A kit for detecting free IgE content in human serum and application thereof

Abstract

The application relates to the technical field of in-vitro detection, and particularly discloses a kit for detecting the content of free IgE in human serum and application thereof. The kit comprises a capture reagent, a combination reagent and a magnetic separation reagent; the capture reagent comprises Fc epsilon R1 protein and a stabilizer; the stabilizer comprises the following components in parts by weight: 0.10-0.50 parts of hyaluronic acid, 0.40-0.80 parts of xanthan gum and 0.15-0.35 parts of sodium lauryl amphoacetate. The kit provided by the application can effectively improve the stability of the kit and the accuracy of detection results.

Description

Technical Field

[0001] This application relates to the technical field of in vitro detection, and more specifically, to a kit for detecting the content of free IgE in human serum and its application. Background Technology

[0002] IgE is a key antibody mediating type I hypersensitivity reactions, and the level of free IgE directly reflects the degree of activation of the body's allergic state. By detecting its concentration, it is possible to distinguish between IgE-mediated immediate allergies (such as food allergies and anaphylactic shock) and non-IgE-mediated allergies (such as contact dermatitis), providing a basis for precision treatment. For example: (1) In acute allergic attacks, for patients suspected of anaphylactic shock or laryngeal edema, detecting free IgE can quickly confirm the IgE-mediated mechanism and guide the use of emergency drugs such as adrenaline; (2) In the differentiation of chronic allergic diseases, about 30% of patients with chronic urticaria are IgE-mediated. Detecting free IgE can distinguish the cause and avoid overuse of glucocorticoids; (3) In asthma patients, allergic asthma (elevated IgE) needs to be differentiated from non-allergic asthma (normal IgE) in order to select anti-IgE treatment (such as omalizumab).

[0003] Meanwhile, free IgE levels are positively correlated with the severity of allergies. For example, when free IgE levels in asthma patients are >100 IU / mL, the risk of severe attacks increases significantly. Therefore, dynamic monitoring of free IgE levels can guide adjustments to treatment plans. In addition, persistently elevated free IgE levels in infancy and early childhood indicate an increased risk of atopic progression and can predict the likelihood of developing asthma and allergic rhinitis in the future.

[0004] Therefore, the detection of free IgE levels in serum is particularly important. However, currently, there are almost no clinical methods for detecting free IgE in human serum. Therefore, there is an urgent need to provide a method for detecting free IgE levels in serum. Summary of the Invention

[0005] This application provides a kit for detecting the level of free IgE in human serum and its application. The kit provided in this application can effectively improve the stability of the kit and the accuracy of the detection results.

[0006] FcεRI protein is a high-affinity receptor for IgE, capable of recognizing and specifically binding to IgE. The specific binding of FcεRI protein to IgE allows for the detection of free IgE levels.

[0007] In a first aspect, this application provides a kit for detecting the content of free IgE in human serum, employing the following technical solution:

[0008] A kit for detecting the content of free IgE in human serum, the kit comprising a capture reagent, a binding reagent, and a magnetic separation reagent; the capture reagent comprising FcεRI protein and a stabilizer; the stabilizer comprising the following components in parts by weight: 0.10-0.50 parts hyaluronic acid, 0.40-0.80 parts xanthan gum, and 0.15-0.35 parts sodium lauryl amphoacetate.

[0009] This application incorporates a combination of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate as a stabilizer into the capture reagent. On one hand, compared to stabilizers used in related technologies in existing kits, the detection results of the kit provided in this application exhibit better stability. On the other hand, using the kit provided in this application to test samples can effectively improve the accuracy of the detection results.

[0010] Hyaluronic acid is a high-molecular-weight polysaccharide, and its sodium salt form, sodium hyaluronate, exhibits good water solubility and stability. Xanthan gum is a microbial extracellular polysaccharide produced by fermentation of *Xanthomonas campestris*, the bacterium causing black rot in cabbage. It is composed of glucose, mannose, and glucuronic acid linked by glycosidic bonds. Its molecular structure contains a large number of negatively charged groups. Sodium lauryl amphoteric acetate is an amphoteric surfactant.

[0011] The hydrophobic chains of sodium lauryl amphoteric acetate can preferentially adsorb onto the hydrophobic regions of antibodies or onto the gas-liquid interface, reducing antibody aggregation and inactivation caused by interfacial adsorption (such as antibody adsorption onto the well walls during detection). Xanthan gum (negatively charged) and sodium lauryl amphoteric acetate (amphoteric ion, negatively charged at neutral pH) may enhance system stability through electrostatic repulsion, especially under neutral or weakly alkaline conditions, where their negative charges synergistically inhibit antibody aggregate formation.

[0012] Through experimental analysis, when using the above-mentioned kit to test the sample, compared with selecting any one or two of hyaluronic acid, xanthan gum and sodium lauryl amphoacetate as stabilizers in the capture reagent of the kit, this application selects a combination of hyaluronic acid, xanthan gum and sodium lauryl amphoacetate as stabilizers in the capture reagent of the kit, and the obtained test results have better stability and accuracy.

[0013] Therefore, this application selects a combination of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate as a stabilizer in the capture reagent. Through the synergistic effect of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate, the stability of the kit is improved, and the accuracy of the detection results is also improved.

[0014] Preferably, the stabilizer comprises the following components in parts by weight: 0.20-0.40 parts of hyaluronic acid, 0.50-0.70 parts of xanthan gum, and 0.20-0.30 parts of sodium lauryl amphoacetate.

[0015] In one specific implementation, the weight parts of hyaluronic acid in the stabilizer may be 0.10 parts, 0.20 parts, 0.30 parts, 0.40 parts, and 0.50 parts.

[0016] In some specific implementations, the weight parts of hyaluronic acid in the stabilizer may be 0.10-0.20 parts, 0.10-0.30 parts, 0.10-0.40 parts, 0.20-0.30 parts, 0.20-0.40 parts, 0.20-0.50 parts, 0.30-0.40 parts, 0.30-0.50 parts, or 0.40-0.50 parts.

[0017] In one specific implementation, the xanthan gum in the stabilizer may be 0.40 parts, 0.50 parts, 0.60 parts, 0.70 parts, 0.75 parts, and 0.80 parts by weight.

[0018] In some specific implementations, the xanthan gum in the stabilizer may be 0.40-0.50 parts, 0.40-0.60 parts, 0.40-0.70 parts, 0.40-0.75 parts, 0.50-0.60 parts, 0.50-0.70 parts, 0.50-0.75 parts, 0.50-0.80 parts, 0.60-0.70 parts, 0.60-0.75 parts, 0.60-0.80 parts, 0.70-0.75 parts, 0.70-0.80 parts, or 0.75-0.80 parts by weight.

[0019] In one specific implementation, the weight parts of sodium lauryl amphoteric acetate in the stabilizer may be 0.15 parts, 0.20 parts, 0.25 parts, 0.30 parts, and 0.35 parts.

[0020] In some specific embodiments, the weight parts of sodium lauryl amphoteric acetate in the stabilizer may be 0.15-0.20 parts, 0.15-0.25 parts, 0.15-0.30 parts, 0.20-0.25 parts, 0.20-0.30 parts, 0.20-0.35 parts, 0.25-0.30 parts, 0.25-0.35 parts, or 0.30-0.35 parts.

[0021] Preferably, the amount of xanthan gum added in the stabilizer is 1.5-2.5 times the amount of hyaluronic acid added.

[0022] In one specific implementation, the amount of xanthan gum added to the stabilizer is 1.5 times, 1.7 times, 2 times, 2.3 times, and 2.5 times the amount of hyaluronic acid added.

[0023] In some specific implementations, the amount of xanthan gum added to the stabilizer is 1.5-1.7 times, 1.5-2 times, 1.5-2.3 times, 1.7-2 times, 1.7-2.3 times, 1.7-2.5 times, 2-2.3 times, 2-2.5 times, or 2.3-2.5 times the amount of hyaluronic acid added.

[0024] Experimental analysis showed that the above method can further and effectively improve the stability of the reagent kit and the accuracy of the free IgE content in serum.

[0025] Preferably, the weight ratio of the FcεRI protein to the stabilizer is (2-4):100.

[0026] Preferably, the weight ratio of the FcεRI protein to the stabilizer is (2.5-3.5):100.

[0027] In one specific implementation, the weight ratio of the FcεRI protein to the stabilizer can be 2:100, 2.5:100, 3:100, 3.5:100, or 4:100.

[0028] In some specific implementations, the weight ratio of the FcεRI protein to the stabilizer can be (2-2.5):100, (2-3):100, (2-3.5):100, (2.5-3):100, (2.5-3.5):100, (2.5-4):100, (3-3.5):100, (3-4):100, or (3.5-4):100.

[0029] Experimental analysis revealed that controlling the weight ratio of FcεRI protein to stabilizer within the above-mentioned range can further improve the stability of the kit and the accuracy of serum free IgE content.

[0030] Preferably, the concentration of the FcεRI protein in the capture reagent is 0.10-0.20 ug / mL.

[0031] Preferably, the FcεRI protein needs to be immobilized on a solid support, and the immobilization method is to directly immobilize the FcεRI protein on the solid support through physical adsorption, non-covalent bonding or chemical bonding.

[0032] Secondly, this application provides a method for using the above-mentioned kit for detecting the content of free IgE in human serum, employing the following technical solution:

[0033] A method for using the above-mentioned kit for detecting the content of free IgE in human serum specifically includes the following steps:

[0034] The capture reagent and the magnetic separation reagent are mixed and incubated to form a magnetic bead-antigen complex;

[0035] The sample to be tested was diluted and mixed with the magnetic bead-antigen complex, and incubated to form a magnetic bead-antigen-IgE complex.

[0036] The binding reagent is mixed with the magnetic bead-antigen-IgE complex and incubated to form a magnetic bead-antigen-IgE-secondary antibody complex.

[0037] The magnetic bead-antigen-IgE-secondary antibody complex was mixed with the reaction substrate, incubated, and the relative luminescence intensity was detected.

[0038] First, the capture reagent and magnetic separation reagent are mixed. The biotinylated FcεRI protein in the capture reagent binds to the streptavidin-coated fluorescent magnetic beads (solid-phase carrier) in the magnetic separation reagent, forming a magnetic bead-antigen complex. Next, the diluted test sample is mixed with the magnetic bead-antigen complex. Free IgE in the test sample is captured by the biotinylated FcεRI protein, allowing it to bind to the magnetic bead-antigen complex, forming a magnetic bead-antigen-IgE complex. Then, the binding reagent is mixed with the magnetic bead-antigen-IgE complex. The enzyme-labeled secondary antibody in the binding reagent binds to the IgE on the magnetic bead-antigen-IgE complex, forming a magnetic bead-antigen-IgE-secondary antibody complex. Finally, the luminescent substrate is mixed with the magnetic bead-antigen-IgE-secondary antibody complex. The enzyme on the enzyme-labeled secondary antibody catalyzes the luminescent substrate to emit photons. The emitted photons are detected by the instrument, and the test results are expressed as relative luminescence intensity. The relative luminescence intensity obtained by the test is directly proportional to the content of free IgE in the sample.

[0039] The relative luminescence intensity of the standard was detected using the method described above, and a standard curve was plotted. The relative luminescence intensity of the sample to be tested, obtained using the same method, was then substituted into the standard curve to determine the content of free IgE in the sample.

[0040] In summary, this application has the following beneficial effects:

[0041] This application uses a combination of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate as a stabilizer. Compared to selecting any one or two of these three as stabilizers in the capture reagent of the kit, this application uses a combination of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate as a stabilizer in the capture reagent of the kit. The relative deviation of the detection results for low-value reference samples is less than 2%, and the relative deviation of the detection results for high-value reference samples is less than 1%. Therefore, using the kit provided in this application to detect free IgE in the test sample yields detection results with good stability and accuracy. Attached Figure Description

[0042] Figure 1 The standard curve was plotted for Embodiment 21 of this application. Detailed Implementation

[0043] Before describing the embodiments of this application in detail, it should be understood that the terminology used herein is for the purpose of describing a particular embodiment only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the term pertains.

[0044] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0045] The endpoints and any values ​​of the ranges disclosed in this application are not limited to the precise ranges or values, and such ranges or values ​​should be understood to include values ​​close to such ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0046] In this application, the terms "comprising" or "including" are open-ended expressions, meaning they include the content specified in this application but do not exclude other aspects.

[0047] This application provides a kit for detecting the content of free IgE in human serum.

[0048] This kit includes a capture reagent, a binding reagent, and a magnetic separation reagent. The capture reagent includes FcεRI protein and a stabilizer. The stabilizer includes hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate. The FcεRI protein is a high-affinity receptor for IgE, capable of recognizing and specifically binding to IgE. The stabilizer comprises the following components in parts by weight: 0.10-0.50 parts hyaluronic acid, 0.40-0.80 parts xanthan gum, and 0.15-0.35 parts sodium lauryl amphoacetate. The FcεRI protein contains the amino acid sequence shown in SEQ ID NO 1.

[0049] The above kit includes the following reagents:

[0050] (1) Capture reagent: including 0.10-0.20ug / mL FcεRI protein, Tris buffer (0.1M, pH 7.4) containing 10mg / mL bovine serum albumin (BSA) and stabilizer.

[0051] Optionally, the FcεRI protein is an enzyme-labeled, chemiluminescently labeled, or biotinylated FcεRI protein.

[0052] In one specific implementation, the FcεRI protein is a biotinylated FcεRI protein.

[0053] Optionally, the weight ratio of FcεRI protein to stabilizer is (2-4):100.

[0054] (2) Binding reagents: including 0.2 ug / mL horseradish peroxidase (HRP) labeled mouse anti-human IgE IgG (enzyme-labeled secondary antibody), MES buffer (0.1 M, pH 6.5) containing 1% BSA and 0.1% (v / v) ProClin 950.

[0055] (3) Standards: Tirs buffer (0.05M, pH 7.4) containing 1% BSA and 0.1% (v / v) ProClin 950, with different concentrations of free IgE antibody added.

[0056] (4) Magnetic separation reagent: 1 mg / mL fluorescent magnetic beads coated with streptavidin, Tris buffer (0.1 M, pH 7.4) containing 1% BSA and 0.1% (v / v) ProClin 950.

[0057] (5) Diluent: 1% human serum albumin (HSB), 0.01M phosphate buffer solution, 0.1% (v / v) ProClin950.

[0058] (6) Luminescent substrate: a mixture of substrate A (2 mol / L hydrogen peroxide, containing 0.1% (v / v) ProClin 950) and substrate B (0.63 mol / L luminol, containing 0.1% (v / v) ProClin 950) in a volume ratio of 1:1.

[0059] (7) Cleaning solution: Sodium phosphate (10mM, pH 7.4), 0.9% NaCl, 0.05% Tween-20, containing 0.1% (v / v) ProClin 950.

[0060] Of the above components, ProClin 950, as a stabilizer, can be replaced with NaN3.

[0061] This application also provides instructions for using the above-mentioned reagent kit, specifically including the following steps:

[0062] (1) Mix the capture reagent and the magnetic separation reagent, incubate to form a magnetic bead-antigen complex. Use magnetic separation and washing solution to wash away unbound substances.

[0063] (2) The sample to be tested is diluted and mixed with the magnetic bead-antigen complex, and incubated to form a magnetic bead-antigen-IgE complex. Unbound substances are washed away using magnetic separation and washing solution.

[0064] (3) Mix the binding reagent with the magnetic bead-antigen-IgE complex and incubate to form a magnetic bead-antigen-IgE-secondary antibody complex. Use magnetic separation and washing solution to wash away unbound substances.

[0065] (4) Mix the reflective substrate with the magnetic bead-antigen-IgE-secondary antibody complex, incubate, detect the relative luminescence intensity (RLU), and obtain the content of IgE in the sample to be tested according to the standard curve.

[0066] This application also provides the application of the above-mentioned kit for detecting the level of free IgE in human serum in the rapid screening and differentiation of allergic and non-allergic diseases.

[0067] To make the objectives, technical solutions, and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0068] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0069] In this embodiment, the hyaluronic acid used has a CAS number of 9004-61-9, the xanthan gum has a CAS number of 11138-66-2, and the sodium lauryl amphoteric acetate has a CAS number of 156028-14-7.

[0070] The present application will be further described in detail below with reference to the embodiments and test results.

[0071] Example 1

[0072] This embodiment provides a kit for detecting the content of free IgE in human serum. The sample to be tested is human serum.

[0073] The above kit includes the following components: capture reagent, binding reagent, magnetic separation reagent, luminescent substrate, diluent, and washing solution. Details are as follows:

[0074] (1) Capture reagent: 0.15ug / mL biotinylated FcεRI protein in Tris buffer (0.1M, pH 7.4) containing 10mg / mL BSA and 5ug / mL stabilizer.

[0075] The biotinylated FcεRI protein is a commercially available FcεRI protein. This commercially available FcεRI protein contains the amino acid sequence shown in SEQ ID NO 1.

[0076] The stabilizers include hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate, all of which are commercially available. The addition details of each component in the stabilizer are shown in Table 1.

[0077] (2) Binding reagent: 0.2 ug / mL enzyme-labeled secondary antibody, MES buffer (0.1 M, pH 6.5) containing 1% BSA and 0.1% (v / v) ProClin 950.

[0078] (3) Magnetic separation reagent: 1 mg / mL fluorescent magnetic beads coated with streptavidin, Tris buffer (0.1 M, pH 7.4) containing 1% BSA and 0.1% (v / v) ProClin 950.

[0079] (4) Diluent: 1% HSB, phosphate buffer solution containing 0.1% (v / v) ProClin 950 (0.01M).

[0080] (5) Luminescent substrate: A mixture of substrate A (2 mol / L hydrogen peroxide, containing 0.1% (v / v) ProClin 950) and substrate B (0.63 mol / L luminol, containing 0.1% (v / v) ProClin 950) in a volume ratio of 1:1.

[0081] (7) Cleaning solution: 10mM sodium phosphate (pH 7.4), 0.9% NaCl, 0.05% Tween-20, containing 0.1% (v / v) ProClin 950.

[0082] The method of using the above reagent kit includes the following steps:

[0083] (1) Mix 40 μL of capture reagent (as shown in Table 1) and 10 μL of magnetic separation reagent, and incubate at 37°C for 3 min to form magnetic bead-antigen complex.

[0084] The magnetic bead-antigen complex is adsorbed onto the sidewall of the container by applying a magnetic field, and the solvent is removed; then 200 μL of washing solution is added for washing, and the washing solution is removed after washing; the magnetic field applied to the sidewall is removed.

[0085] (2) Add 56 μL of diluent to 4 μL of sample to be tested, then mix the diluted solution with the magnetic bead-antigen complex, incubate at 37°C for 13 min, add 150 μL of washing solution to stop the reaction, and form magnetic bead-antigen-IgE complex.

[0086] The magnetic bead-antigen-IgE complex was adsorbed onto the sidewall of the container by applying a magnetic field, and the solvent was removed. Then, 200 μL of washing solution was added for washing, and the process was repeated twice. After washing, the washing solution was removed, and the magnetic field applied to the sidewall was removed.

[0087] (3) Continue to add binding reagent, mix with magnetic bead-antigen-IgE complex, incubate at 37°C for 13 min, add 150 μL washing solution to stop the reaction, and form magnetic bead-antigen-IgE-secondary antibody complex;

[0088] The magnetic bead-antigen-IgE-secondary antibody complex was adsorbed onto the sidewall of the container by applying a magnetic field, and the solvent was removed. Then, 200 μL of washing solution was added for washing, and the process was repeated twice. After washing, the washing solution was removed, and the magnetic field applied to the sidewall was removed.

[0089] (4) Add 50 μL of luminescent substrate (the volume ratio of substrate A to substrate B is 1:1), mix with the magnetic bead-antigen-IgE-secondary antibody complex, incubate at 37°C for 3.5 min, measure the relative luminescence intensity (RLU) of the solution using an ELISA reader, and then calculate the concentration of free IgE in the sample to be tested according to the standard curve provided in Example 21.

[0090] Example 2-14

[0091] Examples 2-14 provide kits for detecting the content of free IgE in human serum. The difference between these examples and Example 1 lies in the amount of each component added to the stabilizer of the capture reagent, as shown in Table 1. All other operating procedures remain the same as in Example 1.

[0092] Specifically, the differences in Examples 1-14 are as follows:

[0093] The difference between Examples 1-5 lies in the amount of hyaluronic acid added to the stabilizer.

[0094] The difference between Examples 3 and 6-10 lies in the amount of xanthan gum added to the stabilizer.

[0095] The difference between Examples 3 and 11-14 lies in the amount of sodium lauryl amphoteric acetate added to the stabilizer.

[0096] Table 1. Addition of stabilizer and FcεRI protein in the capture reagent of the kit.

[0097]

[0098] Examples 15-20

[0099] Examples 15-20 provide kits for detecting the content of free IgE in human serum. The difference between these examples and Example 3 is the weight ratio of FcεRI protein to stabilizer, as shown in Table 1. All other operating steps are the same as in Example 3.

[0100] Example 21

[0101] This embodiment uses the kit provided in Example 3 to create a standard curve corresponding to the IgE content. The difference between this embodiment and Example 3 is that the test samples are standards of different concentrations. All other operating steps are the same as in Example 3.

[0102] The standards included Tirs buffer (0.05M, pH 7.4) containing 1% BSA, 0.1% (v / v) ProClin 950, and different concentrations of free IgE antibody.

[0103] The specific concentrations of the added free IgE antibody are shown in Table 2.

[0104] The relative luminescence intensity (RLU) of standards at different concentrations was measured, and the results are shown in Table 2. A standard curve was plotted with the concentration of free IgE in the standards on the x-axis and the measured relative luminescence intensity on the y-axis, as shown in Table 2. Figure 1 As shown.

[0105] Table 2. Data related to the standard curve

[0106]

[0107] Combine Table 2 and Figure 1 It can be seen that the standard curve prepared in Example 20 conforms to Y=A×X 2 The equation is: A = 198.63, B = 81196, C = 20266, R ... and R = 198.63. 2 =0.9999. This indicates that the standard curve provided in this application has a good fit.

[0108] Comparative Example

[0109] Comparative Examples 1-6

[0110] Comparative Examples 1-6 each provide a kit for detecting the content of free IgE in human serum. The difference between the above comparative examples and Example 3 is that the amount of each component added in the stabilizer of the capture reagent is shown in Table 3. Everything else is the same as in Example 3.

[0111] Table 3. Comparative Examples 1-6—Addition of Stabilizer and FcεRI Protein in the Capture Reagent

[0112]

[0113] Comparative Example 7

[0114] This comparative example provides a kit for detecting the level of free IgE in human serum. The difference between this kit and Example 3 is that the stabilizer in the capture reagent in this comparative example is 1% Proclin 950. All other aspects are the same as in Example 3.

[0115] Comparative Example 8

[0116] This comparative example provides a kit for detecting the content of free IgE in human serum. The difference between this kit and Example 3 is that the stabilizer in the capture reagent in this comparative example is 1% NaN3. All other aspects are the same as in Example 3.

[0117] Comparative Example 9

[0118] This comparative example provides a kit for detecting the level of free IgE in human serum. It differs from Example 3 in that the stabilizer in the capture reagent is 1% Proclin 950, and the stabilizer in the binding reagent is the same as that used in the capture reagent in Example 3. All other aspects are the same as in Example 3.

[0119] Comparative Example 10

[0120] This comparative example provides a kit for detecting the level of free IgE in human serum. It differs from Example 3 in that the stabilizer in the capture reagent is 1% Proclin 950, and the stabilizer in the magnetic separation reagent is the same as that used in the capture reagent in Example 3. All other aspects are the same as in Example 3.

[0121] Performance testing

[0122] I. Accuracy Testing of the Reagent Kit

[0123] The kits provided in the above examples and comparative examples were used to detect reference samples with known IgE antibody content. The testing period was within 15 days after the kits were prepared. The accuracy of the kits was determined by calculating the relative deviation of the test results. The specific test results are shown in Table 4.

[0124] In this experiment, the reference materials included a low-value reference material and a high-value reference material. The IgE concentration in the low-value reference material was 35.50 u / mL, and the IgE concentration in the high-value reference material was 175.50 u / mL.

[0125] Table 4. Accuracy test results of the reagent kit

[0126]

[0127] As shown in Table 4, when the kit provided in this application was used to detect reference samples containing free IgE antibodies, the relative deviation of the detection results for low-value reference samples was less than 2%, and the relative deviation of the detection results for high-value reference samples was less than 1%. The kit provided in this application can effectively improve the accuracy of detecting the content of free IgE antibodies in human serum samples.

[0128] Comparing the detection results of Example 3 with those of Comparative Examples 1-6, it can be seen that when hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate were used simultaneously as stabilizers in the capture reagent in Example 3, the relative deviation of the detection results for low-value references was 0.82%, and the relative deviation of the detection results for high-value references was 0.42%, indicating that the accuracy of the detection results was relatively high. However, when any one or two of the three were used as stabilizers in the capture reagent, the relative deviation of the obtained detection results was larger, and the accuracy of the detection results was lower. Therefore, this application selects to use hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate simultaneously as stabilizers in the capture reagent, thereby improving the accuracy of the detection results.

[0129] Comparing the detection results of Example 3 with those of Comparative Examples 7-10, it is evident that, compared to the use of NaN3 or Proclin 950 as stabilizers in related technologies for the capture reagent, Example 3's simultaneous use of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate as stabilizers for the capture reagent reduces the relative bias of the detection results, thereby improving their accuracy. Furthermore, compared to using these three components as stabilizers in binding reagents or magnetic separation reagents, the combination of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate as stabilizers for the capture reagent results in a lower relative bias, effectively improving the accuracy of the detection results.

[0130] By comparing the test results of Examples 1-5, it can be seen that the present application controls the amount of hyaluronic acid added within the range of 0.10-0.50 parts. The relative deviation of the test results for low-value reference materials is less than 2%, and the relative deviation of the test results for high-value reference materials is less than 1%, indicating that it can effectively improve the accuracy of the test results.

[0131] By comparing the test results of Examples 3 and 6-10, it can be seen that the application selects to control the amount of xanthan gum added within the range of 0.40-0.80 parts. The relative deviation of the test results for low-value reference materials is less than 2%, and the relative deviation of the test results for high-value reference materials is less than 1%, indicating that the accuracy of the test results can be effectively improved.

[0132] By comparing the test results of Examples 3 and 11-14, it can be seen that when the amount of sodium lauryl amphoacetate added in this application is controlled within the range of 0.15-0.35 parts, the relative deviation of the test results for low-value reference materials is less than 2%, and the relative deviation of the test results for high-value reference materials is less than 1%, indicating that the accuracy of the test results can be effectively improved.

[0133] By comparing the detection results of Examples 3 and 15-20, it can be seen that this application further limits the weight ratio of FcεRI protein to stabilizer in the capture reagent. This application controls the weight ratio of FcεRI protein to stabilizer in the capture reagent within the range of (2-4):100, and the obtained detection results are relatively accurate.

[0134] In summary, the kit provided in this application selects a combination of hyaluronic acid, xanthan gum, and sodium lauryl amphoacetate as the stabilizer for the capture reagent, thereby improving the accuracy of the detection results. Furthermore, the ratio of these three components has been optimized, further enhancing the accuracy of the detection results. In addition, the weight ratio of FcεRI protein to stabilizer in the capture reagent and the type of FcεRI protein were screened, further improving the accuracy of the detection results.

[0135] II. Repeatability testing of the reagent kit

[0136] The kit provided in Example 3 was used to test reference samples with known IgE antibody content, using 10 reference samples from the same batch. The reproducibility of the kit was determined by calculating the relative deviation of the test results.

[0137] The experiment was conducted within 15 days after the kit was prepared. The specific test results are shown in Table 5.

[0138] In this experiment, the reference materials included a low-value reference material and a high-value reference material. The IgE concentration in the low-value reference material was 35.50 u / mL, and the IgE concentration in the high-value reference material was 175.50 u / mL.

[0139] Table 5. Repeatability test results of the kit

[0140]

[0141] As shown in Table 5, when the kit provided in Example 3 of this application was used to detect reference samples containing free IgE antibodies, the relative deviation of the detection results for low-value reference samples was within the range of 0.75-0.85%, while the relative deviation of the detection results for high-value reference samples was close to 0.4%. Based on the above detection results, it can be seen that the kit provided in this application has good repeatability, and the detection results have good accuracy.

[0142] III. Stability testing of the reagent kit

[0143] The kits provided in the above examples and comparative examples were used to detect reference samples with known IgE antibody content. The testing period was within 24 months after the kit was prepared (stored sealed at 2-8℃). The stability of the kit was determined by calculating the relative deviation of the test results. The specific test results are shown in Table 6.

[0144] In this experiment, the reference materials included a low-value reference material and a high-value reference material. The IgE concentration in the low-value reference material was 35.50 u / mL, and the IgE concentration in the high-value reference material was 175.50 u / mL.

[0145] Table 6. Results of stability testing of the reagent kit

[0146]

[0147] As shown in Table 6, the results of the test in "I. Accuracy Test of the Reagent Kit" indicate that the relative deviation of the test results of Comparative Example 2 and Comparative Example 3 is greater than 10%, which does not meet the industry requirement that the relative deviation value be less than or equal to 10%. Therefore, no further tests will be conducted.

[0148] The test results of Comparative Examples 1 and 4-10 show that the relative deviation of the test results is greater than 10% as required by the industry, whether for low-value or high-value reference materials. This indicates that the stability of the reagent kit after 24 months of preparation no longer meets the industry requirements.

[0149] By comparing the test results of Examples 1-5, it can be seen that when the amount of hyaluronic acid added is controlled within the range of 0.10-0.50 parts, the maximum relative deviation of the test results for low-value reference materials is close to 2.5%, and the relative deviation of the test results for high-value reference materials is less than 1.5%.

[0150] By comparing the test results of Examples 3 and 6-10, it can be seen that when the amount of xanthan gum added is controlled within the range of 0.40-0.80 parts, the relative deviation of the test results for low-value reference materials is less than 2.5%, and the relative deviation of the test results for high-value reference materials is less than or equal to 1.5%.

[0151] By comparing the test results of Examples 3 and 11-14, it can be seen that when the amount of sodium lauryl amphoacetate added in this application is controlled within the range of 0.15-0.35 parts, the relative deviation of the test results for low-value reference materials is less than 2.5%, and the relative deviation of the test results for high-value reference materials is less than 1.5%.

[0152] By comparing the detection results of Examples 3 and 15-20, it can be seen that this application further limits the weight ratio of FcεRI protein to stabilizer in the capture reagent. This application controls the weight ratio of FcεRI protein to stabilizer in the capture reagent within the range of (2-4):100. The relative deviation of the detection results for low-value reference materials is close to 2.5%, and the relative deviation of the detection results for high-value reference materials is less than 1.5%.

[0153] As can be seen from the above, the relative deviation of the test results of the kit provided in this application to the reference sample can still be maintained at a relatively low level within 24 months, indicating good stability.

[0154] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0155] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. A kit for detecting the content of free IgE in human serum, characterized in that, The kit includes a capture reagent, a binding reagent, and a magnetic separation reagent; the capture reagent includes FcεRI protein and a stabilizer; the stabilizer includes the following components in parts by weight: 0.10-0.50 parts hyaluronic acid, 0.40-0.80 parts xanthan gum, and 0.15-0.35 parts sodium lauryl amphoacetate.

2. The reagent kit according to claim 1, characterized in that, The stabilizer comprises the following components in parts by weight: 0.20-0.40 parts hyaluronic acid, 0.50-0.70 parts xanthan gum, and 0.20-0.30 parts sodium lauryl amphoacetate.

3. The reagent kit according to claim 1, characterized in that, The amount of xanthan gum added in the stabilizer is 1.5-2.5 times the amount of hyaluronic acid added.

4. The reagent kit according to claim 1, characterized in that, The weight ratio of the FcεRI protein to the stabilizer is (2-4):

100.

5. The reagent kit according to claim 4, characterized in that, The weight ratio of the FcεRI protein to the stabilizer is (2.5-3.5):

100.

6. The reagent kit according to claim 1, characterized in that, The concentration of the FcεRI protein in the capture reagent is 0.10-0.20 ug / mL.

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