includes microspheres conjugated with food antigens and quality control microspheres. The food antigens consist of a first group of food antigens, a second group of food antigens, a third group of food antigens, and a fourth group of food antigens. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens, but there is no cross-reaction between any antigen in any group of food antigens and the antibody corresponding to any other antigen. The first group of food antigens includes at least three antigens selected from eggs, milk, soybeans, wheat, rice, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, pork, potatoes, and beef. The second group of food antigens includes at least three antigens selected from sesame, peanuts, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, lamb, cashews, sweet potatoes, chocolate, black tea, and honey. The third group of food antigens includes scallops, clams, oysters, lobsters, salmon, sardines, ribbonfish, grass carp, and tuna. The kit includes at least three food antigens selected from fish, trout, sole, and salmon antigens; the fourth group of food antigens includes at least three food antigens selected from mango, orange, pineapple, watermelon, olive, peach, durian, banana, cantaloupe, grape, grapefruit, strawberry, blueberry, apple, and lemon antigens; a blocking agent, PEG6000, is added to the microsphere mixture conjugated with beef, green bean, scallop, and peach antigens; the quality control microspheres for the first, second, third, and fourth groups of food antigens are microspheres conjugated with anti-human IgG2 antibodies; the capture microsphere mixture also includes an blocking agent, inactivated rabbit IgG or HAMA blocking agent; the kit also includes a washing buffer, sample dilution buffer, calibration microsphere mixture, calibration-specific fluorescent detection antibody, and calibrator; the calibration microsphere mixture is a mixture of microspheres labeled with different levels of anti-human IgG3 antibody, the calibration-specific fluorescent detection antibody is an anti-human IgG antibody labeled with phycoerythrin, and the calibrator is a human IgG3 antibody; The quantitative method of the kit is as follows: calibrate once, obtain the MFI value of each food-specific IgG antibody by testing serum, and then obtain the concentration of each food-specific IgG antibody by regression of calibration curve.

2. The use of PEG6000 in the preparation of a food-specific IgG antibody detection kit that divides 57 food antigens into 4 groups to eliminate cross-interference, characterized in that... The kit includes a microsphere capture mixture and a fluorescent detection antibody reagent. The microsphere capture mixture includes microspheres conjugated with food antigens and quality control microspheres. The food antigens consist of a first group of food antigens, a second group of food antigens, a third group of food antigens, and a fourth group of food antigens. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens, but there is no cross-reaction between any antigen in any group of food antigens and the antibody corresponding to any other antigen. The first group of food antigens includes at least three antigens selected from eggs, milk, soybeans, wheat, rice, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, pork, potatoes, and beef. The second group of food antigens includes at least three antigens selected from sesame, peanuts, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, lamb, cashews, sweet potatoes, chocolate, black tea, and honey. The third group of food antigens includes scallops, clams, oysters, lobsters, salmon, sardines, ribbonfish, grass carp, and tuna. The kit includes at least three food antigens selected from fish, trout, sole, and salmon antigens; the fourth group of food antigens includes at least three food antigens selected from mango, orange, pineapple, watermelon, olive, peach, durian, banana, cantaloupe, grape, grapefruit, strawberry, blueberry, apple, and lemon antigens; a blocking agent, PEG6000, is added to the microsphere mixture conjugated with beef, green bean, scallop, and peach antigens; the quality control microspheres for the first, second, third, and fourth groups of food antigens are microspheres conjugated with anti-human IgG2 antibodies; the capture microsphere mixture also includes an blocking agent, inactivated rabbit IgG or HAMA blocking agent; the kit also includes a washing buffer, sample dilution buffer, calibration microsphere mixture, calibration-specific fluorescent detection antibody, and calibrator; the calibration microsphere mixture is a mixture of microspheres labeled with different levels of anti-human IgG3 antibody, the calibration-specific fluorescent detection antibody is an anti-human IgG antibody labeled with phycoerythrin, and the calibrator is a human IgG3 antibody; The quantitative method of the kit is as follows: calibrate once, obtain the MFI value of each food-specific IgG antibody by testing serum, and then obtain the concentration of each food-specific IgG antibody by regression of calibration curve.

3. The use of rabbit IgG and HAMA blocking agents in the preparation of a food-specific IgG antibody detection kit that divides 57 food antigens into 4 groups to eliminate false positives, characterized in that... The kit includes a microsphere capture mixture and a fluorescent detection antibody reagent. The microsphere capture mixture includes microspheres conjugated with food antigens and quality control microspheres. The food antigens consist of a first group of food antigens, a second group of food antigens, a third group of food antigens, and a fourth group of food antigens. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens, but there is no cross-reaction between any antigen in any group of food antigens and the antibody corresponding to any other antigen. The first group of food antigens includes at least three antigens selected from eggs, milk, soybeans, wheat, rice, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, pork, potatoes, and beef. The second group of food antigens includes at least three antigens selected from sesame, peanuts, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, lamb, cashews, sweet potatoes, chocolate, black tea, and honey. The third group of food antigens includes scallops, clams, oysters, lobsters, salmon, sardines, ribbonfish, grass carp, and tuna. The kit includes at least three food antigens selected from fish, trout, sole, and salmon antigens; the fourth group of food antigens includes at least three food antigens selected from mango, orange, pineapple, watermelon, olive, peach, durian, banana, cantaloupe, grape, grapefruit, strawberry, blueberry, apple, and lemon antigens; a blocking agent, PEG6000, is added to the microsphere mixture conjugated with beef, green bean, scallop, and peach antigens; the quality control microspheres for the first, second, third, and fourth groups of food antigens are microspheres conjugated with anti-human IgG2 antibodies; the capture microsphere mixture also includes an blocking agent, inactivated rabbit IgG or HAMA blocking agent; the kit also includes a washing buffer, sample dilution buffer, calibration microsphere mixture, calibration-specific fluorescent detection antibody, and calibrator; the calibration microsphere mixture is a mixture of microspheres labeled with different levels of anti-human IgG3 antibody, the calibration-specific fluorescent detection antibody is an anti-human IgG antibody labeled with phycoerythrin, and the calibrator is a human IgG3 antibody; The quantitative method of the kit is as follows: calibrate once, obtain the MFI value of each food-specific IgG antibody by testing serum, and then obtain the concentration of each food-specific IgG antibody by regression of calibration curve.

4. A food-specific IgG antibody detection kit, characterized in that, The reagent includes a microsphere capture mixture and a fluorescent detection antibody reagent. The microsphere capture mixture comprises microspheres conjugated with food antigens and control microspheres. The food antigens consist of a first group of food antigens, a second group of food antigens, a third group of food antigens, and a fourth group of food antigens. Any one of the four groups of food antigens will cross-react with the antibody corresponding to any other group of food antigens, but there is no cross-reaction between any antigen in any group of food antigens and the antibody corresponding to any other antigen. The first group of food antigens includes at least three antigens selected from eggs, milk, soybeans, wheat, rice, cod, shrimp, crab, tomatoes, mushrooms, corn, chicken, pork, potatoes, and beef. The second group of food antigens includes sesame seeds, peanuts, tobacco, coffee, cottage cheese, sucrose, green beans, spinach, oats, lamb, cashews, sweet potatoes, and chocolate. The third group of food antigens includes at least three antigens from the following groups: black tea, honey, scallop, oyster, lobster, salmon, sardine, ribbonfish, grass carp, tuna, trout, sole, and salmon. The fourth group of food antigens includes at least three antigens from the following groups: mango, orange, pineapple, watermelon, olive, peach, durian, banana, cantaloupe, grape, grapefruit, strawberry, blueberry, apple, and lemon. A blocking agent, PEG6000, is added to the microsphere mixture conjugated with beef, green bean, scallop, and peach antigens. The quality control microspheres for the first, second, third, and fourth groups of food antigens are microspheres conjugated with anti-human IgG2 antibodies. The microsphere mixture also includes an blocking agent, which is an inactivated rabbit IgG and HAMA blocking agent.

5. The food-specific IgG antibody detection kit as described in claim 4, characterized in that, It also includes washing buffer, sample dilution buffer, calibration microsphere mixture, calibration-specific fluorescent detection antibody and calibrator. The calibration microsphere mixture is a mixture of microspheres labeled with different levels of anti-human IgG3 antibody, the calibration-specific fluorescent detection antibody is an anti-human IgG antibody labeled with phycoerythrin, and the calibrator is a human IgG3 antibody.

6. The quantitative method of the food-specific IgG antibody detection kit according to any one of claims 4-5, characterized in that, The calibration is performed once, and the MFI value of each food-specific IgG antibody is obtained by testing the serum. Then, the concentration of each food-specific IgG antibody is obtained by regression through calibration curve.

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