A milk allergen-specific IgE antibody enzyme-linked immunosorbent assay method

Abstract

The present invention provides a milk allergen-specific IgE antibody enzyme-linked immunosorbent assay method. In order to solve the problem of false positives and the like in the milk allergen-specific IgE antibody enzyme-linked immunosorbent assay method, the present invention combines bovine serum albumin and Roche blocker with item number 11922122103 to prepare a blocker, and successfully blocks the binding of anti-bovine serum albumin IgG antibodies in a sample to be detected with the bovine serum albumin used for blocking on the microplate and the non-specific binding of heterophilic antibodies to the microplate, without affecting the capture of the milk allergen-specific IgE antibodies by anti-human IgE antibodies on the microplate and the activity of biotin, thereby reducing the false positive results caused by the influence of anti-bovine serum albumin IgG antibodies and heterophilic antibodies. The present invention adopts the form of a freeze-dried product, which is convenient for storage and reduces the problem of low test results caused by the dilution of the sample to be detected.

Description

Technical Field

[0001] The invention belongs to the field of antibody detection, and in particular relates to an enzyme-linked immunosorbent assay method for detecting milk allergen-specific IgE antibodies. Background Art

[0002] Since its establishment, Enzyme-Linked Immunosorbent Assays (ELISA) has developed rapidly and has been widely used in clinical practice due to its advantages such as rapidity, sensitivity, simplicity, and ease of standardization.

[0003] Since the content of IgE immunoglobulin in patient samples is usually only about one forty thousandth of that of IgG immunoglobulin, conventional allergy IgE detection methods will be interfered by a large amount of IgG, with low sensitivity and specificity, and a large amount of allergen consumption. The use of ELISA enzyme-linked immunosorbent capture method to establish a detection method for allergen-specific IgE antibodies in human serum can greatly reduce the interference of IgG. Since the ELISA enzyme-linked immunosorbent capture method is coated with anti-IgE monoclonal antibodies on the microplate, in theory, only the IgE antibodies in the sample are captured, thereby reducing the interference of IgG antibodies and improving the sensitivity and specificity of specific IgE antibody detection.

[0004] However, the existing ELISA enzyme-linked immunosorbent capture method for establishing the detection method of allergen-specific IgE antibodies in human serum is not suitable for the detection of milk allergen-specific IgE antibodies. After the microplate is coated with anti-human IgE antibodies, bovine serum albumin (BSA) needs to be used to block the microplate to reduce the nonspecific adsorption of the microplate to reduce interference, but BSA is also a component of milk allergens. The BSA fixed on the microplate will bind to the anti-BSA IgG antibody in the patient sample, and the anti-BSA IgG antibody will bind to the biotinylated milk allergen, resulting in a false positive. In addition, the interference of other heterophilic antibodies in human serum will also cause false positives in the test results. These false positive test results have basically not been taken seriously, and some existing blockers have no obvious effect on these false positive interferences. For example, we tried to use conventional mouse-derived blockers, but no obvious blocking effect was shown. In addition, since the IgE antibody content in the serum sample is low, the addition of conventional blockers will cause the test sample to be diluted, and the test results will be reduced, thereby affecting the accuracy of the test results. Therefore, there is currently no better solution to the problem of inaccurate test results in the milk allergen-specific IgE antibody enzyme-linked immunosorbent assay. Summary of the invention

[0005] The purpose of the present invention is to provide a blocking agent for milk allergen-specific IgE antibody enzyme-linked immunosorbent assay, which can block the interference of anti-bovine serum albumin antibodies and heterophile antibodies in the sample to be tested, effectively avoid false positive results, and improve the accuracy of the test results.

[0006] Another object of the present invention is to provide an enzyme-linked immunosorbent assay method for detecting milk allergen-specific IgE antibodies for non-diagnostic and therapeutic purposes with a low false positive rate.

[0007] Another object of the present invention is to provide a kit for detecting milk allergen-specific IgE antibodies, which can improve the accuracy of milk allergen-specific IgE antibody detection results.

[0008] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0009] A blocking agent for milk allergen-specific IgE antibody enzyme-linked immunosorbent assay, comprising bovine serum albumin and Roche blocking agent with item number 11922122103.

[0010] Specifically, the bovine serum albumin can bind to the anti-bovine serum albumin antibodies in the sample to be tested. When the sample to be tested is added to a microplate coated with anti-human IgE antibodies and blocked with bovine serum albumin for incubation, the anti-bovine serum albumin antibodies in the sample to be tested no longer bind to the bovine serum albumin on the microplate, thereby avoiding false positive results caused by the anti-bovine serum albumin antibodies.

[0011] Specifically, the Roche blocker with the catalog number 11922122103 can bind to heterophilic antibodies in the sample to be tested. When the sample to be tested is added to a microplate coated with anti-human IgE antibodies and blocked with bovine serum albumin for incubation, the probability of non-specific binding of heterophilic antibodies in the sample to be tested to the microplate is greatly reduced, thereby reducing false positive results caused by heterophilic antibodies. In addition, the Roche blocker with the catalog number 11922122103 will not interfere with the activity of biotin.

[0012] Preferably, the sample to be detected is serum.

[0013] Preferably, the blocking agent is a lyophilized product.

[0014] When in use, the sample to be tested is directly added to the centrifuge tube containing the freeze-dried product. After the freeze-dried product is completely dissolved, the enzyme-linked immunosorbent assay can be performed. This will not dilute the sample to be tested, will not cause the sample detection amount to be low, and the test result will be more accurate.

[0015] Further preferably, a blocking agent buffer is first prepared, and then freeze-drying is used to obtain the blocking agent, wherein the blocking agent buffer comprises: 0.05-10 g bovine serum albumin, 50-300 μL Roche blocking agent with catalog number 11922122103, and 0.01-1 mL preservative per 100 mL Tris-HCl buffer with a pH value of 7.0-8.0.

[0016] Still further preferably, the blocking agent buffer is: every 100 mL of Tris-HCl buffer with a pH value of 7.0 to 8.0 contains 0.05 to 5 g of bovine serum albumin, 50 to 250 μL of Roche blocking agent with product number 11922122103 and 0.01 to 0.5 mL of preservative.

[0017] According to some specific and preferred embodiments, the blocking agent buffer is: every 100 mL of Tris-HCl buffer with a pH value of 7.0 to 7.5 contains 0.1 to 1 g of bovine serum albumin, 50 to 250 μL of Roche blocking agent with product number 11922122103 and 0.01 to 0.2 mL of preservative.

[0018] Still more preferably, the preservative is Proclin-300.

[0019] The present invention also provides an enzyme-linked immunosorbent assay method for detecting milk allergen-specific IgE antibodies for non-diagnostic and therapeutic purposes, wherein the blocking agent is mixed with a sample to be detected to obtain a mixed solution, and then an enzyme-linked immunosorbent assay is used to detect the milk allergen-specific IgE antibodies in the mixed solution. The microplate used in the enzyme-linked immunosorbent assay is coated with anti-human IgE antibodies and blocked with bovine serum albumin.

[0020] Preferably, the milk allergen-specific IgE antibody enzyme-linked immunosorbent assay method comprises the following steps in sequence:

[0021] (1) mixing the sample to be tested with the blocking agent to obtain a mixed solution;

[0022] (2) adding the mixed solution of step (1) into the microwells of the microwell plate, and incubating at room temperature to obtain a conjugate of anti-human IgE antibody-milk allergen-specific IgE antibody;

[0023] (3) after washing the plate, adding milk allergen-biotin, and incubating at room temperature to obtain a conjugate of anti-human IgE antibody-milk allergen-specific IgE antibody-milk allergen-biotin;

[0024] (4) after washing the plate, adding horseradish peroxidase-streptavidin binding solution, and incubating at room temperature to obtain a conjugate of anti-human IgE antibody-milk allergen-specific IgE antibody-milk allergen-biotin-horseradish peroxidase-streptavidin;

[0025] (5) After washing the plate, add a color developing solution for color development, add a stop solution to terminate the reaction, and then detect the absorbance.

[0026] Further preferably, the milk allergen-biotin is Hao Ou Bo Milk Allergen-Biotin with the product number of MC00152

[0027] Further preferably, the horseradish peroxidase-streptavidin binding solution is Haooubo horseradish peroxidase-streptavidin binding solution with product number MC00128.

[0028] Further preferably, the ratio of the feed volumes of the mixed solution, the milk allergen-biotin and the horseradish peroxidase-streptavidin binding solution is 1:(1.8-2.2):(1.8-2.2).

[0029] According to a specific embodiment, the mixed solution is added in an amount of 50 μL per well, the milk allergen-biotin is added in an amount of 100 μL per well, and the horseradish peroxidase-streptavidin binding solution is added in an amount of 100 μL per well.

[0030] Still more preferably, the color developing solution used in step (5) is 3,3',5,5'-tetramethylbenzidine substrate color developing solution, and the stop solution is 0.2-0.8 mol / L H 2 SO 4 , the detection wavelength is 420~480nm.

[0031] 3,3',5,5'-Tetramethylbenzidine substrate colorimetric solution is referred to as TMB substrate colorimetric solution. After adding TMB substrate for incubation, the reaction solution turns blue, and then sulfuric acid is added to terminate the reaction, after which the reaction solution turns yellow. The IgE antibody concentration in the sample to be tested is proportional to the optical density (OD value) when reading at 450nm on an ELISA reader (reference wavelength 620nm or 630nm, eliminating the influence of non-specific absorbance values).

[0032] The present invention also provides a kit for detecting milk allergen-specific IgE antibodies, wherein the kit comprises the blocking agent.

[0033] Preferably, the kit further comprises a microplate coated with anti-human IgE antibody and blocked with bovine serum albumin, milk allergen-biotin, horseradish peroxidase-streptavidin binding solution, 3,3',5,5'-tetramethylbenzidine substrate color development solution, stop solution and optionally anti-IgE antibody-biotin and / or calibrator.

[0034] Compared with the prior art, the present invention has the following advantages:

[0035] The present invention is the first to address the problems existing in the milk allergen-specific IgE antibody enzyme-linked immunosorbent capture detection method, and combines bovine serum albumin and Roche blocker with item number 11922122103 to prepare a blocker, which successfully blocks the binding of anti-bovine serum albumin IgG antibodies in the sample to be detected with the bovine serum albumin used for blocking on the microplate and the non-specific binding of heterophilic antibodies to the microplate without affecting the capture of the milk allergen-specific IgE antibodies by anti-human IgE antibodies on the microplate, thereby reducing false positive results caused by the influence of anti-bovine serum albumin IgG antibodies and heterophilic antibodies. The present invention adopts the form of a freeze-dried product, which is easy to store and reduces the problem of low test results caused by the dilution of the sample to be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 This is a schematic diagram of the detection principle of the enzyme-linked immunosorbent capture method for the sample to be detected without adding the impedance agent of Example 1.

[0037] Figure 2 This is a schematic diagram of the detection principle of the enzyme-linked immunosorbent capture method of the sample to be detected to which the impedance agent of Example 1 is added.

[0038] Figure 3 This is the four-parameter curve of Example 2.

[0039] Figure 4 The results of the enzyme-linked immunosorbent assay of samples with different degrees of interference in Example 2 are shown in FIG. DETAILED DESCRIPTION

[0040] The present invention is further described below in conjunction with the examples. However, the present invention is not limited to the following examples. The implementation conditions used in the examples can be further adjusted according to the different requirements of specific use, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in each embodiment of the present invention can be combined with each other as long as they do not conflict with each other.

[0041] In practice, the inventors found that the anti-human IgE antibody-coated microplates with good effects in the existing kits for milk allergen-specific IgE antibody immunoassay were all blocked with bovine serum albumin, which is also a kind of milk allergen. Therefore, the anti-bovine serum albumin IgG antibody (anti-BSA IgG antibody) in the test sample will bind to the bovine serum albumin on the microplate, thereby causing a false positive result in the milk allergen-specific IgE antibody test, and the non-specific binding of heterophilic antibodies in the sample to the microplate will also cause a false positive result in the milk allergen-specific IgE antibody test. Therefore, the inventors conducted a lot of research and first proposed to prepare a blocker by combining bovine serum albumin and Roche blocker with item number 11922122103, which can simultaneously block anti-bovine serum albumin IgG antibodies and heterophilic antibodies without affecting the capture of milk allergen-specific IgE antibodies by anti-human IgE antibodies on the microplate, effectively avoiding false positive results. Furthermore, the inventor prepared a lyophilized product by preparing a blocking agent buffer solution made of bovine serum albumin and Roche blocking agent with product number 11922122103. When using, it can be directly added to the sample to dissolve. The sample volume changes very little and the sample is basically not diluted, thereby obtaining more accurate test results.

[0042] According to the embodiment, the blocker of the present invention is a lyophilized product of Roche blocker with bovine serum albumin and product number 11922122103 as main components.

[0043] Specifically, the preparation method of the blocking agent of the present invention is:

[0044] Firstly, a blocking agent buffer is prepared, and then freeze-dried to obtain the blocking agent, wherein the blocking agent buffer comprises: 0.05-10 g bovine serum albumin, 50-300 μL Roche blocking agent with a product number of 11922122103, and 0.01-1 mL preservative in every 100 mL Tris-HCl buffer with a pH value of 7.0-8.0.

[0045] Specifically, the method for using the blocker of the present invention is to directly mix the blocker with the test sample. For example, the serum sample to be tested can be added to a test tube containing a freeze-dried blocker. After the freeze-dried blocker is completely dissolved, it is directly added to a microplate for enzyme-linked immunosorbent assay.

[0046] 1. The present invention blocks the binding of anti-BSA IgG antibodies in the sample to BSA on the microplate, reducing the false positives caused by the influence of anti-BSA IgG antibodies; it also blocks the non-specific binding of heterophilic antibodies in the sample to the microplate, reducing the false positives caused by the influence of heterophilic antibodies.

[0047] 2. The blocking agent of the present invention will not affect the capture of milk allergen-specific IgE antibodies by anti-human IgE antibodies on the microplate, nor will it interfere with the activity of biotin.

[0048] 3. The blocking agent of the present invention is in the form of a freeze-dried product, which is convenient for storage and reduces the problem of low test results caused by volume changes during serum processing.

[0049] 4. During use, the present invention does not affect the normal detection process, is simple to operate, has a stable product, and has obvious effects.

[0050] The technical scheme and technical effects of the present invention are further described below through examples and comparative examples.

[0051] In the following examples, the implementation conditions not specified are conventional conditions in the industry, and the reagents can be purchased from the market.

[0052] Example 1

[0053] Prepare blocking buffer: weigh 0.6 mg of tris-hydrochloric acid, dissolve in 100 mL of purified water, adjust pH to 7.0-7.5, then add 0.05 mL of Proclin-300, 0.5 g of BSA, and 150 μL of Roche blocking agent (catalog number 11922122103), mix well, and completely dissolve the solid matter.

[0054] The blocking agent is obtained by packaging and lyophilizing in a volume of 50 μL / vial.

[0055] Comparative Example 1

[0056] Prepare blocking buffer: weigh 0.6 mg of tris-hydrochloric acid, dissolve in 100 mL of purified water, adjust pH to 7.0-7.5, then add 0.05 mL of Proclin-300, 0.5 g of BSA, and 50 mg of Roche MAB33 IgG1 / IgG1 multimer blocking agent (catalog number 11939661103), mix well, and completely dissolve the solid matter.

[0057] The blocking agent is obtained by packaging and lyophilizing in a volume of 50 μL / vial.

[0058] Comparative Example 2

[0059] Prepare blocking buffer: weigh 0.6 mg of tris(hydroxymethyl)aminomethane-hydrochloric acid) and dissolve it in 100 mL of purified water, adjust the pH to 7.0-7.5, then add 0.05 mL of Proclin-300 and 50 mg of Roche MAB33IgG1 / IgG1 multimer blocking agent (catalog number 11939661103), mix well, and completely dissolve the solid matter.

[0060] The blocking agent is obtained by packaging and lyophilizing in a volume of 50 μL / vial.

[0061] Example 2

[0062] The performance was verified using the blockers of Example 1 and Comparative Examples 1 and 2, respectively, with the test results without the blockers being used as a control.

[0063] The 24 samples to be tested were all from clinical sera, including 16 negative samples with varying degrees of interference (0.41 IU / mL-86.7 IU / mL), 4 positive samples without interference, and 4 negative samples without interference.

[0064] Detection method:

[0065] Take 50 μL of each of the 24 samples to be tested and add them to the cryopreservation tube containing the freeze-dried product of the blocking agent of Example 1, the cryopreservation tube containing the freeze-dried product of the blocking agent of Comparative Example 1, and the cryopreservation tube containing the freeze-dried product of the blocking agent of Comparative Example 2. After the freeze-dried products are completely dissolved, they are used as samples for testing according to the following experimental steps:

[0066] Step 1: Pipette 50 μL of calibrator, quality control and sample and add them into the corresponding microwells.

[0067] Step 2: Incubate at room temperature for 60 minutes.

[0068] Step 3: Wash the microplate three times using a plate washer (or manually), using 300 μL of washing solution each time. Pat the remaining liquid on absorbent paper to dry the microplate.

[0069] Step 4: Add 100 μL of milk allergen-biotin to the sample and 100 μL of anti-IgE antibody-biotin to the calibrator. Seal the plate and incubate at room temperature for 60 minutes.

[0070] Step 5: After incubation, wash as in step 3.

[0071] Step 6: Add 100 μL of horseradish peroxidase-streptavidin conjugate to each well, seal the plate, and incubate at room temperature for 30 minutes.

[0072] Step 7: Clean as in step 3.

[0073] Step 8: Add 100 μL TMB substrate solution to each well, seal the plate, protect from light, and incubate at room temperature for 30 minutes.

[0074] Step 9: Add 100 μL of stop solution to each well in the order of adding substrate solution, tap the rack to mix the liquid in the microwells. Read the result at 450 nm (reference wavelength 620 nm or 630 nm) with a microplate reader within 5 minutes.

[0075] In this embodiment, the enzyme-linked immunosorbent assay kit used is from the product of Ho-O-Bo with the item number MB00141, and the matching calibration kit is from the product of Ho-O-Bo with the item number MB00132.

[0076] According to the OD value of the calibration sample (Table 1), a four-parameter curve was drawn ( Figure 3 ), the y-axis is the OD value, the x-axis is the concentration, and the sample concentration is calculated according to the four-parameter curve.

[0077] Table 1

[0078]

[0079] The test results of 16 interfered negative samples (0.41 IU / mL-86.7 IU / mL), 4 non-interfered positive samples, and 4 non-interfered negative samples are shown in Tables 2 and Figure 4 .

[0080] Table 2

[0081]

[0082] Table 1 and Figure 4 The results showed that after the 16 interfered negative samples were treated with the blockers of Comparative Examples 1 and 2, there was no complete blocking effect, and the test results were all positive; while after the blockers of Example 1 were treated, the test results were all negative. The use of blockers had no effect on the test results of the 4 non-interfered positive samples and the 4 non-interfered negative samples.

[0083] Example 3

[0084] The performance was verified using Example 1, and then 100 clinical negative samples and 36 clinical positive samples were tested. The test results without blockers were used as controls. The negative compliance rate without blockers was 78%, and the positive compliance rate was 96%. The negative compliance rate with Example 1 was 92%, which was increased by 14%, and the positive compliance rate remained unchanged at 96%. The test results are shown in Table 3.

[0085] Table 3

[0086]

[0087]

[0088] Example 4

[0089] Stability test:

[0090] The lyophilized blocker of Example 1 was destroyed by heating at 37°C for 10 days, and then the samples of Example 2 (only the samples containing the blocker of Example 1) were used for detection according to the method of Example 2, and the lyophilized product stored at 2-8°C for 10 days was used as a control. The detection results are shown in Table 4.

[0091] Table 4

[0092]

[0093] Table 4 shows that the lyophilized blocking agent of Example 1 has high stability and is easy to store.

[0094] The above detailed description of the present invention is intended to enable persons familiar with the art to understand the contents of the present invention and implement them. It does not limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A kit for detecting milk allergen-specific IgE antibodies, characterized in that: It includes a blocker, which is a freeze-dried product, and includes bovine serum albumin and Roche blocker with a product number of 11922122103. The bovine serum albumin can bind to the anti-bovine serum albumin antibody in the sample to be detected, and the Roche blocker with the product number 11922122103 can bind to the heterophilic antibody in the sample to be detected. The preparation method of the blocker is as follows: firstly prepare a blocker buffer, and then freeze-dry to obtain the blocker, wherein the blocker buffer comprises: 0.05-10 g bovine serum albumin, 50-300 μL Roche blocker with a product number of 11922122103, and 0.01-1 mL and / or 0.01-1 g preservative per 100 mL Tris-HCl buffer with a pH value of 7.0-8.0, The kit also includes a microplate coated with anti-human IgE antibody and blocked with bovine serum albumin, milk allergen-biotin, horseradish peroxidase-streptavidin binding solution, 3,3',5,5'-tetramethylbenzidine substrate color development solution, stop solution, and anti-IgE antibody-biotin and / or calibration substance.

2. The kit according to claim 1, characterized in that The sample to be detected is serum.

3. The kit according to claim 1, characterized in that The blocking agent buffer is: every 100 mL of Tris-HCl buffer with a pH value of 7.0 to 8.0 contains 0.01 to 5 g of bovine serum albumin, 50 to 250 μL of Roche blocking agent with a product number of 11922122103, and 0.01 to 0.5 mL of preservative.

4. The kit according to claim 3, characterized in that The preservative is one or more of Proclin-300, 4-aminoantipyrine, and gentamicin sulfate.

5. A method for detecting milk allergen-specific IgE antibodies for non-diagnostic and therapeutic purposes, characterized in that: The test kit according to any one of claims 1 to 4 is used for detection, the blocking agent is mixed with the sample to be detected to obtain a mixed solution, and then the milk allergen-specific IgE antibody in the mixed solution is detected by enzyme-linked immunosorbent assay.

6. The method for detecting milk allergen-specific IgE antibodies by enzyme-linked immunosorbent assay according to claim 5, characterized in that: The milk allergen-specific IgE antibody enzyme-linked immunosorbent assay method comprises the following steps in sequence: (1) mixing the sample to be tested with the blocking agent to obtain a mixed solution; (2) adding the mixed solution of step (1) into the microwells of the microwell plate, and incubating at room temperature to obtain a conjugate of anti-human IgE antibody-milk allergen-specific IgE antibody; (3) After washing the plate, adding the milk allergen-biotin, and incubating at room temperature to obtain a conjugate of anti-human IgE antibody-milk allergen-specific IgE antibody-milk allergen-biotin; (4) After washing the plate, the horseradish peroxidase-streptavidin binding solution is added, and incubated at room temperature to obtain a conjugate of anti-human IgE antibody-milk allergen-specific IgE antibody-milk allergen-biotin-streptavidin-horseradish peroxidase; (5) After washing the plate, add the color developing solution to develop color, add the stop solution to terminate the reaction, and then detect the absorbance.

7. The method for detecting milk allergen-specific IgE antibodies by enzyme-linked immunosorbent assay according to claim 6, characterized in that: The stop solution used in step (5) is 0.2-0.8 mol / L H2SO4, and the detection wavelength is 420-480 nm.

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