A sample treatment agent for reducing interference in allergen-specific IgE antibody detection

Abstract

The present invention relates to a sample treatment agent for reducing interference in allergen-specific IgE antibody detection. The sample treatment agent of the present invention comprises a magnetic particle complex formed by coupling streptavidin magnetic particles and a biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides, wherein the biotin in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides is hydrazide-PEG-biotin or alkoxyamine-PEG-biotin. The present invention develops for the first time a sample treatment agent capable of reducing interference in allergen-specific IgE antibody detection. By using the sample treatment agent of the present invention to treat the sample to be detected and then performing allergen-specific IgE antibody detection, higher sensitivity and fewer false positive and false negative results can be achieved, that is, the accuracy of the test result is higher.

Description

Technical Field

[0001] The present invention belongs to the technical field of in vitro diagnosis, and in particular relates to a sample treatment agent for reducing interference in detection of allergen-specific IgE antibodies. Background Art

[0002] The importance of allergen-specific IgE (SIgE) detection in the diagnosis of allergic diseases has been widely recognized. Currently, SIgE detection mainly relies on immunoassays, including qualitative and semi-quantitative tests such as enzyme immunoblotting and linked immunosorbent assay, and quantitative tests such as fluorescent enzyme immunoassay and magnetic microparticle chemiluminescence. Regardless of the immunoassay method, there are three factors that greatly interfere with the test results.

[0003] The first is allergen-specific IgG antibodies. When allergens enter the human body, they not only induce the production of IgE, but also induce the production of a large amount of IgG. During immunoassays, these IgGs in the serum will compete with IgE for binding to allergens on the solid phase, resulting in false low values ​​or false negatives. The second is cross-reactive carbohydrate determinants (CCD). CCD is a sugar-modified part common to cell surface molecules of many plant and insect species. After entering the human body, IgE antibodies against them will be produced. These antibodies usually do not cause clinical allergic symptoms. Since CCD is widely present in a variety of allergens and produces cross-reactions, Anti-CCD-IgE will cause multiple serum SIgE test results to be positive, while there is no clinical allergy, that is, "false positives". The third is antibodies or non-specific adsorption produced against the surface groups of the immunoassay solid phase. If the non-specific antibody in the sample is IgE, it will be recognized by the labeled secondary antibody in the IgE detection reagent, resulting in a false increase in the test results. Summary of the invention

[0004] The object of the present invention is to provide a sample treatment agent for reducing interference in allergen-specific IgE antibody detection.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A sample processing agent for reducing interference in allergen-specific IgE antibody detection, the sample processing agent comprising a magnetic particle complex, the magnetic particle complex being formed by coupling streptavidin magnetic particles with a biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides, the biotin in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides being hydrazide-PEG-biotin or alkoxyamine-PEG-biotin.

[0007] Preferably, the feed ratio of the CCD glycopeptide to the biotinylated anti-human IgG antibody in the biotinylated anti-human IgG antibody linked with multiple CCD glycopeptides is greater than 5:1, more preferably 5 to 20:1, and even more preferably 5 to 10:1.

[0008] Preferably, the molar ratio of the anti-human IgG antibody and biotin in the biotinylated anti-human IgG antibody linked to multiple CCD glycopeptides is 1:(1-10), for example 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10.

[0009] Preferably, the CCD glycopeptide in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides contains an active group, and the active group is an amino group or a carboxyl group.

[0010] Preferably, the CCD glycopeptide in the biotinylated anti-human IgG antibody linked to multiple CCD glycopeptides has no more than 3 amino acid residues.

[0011] Preferably, the CCD glycopeptide in the biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides is obtained by hydrolysis of glycoprotein under the action of non-specific protease, the glycoprotein is selected from one or more of bromelain, horseradish peroxidase, potato storage protein, bee venom phospholipase, and the non-specific protease is selected from one or more of subtilisin, proteinase K, pronase or pepsin.

[0012] Preferably, the degree of polymerization of PEG in the biotin is 4-8, for example, the degree of polymerization is 4, 5, 6, 7 or 8.

[0013] Preferably, the sample processing agent is a lyophilized agent, and the lyophilization buffer consists of an inorganic buffer salt and an excipient.

[0014] Preferably, the sample processing agent further comprises an inorganic buffer salt and an excipient.

[0015] More preferably, the inorganic buffer salt is tris(hydroxymethyl)aminomethane hydrochloride.

[0016] More preferably, the excipient is one or more of sucrose, trehalose or mannitol.

[0017] The present invention also provides a method for preparing the sample treatment agent for reducing interference in allergen-specific IgE antibody detection, which comprises the following steps:

[0018] (1) reacting the anti-human IgG antibody with sodium metaperiodate to obtain aldehyde-activated anti-human IgG;

[0019] (2) reacting the biotin and the aldehyde-activated anti-human IgG in the presence of sodium cyanoborohydride to obtain a biotinylated anti-human IgG antibody after purification;

[0020] (3) activating the biotinylated anti-human IgG antibody with Sulfo-SMCC to obtain a maleimide-activated biotinylated anti-human IgG antibody, treating the CCD glycopeptide with 2-iminothiolane to obtain a sulfhydryl-activated CCD glycopeptide, coupling the maleimide-activated biotinylated anti-human IgG antibody with the sulfhydryl-activated CCD glycopeptide, and purifying to obtain the biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides;

[0021] (4) coupling the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides with the streptavidin magnetic particles to obtain the sample processing agent.

[0022] Preferably, the reactions of step (1), step (2), step (3) and step (4) are all carried out at room temperature.

[0023] Preferably, the molecular weight of the anti-human IgG antibody in step (1) is 8000-14000D, and the amount of sodium metaperiodate used is 8-12mM.

[0024] Preferably, in step (2), the molar ratio of biotin to the aldehyde-activated anti-human IgG is 1.5 to 10:1, the sodium cyanoborohydride is added in the form of a sodium cyanoborohydride solution with a concentration of 0.8 to 1.2 mM, and the amount of the sodium cyanoborohydride solution is 0.8 to 1.2 wt%.

[0025] Preferably, in step (3), the molar ratio of the thiol-activated CCD to the maleimide-activated biotinylated anti-human IgG antibody is (8-12):1.

[0026] Preferably, the purification in step (2) and step (3) is performed independently by dialysis, desalting column elution or ultrafiltration using a PBS buffer having a pH value of 7.0 to 7.8.

[0027] Preferably, in step (4), the streptavidin magnetic particles and the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides are coupled in a lyophilization buffer, and then lyophilized to obtain the sample treatment agent, the lyophilization buffer has a pH value of 7.2-7.6, contains 10-50 mM Tris-HCl, 0.8-1.0 wt% NaCl, 10-20 wt% mannitol and 0.04-0.06 wt% ProClin 300, the amount of the streptavidin magnetic particles in the coupling reaction system is 50-100 mg / mL, and the amount of the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides in the coupling reaction system is 20-50 μg / mg.

[0028] Preferably: the preparation method of the CCD glycopeptide comprises the following steps:

[0029] (1) using a non-specific protease to hydrolyze the CCD glycopeptide in a buffer having a pH value of 7.5 to 8.0 to obtain a first digestion mixture from the glycoprotein;

[0030] (2) separating and collecting glycopeptides in the digested mixture using a dextran gel column;

[0031] (3) hydrolyzing the glycopeptide with the non-specific protease again to obtain a second digestion mixture;

[0032] (4) Purifying the second digestion mixture using an anion exchange chromatography column to obtain the CCD glycopeptide.

[0033] The present invention also provides the use of the sample treatment agent for reducing interference in allergen-specific IgE antibody detection in allergen-specific IgG antibody detection.

[0034] The present invention also provides an allergen-specific IgG antibody detection kit, which comprises the sample treatment agent for reducing interference in allergen-specific IgE antibody detection.

[0035] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0036] The present invention has developed for the first time a sample treatment agent that can reduce interference in allergen-specific IgE antibody detection. Using the sample treatment agent of the present invention to treat the sample to be detected before performing allergen-specific IgE antibody detection can achieve higher sensitivity, fewer false positive and false negative results, that is, the accuracy of the test results is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 It is a schematic diagram of the structure of the sample processing agent of the present invention. DETAILED DESCRIPTION

[0038] Allergen-specific IgE antibody detection is of great significance for the accurate diagnosis of allergic diseases. However, due to the complexity of the components of the sample to be detected, false low values, false negatives and false positives are inevitable, that is, the sensitivity and accuracy are not high. In view of this situation, the inventors have developed a sample treatment agent that can reduce the interference of allergen-specific IgE antibody detection for the first time after a lot of research and experimental verification.

[0039] Specifically, the sample processing agent includes a magnetic particle complex formed by coupling streptavidin magnetic particles and a biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides, wherein the biotin in the biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides is hydrazide-PEG-biotin or alkoxyamine-PEG-biotin.

[0040] The preparation method of the sample treatment agent is as follows:

[0041] (1) reacting the anti-human IgG antibody with sodium metaperiodate to obtain aldehyde-activated anti-human IgG;

[0042] (2) reacting the biotin and the aldehyde-activated anti-human IgG in the presence of sodium cyanoborohydride to obtain a biotinylated anti-human IgG antibody after purification;

[0043] (3) activating the biotinylated anti-human IgG antibody with Sulfo-SMCC to obtain a maleimide-activated biotinylated anti-human IgG antibody, treating the CCD glycopeptide with 2-iminothiolane to obtain a sulfhydryl-activated CCD glycopeptide, coupling the maleimide-activated biotinylated anti-human IgG antibody with the sulfhydryl-activated CCD glycopeptide, and purifying to obtain the biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides;

[0044] (4) coupling the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides with the streptavidin magnetic particles to obtain the sample processing agent.

[0045] Wherein, the preparation method of the CCD glycopeptide comprises the following steps:

[0046] (1) using a non-specific protease to hydrolyze the CCD glycopeptide in a buffer having a pH value of 7 / 5 to 8.0 to obtain a first digestion mixture from the glycoprotein;

[0047] (2) separating and collecting glycopeptides in the digested mixture using a dextran gel column;

[0048] (3) hydrolyzing the glycopeptide with the non-specific protease again to obtain a second digestion mixture;

[0049] (4) Purifying the second digestion mixture using an anion exchange chromatography column to obtain the CCD glycopeptide.

[0050] More specifically, according to some preferred embodiments, the sample treatment agent preparation method is as follows:

[0051] 1. Preparation of CCD glycopeptide:

[0052] 1.1. Use non-specific protease to completely hydrolyze glycoproteins containing a large number of CCD structures to obtain a mixture of CCD glycopeptides, free amino acids, and polypeptide fragments;

[0053] 1.2. The mixture is subjected to gel chromatography column chromatography to collect CCD glycopeptides and polypeptides with larger molecular weight;

[0054] 1.3. Concentrate the above-preliminarily purified CCD glycopeptide by rotary evaporation, and repeat steps (1) and (2) several times;

[0055] 1.4. The larger polypeptides in the products of multiple hydrolysis are basically decomposed into small peptides and amino acids. The molecular weight of the glycopeptide containing CCD structure is above 1000 Daltons, and it is separated and purified again by gel chromatography column chromatography;

[0056] 1.5. The above product is concentrated again by rotary evaporator and further purified by anion exchange chromatography to obtain CCD glycopeptide with a purity greater than or equal to 95%.

[0057] 2. Conjugated anti-human IgG antibody

[0058] 2.1. Humanized anti-human IgG antibody coupled with biotin. In this step, the activated group selected is the aldehyde group, the anti-human IgG antibody is treated with sodium metaperiodate, the selected biotin is hydrazide-PEG-biotin or alkoxyamine-PEG-biotin, and the PEG polymerization amount in the biotin is 4 to 8.

[0059] 2.2. Coupling of CCD glycopeptide with biotinylated humanized anti-human IgG antibody. The two-step method of glutaraldehyde, or a homobifunctional cross-linker with two succinimides / sulfonyl succinimides, or a heterobifunctional cross-linker with succinimide and maleimide can be used to covalently couple CCD glycopeptide with biotinylated humanized anti-human IgG antibody. The feed ratio of CCD glycopeptide to biotinylated humanized anti-human IgG antibody must be greater than or equal to 5:1, so that multiple CCD glycopeptides can be connected to one anti-human IgG antibody.

[0060] 3. Connecting magnetic particles

[0061] The prepared biotinylated anti-human IgG antibody linked to multiple CCD glycopeptides was coated onto streptavidin (SA) magnetic particles to form a magnetic particle complex of magnetic particle-streptavidin-biotin-anti-human IgG antibody-CCD glycopeptide (eg Figure 1 This complex can then be used for sample processing.

[0062] 4. Freeze-drying

[0063] For easier storage and use, the magnetic particle complex is freeze-dried. After washing the magnetic particle complex three times with freeze-drying buffer, dilute it to a fixed concentration, mix it evenly, and then put it into -80℃ for pre-freezing. After the freeze dryer is turned on, pre-freeze it first, wait for the freeze-drying chamber to cool down to below -50℃, and then put the pre-frozen magnetic particle complex into the freeze dryer for freeze drying to obtain the final sample treatment agent. The freeze-drying buffer contains only buffer salts and excipients, and does not contain other metal ions, chelating agents and protein components.

[0064] The present invention uses a glycoprotein antigen containing a CCD structure as a raw material, and uses a non-specific protease to hydrolyze it to obtain a CCD glycopeptide. Glycosidase cannot be used to directly cleave the glycoside. If a glycosidase is used to break the glycosidic bond, the CCD glycoside will have no extra active groups for the next step of coupling (coupling monosaccharides will cause changes in CCD immunity). Therefore, a non-specific protease is selected, such as subtilisin, proteinase K, pronase, pepsin, etc., preferably pronase, to hydrolyze the glycoprotein into a CCD glycopeptide containing a peptide segment, and the amino or carboxyl group on the peptide chain can be activated for coupling.

[0065] The present invention uses glycoprotein antigens containing CCD structures as hydrolysis raw materials. It is currently recognized that CCD is an N-terminal oligosaccharide of plant and animal glycoproteins, and its structural characteristics are α (1,3) fucose and β (1,2) xylose. These two antigenic epitopes and other parts of the glycosyl group synergistically induce the body to produce Anti-CCD-IgE, which is the structural basis for causing multiple cross reactions. Therefore, it is necessary to select polysaccharide glycoproteins containing these two glycosyl structures at the same time, including bromelain from pineapple stems, horseradish peroxidase from horseradish, potato tuber storage protein from potatoes, phospholipase from bee venom, etc., preferably horseradish peroxidase.

[0066] The glycoprotein containing the CCD structure of the present invention cannot be used directly and needs to be hydrolyzed by protease several times. Macromolecular proteins or incompletely hydrolyzed long-chain glycopeptides may contain other IgE antibody binding sites, and as adsorbents, new interference may be introduced. Preferably, the CCD glycopeptide obtained by hydrolysis twice is purified and analyzed by mass spectrometry, and 95% or more of the CCD glycopeptides have no more than 3 amino acid residues.

[0067] The immunoreactivity of the CCD glycopeptide small molecule monomer of the present invention is relatively weak, and the reactivity can be improved by multivalent coupling to a macromolecule.

[0068] In order to prevent cross-reaction of proteins from other sources, the macromolecule selected in the present invention should be human or other antigens that do not interfere with IgE detection. Anti-human IgG antibodies can be used to bind and separate IgG in samples, and anti-human IgG is humanized, avoiding interference from proteins from other sources. It is preferably directly connected to humanized anti-human IgG, and of course, the CCD glycopeptide can also be coupled to other human proteins such as human serum albumin, and then connected to the solid phase with the anti-human IgG antibody to achieve the same effect.

[0069] In order to facilitate the coupling of SA magnetic particles, the humanized anti-human IgG antibody of the present invention is first coupled with biotin before coupling with CCD glycopeptide. The activated group selected in this step is an aldehyde group, and the selected biotin is hydrazide-PEG-biotin or alkoxyamine-PEG-biotin. NHS-biotin conventionally used in the art uses primary amine as the activating group, and the subsequent CCD glycopeptide coupling step also requires the use of primary amine groups. The use of NHS-biotin will result in extremely low titer of CCD glycopeptide coupling, and coupling with CCD glycopeptide will also change the hydrophilicity of the protein, which will easily lead to precipitation. Therefore, the present invention uses activated aldehyde coupling, and long-chain PEG adds hydrophilicity. Preferably, the degree of polymerization of PEG in biotin is 4 to 8.

[0070] Conventionally used blocking agents or adsorbents are added to reagents or samples to be tested, and the interfering substances still exist after treatment and are not completely removed. However, the present invention uses magnetic particles, which combine with the interfering substances and are separated from the samples by magnetic separation or centrifugation to completely remove the interfering substances.

[0071] The present invention takes into account that individuals infected with Streptomyces have a probability of producing anti-streptavidin IgE antibodies (Anti-SA-IgE), and the sIgE detection method using SA magnetic particles as a solid phase may produce false positive results when detecting samples of such individuals. Therefore, streptavidin magnetic particles are selected, and not only can CCD-anti-human IgG antibodies be conveniently connected to magnetic particles by coupling biotin, but the streptavidin group on the magnetic beads can also be used to adsorb Anti-SA-IgE in the sample, avoiding the false positive test results caused by the above interference.

[0072] The present invention uses a freeze-drying process, and the freeze-drying buffer contains only inorganic buffer salts and excipients, so that the treatment agent does not contain liquid components and protein components that interfere with detection, and does not dilute the sample to be detected during use, thereby not reducing the sensitivity of IgE detection. The inorganic buffer salt is preferably 50mM Tris, and the excipient can be sucrose, trehalose, mannitol, etc., preferably 20wt% mannitol.

[0073] Method of using the sample treatment agent of the present invention:

[0074] (1) directly adding the sample to be processed into a bottle of the sample treatment agent, gently mixing until the solid is evenly dispersed, and leaving it at room temperature to react for a period of time;

[0075] (2) Use a magnetic separation device or centrifuge to separate the magnetic particles from the sample. When the magnetic particles settle to the bottom, carefully aspirate the supernatant;

[0076] (3) The treated samples can be used for subsequent allergen-specific IgE detection. Allergen-specific IgE detection can be performed using conventional methods in the art.

[0077] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0078] The reagents and the like used in the present invention are all commercially available.

[0079] The room temperature in the present invention refers to 25±5°C.

[0080] In the present invention, CCD glycopeptide refers to a short peptide containing a CCD structure.

[0081] Example 1

[0082] This embodiment provides a sample treatment agent, and the preparation method thereof is as follows:

[0083] 1. Preparation of CCD glycopeptide:

[0084] 1.1. Dissolve horseradish peroxidase (from horseradish) powder with purity >99% in 0.15M Tris-HCl pH 7.8, 1mM CaCl2 at a ratio of 1% (W / V);

[0085] 1.2. Add pronase (from Sigma-Aldrich) to the above horseradish peroxidase solution at a ratio of 2% (W / V) and incubate at 37°C overnight;

[0086] 1.3. The digested mixture was passed through a Sephadex G-25 (GE Healthcare 2.5×120 cm) column with 1% (volume percentage) acetic acid as the mobile phase to preliminarily separate the CCD glycopeptides from macromolecular proteins, small peptides, and amino acids. The glycopeptide fraction was collected and concentrated using a rotary evaporator;

[0087] 1.4. Add 0.5% pronase by mass, digest again, and incubate at 37°C overnight.

[0088] 1.5. The glycopeptide was further purified using an anion exchange chromatography column. Sephadex DEAE A-25 (GE Healthcare, 2.5×20 cm) was equilibrated with 50 mM ammonium acetate at pH 8.8, and the CCD glycopeptide fraction was eluted last.

[0089] 2. Preparation of biotinylated anti-human IgG antibody:

[0090] The anti-human IgG antibody was dialyzed with 0.02M PBS at pH 6.5 for 6 hours, the molecular cutoff of the dialysis bag was 8000-14000D, and the liquid was changed once in the middle. After dialysis, it was treated with 10mM sodium metaperiodate and reacted at room temperature for 20 minutes to activate the aldehyde group on IgG; hydrazide-PEG-biotin (PEG polymerization degree is 4) was added, and the reaction molar ratio of anti-human IgG antibody to biotin was 1:2. After reacting at room temperature for 20 minutes, 1wt% of 1mM sodium cyanoborohydride was added. After reacting at room temperature for 10 minutes, it was dialyzed with 0.02M PBS at pH 7.4 for 6 hours, and the liquid was changed once in the middle to obtain biotinylated anti-human IgG antibody.

[0091] 3. IgG adsorbent coupled to CCD

[0092] 3.1. Treat CCD with 2-iminothiolane to obtain thiol-activated CCD;

[0093] 3.2. Activate the above biotinylated anti-human IgG with Sulfo-SMCC to obtain maleimide-activated biotinylated anti-human IgG antibody;

[0094] 3.3 The above-mentioned thiol-activated CCD and maleimide-activated biotinylated anti-human IgG antibody were coupled at a molar ratio of 10:1, mixed and reacted at room temperature for 30 minutes; dialyzed with 0.02M PBS at pH 7.4 for 6 hours, changing the solution once in the middle to obtain biotinylated anti-human IgG antibody-CCD glycopeptide. The same effect can be achieved by using a desalting column or ultrafiltration tube instead of dialysis.

[0095] 4. Connecting magnetic particles:

[0096] Streptavidin magnetic particles (Sichuan Xigerui Biotechnology Co., Ltd.) were diluted to 50 mg / mL with lyophilization buffer (20 mM Tris-HCl, 0.9 wt% NaCl, 20 wt% mannitol, 0.05 wt% ProClin 300, pH 7.2-7.6), and the above biotinylated anti-human IgG antibody-CCD glycopeptide was added at a dosage of 20 μg / mg, and mixed and reacted at room temperature for 30 minutes. After washing three times with lyophilization buffer, the volume was adjusted to 50 mg / mL.

[0097] 5. Freeze-drying:

[0098] Mix the solution in step 4 evenly and pre-freeze it in a -80℃ refrigerator for 8 hours. Turn on the freeze dryer, pre-freeze until the temperature in the freeze drying room is below -55℃, put in the pre-frozen treatment agent, and start freeze drying. Adjust the temperature to below the eutectic temperature of 10-20℃ and keep it for 1 hour; heat it to near the eutectic temperature and keep it for 1 hour; then cool it down to below the eutectic temperature of 10-20℃ and keep it for 2 hours; sublimation: heat it up and maintain it below the eutectic temperature by 5℃ for 15 hours, and control the vacuum degree at 2.5Pa; desorption: heat it up to room temperature and keep it under high vacuum for 6 hours, and finally get a finished product that can be used directly.

[0099] The finished product is a brown freeze-dried powder, which becomes a brown (yellow) liquid after reconstitution. After standing for a long time, the supernatant becomes clear and the magnetic particles settle at the bottom.

[0100] Comparative Example 1

[0101] This comparative example provides a sample treatment agent, and its preparation method is basically the same as that of Example 1, except that 1 mM sodium metaperiodate is used in step 2, and the prepared sample treatment agent has no adsorption effect on Anti-SA-IgE.

[0102] Comparative Example 2

[0103] This comparative example provides a sample treatment agent, and its preparation method is basically the same as that of Example 1, except that NHS-biotin without a spacer arm is used in step 2. A large amount of white precipitate appears after the reaction. It is speculated that the change in hydrophobicity causes protein precipitation, resulting in a low titer.

[0104] Comparative Example 3

[0105] This comparative example provides a sample treatment agent, and its preparation method is basically the same as that of Example 1, except that the reaction molar ratio of the anti-human IgG antibody and biotin in step 2 is selected to be 1:20.

[0106] Effect test:

[0107] Experiment 1:

[0108] The test samples are from the remaining clinical samples of the hospital test, and the test results of the samples without sample treatment, using the sample treatment agent of Example 1, the sample treatment agent of Comparative Example 1 and the sample treatment of Comparative Example 3, that is, after the sample treatment, are compared.

[0109] The sample processing method is:

[0110] 1. Mixing reaction: Add 500 μL of the sample to be treated directly into a bottle of allergy sample treatment agent, mix gently until the solid is evenly dispersed, and leave it at room temperature for 15 minutes;

[0111] 2. Separation of magnetic beads and samples:

[0112] (1) Method 1: Use a magnetic separation device. Place the mixture on the magnetic separation device and let it stand for 3 minutes until the magnetic particles are adsorbed by the magnetic field. Carefully aspirate the supernatant.

[0113] (2) Method 2: Use a centrifuge to transfer the entire mixture into a centrifuge tube and centrifuge at 3000 RPM for 3 minutes. When the magnetic particles settle to the bottom, carefully aspirate the supernatant.

[0114] 3. When aspirating the supernatant, if the magnetic particles are accidentally carried out, repeat step 2. The sample can be used for subsequent allergen-specific IgE testing after processing.

[0115] Allergen-specific IgE was detected using the indirect method of magnetic particle chemiluminescent immunoassay.

[0116] The experimental method is: the sample to be tested is mixed with magnetic particles and biotin-labeled house dust mite allergens and incubated for 10 minutes. After washing, alkaline phosphatase-labeled mouse anti-human IgE secondary antibody is added to react for 10 minutes to form a solid phase antigen-antibody-enzyme-labeled secondary antibody complex. The unbound enzyme-labeled antibody and other substances are removed by washing. The chemiluminescent substrate 3-(2-spiroadamantane)-4-methoxy-4-(3-phosphoinosyl)-phenyl-1,2-dioxetane disodium salt (AMPPD) is added. The luminescent substrate emits photons under the catalysis of alkaline phosphatase, and the number of photons generated is proportional to the concentration of house dust mite-specific IgE in the sample.

[0117] The test results are shown in Table 1.

[0118] Table 1

[0119] Sample No. No RLU processing Example 1 Comparative Example 1 Comparative Example 3 1 150435 12284 142701 92415 2 183204 13103 191462 102079 3 241907 12846 233269 129750 4 364354 13302 365328 179744 5 863931 12183 859702 410326

[0120] Table 1 shows that for 5 samples with negative clinical diagnosis but high signal values, after being treated with the sample treatment agent prepared in Example 1 (final sodium metaperiodate concentration of 10 mM, anti-human IgG:biotin=1:2), the signal values ​​significantly decreased to the background level, while the signal values ​​of the sample treatment agents prepared in Comparative Examples 1 and 3 did not decrease significantly. Therefore, the sample treatment agent prepared in Example 1 has the best effect in reducing false positives.

[0121] Experiment 2:

[0122] A group of CCD positive samples were treated with the sample treatment agent of Example 1 and then tested, and the data were compared with those of direct testing without treatment. The treatment and testing methods were the same as those of Experiment 1. The results are shown in Table 2.

[0123] Table 2

[0124]

[0125]

[0126] Table 2 shows that two samples with negative clinical diagnosis but weak positive results in multiple tests were negative after treatment, which is consistent with the clinical diagnosis results. It can be seen that the sample treatment agent of Example 1 can effectively inhibit CCD false positives.

[0127] Experiment 3:

[0128] A group of multi-item positive samples containing anti-streptavidin antibodies were treated with the sample treatment agent of Example 1, and the magnetic beads were removed after 15 minutes of reaction. The data were compared with those of the untreated samples directly tested. The processing and testing methods were the same as those of Experiment 1. The results are shown in Table 3.

[0129] Table 3

[0130]

[0131] Table 3 shows that after two samples with negative clinical diagnosis but positive test items were treated with the sample treatment agent, the results were both negative. It can be seen that the sample treatment agent of Example 1 can effectively inhibit false positives caused by the interference of anti-CCD antibodies and anti-streptavidin antibodies. In addition, the form of lyophilized powder will not affect the concentration of other substances to be tested in the sample, and the magnetic beads also have the advantage of being easy to remove.

[0132] Experiment 4: A group of samples containing a large amount of IgG antibody interference were treated with the sample treatment agent of Example 1, and the data were compared with those of the untreated samples directly tested, and compared with the Phadia test results. The treatment and testing methods were the same as those of Experiment 1. The results are shown in Table 4.

[0133] Table 4

[0134]

[0135] Table 4 shows that the sample treatment agent of Example 1 can adsorb a large amount of IgG antibodies that interfere with IgE detection, so that samples that were missed due to the interference of IgG antibodies can be detected as positive, which is consistent with the Phadia test results.

[0136] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to 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 sample treatment agent for reducing interference in allergen-specific IgE antibody detection, characterized in that: The sample treatment agent comprises a magnetic particle complex, wherein the magnetic particle complex is formed by coupling streptavidin magnetic particles with a biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides, wherein the biotin in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides is hydrazide-PEG-biotin or alkoxyamine-PEG-biotin, wherein the molar ratio of the anti-human IgG antibody to biotin in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides is 1:1-10, wherein the CCD glycopeptides in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides are obtained by hydrolysis of glycoproteins under the action of non-specific proteases, wherein the glycoproteins are selected from one or more of bromelain, horseradish peroxidase, potato storage protein, and bee venom phospholipase, and the non-specific proteases are selected from one or more of subtilisin, proteinase K, pronase, or pepsin, wherein the sample treatment agent further comprises an inorganic buffer salt and an excipient, and the sample treatment agent is a lyophilizer, wherein the lyophilization buffer is composed of an inorganic buffer salt and an excipient.

2. The sample processing agent according to claim 1, characterized in that: The feed ratio of the CCD glycopeptide to the biotinylated anti-human IgG antibody in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides is greater than 5:1; and / or, the CCD glycopeptide in the biotinylated anti-human IgG antibody connected with a plurality of CCD glycopeptides contains an active group, and the active group is an amino group or a carboxyl group; And / or, the CCD glycopeptide in the biotinylated anti-human IgG antibody linked to a plurality of CCD glycopeptides has no more than 3 amino acid residues.

3. The sample processing agent according to claim 1, characterized in that: The degree of polymerization of PEG in the biotin is 4-8.

4. The sample processing agent according to claim 1, characterized in that: The inorganic buffer salt is tris(hydroxymethyl)aminomethane hydrochloride; and / or the excipient is one or more of sucrose, trehalose or mannitol.

5. The method for preparing a sample treatment agent for reducing interference in allergen-specific IgE antibody detection according to any one of claims 1 to 4, characterized in that: It includes the following steps: (1) reacting the anti-human IgG antibody with sodium metaperiodate to obtain aldehyde-activated anti-human IgG, wherein the amount of sodium metaperiodate used is 8-12 mM; (2) reacting the biotin and the aldehyde-activated anti-human IgG in the presence of sodium cyanoborohydride to obtain a biotinylated anti-human IgG antibody after purification; (3) activating the biotinylated anti-human IgG antibody with Sulfo-SMCC to obtain a maleimide-activated biotinylated anti-human IgG antibody, treating the CCD glycopeptide with 2-iminothiolane to obtain a sulfhydryl-activated CCD glycopeptide, coupling the maleimide-activated biotinylated anti-human IgG antibody with the sulfhydryl-activated CCD glycopeptide, and purifying to obtain the biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides; (4) The biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides is coupled to the streptavidin magnetic particles to obtain the sample processing agent.

6. The preparation method according to claim 5, characterized in that: The reactions of step (1), step (2), step (3) and step (4) are all carried out at room temperature; and / or, the molecular weight of the anti-human IgG antibody in step (1) is 8000-14000D; And / or, in step (2), the molar ratio of biotin to the aldehyde-activated anti-human IgG is 1.5-10:1, the sodium cyanoborohydride is added in the form of a sodium cyanoborohydride solution with a concentration of 0.8-1.2 mM, and the amount of the sodium cyanoborohydride solution is 0.8-1.2 wt %; and / or, in step (3), the molar ratio of the thiol-activated CCD to the maleimide-activated biotinylated anti-human IgG antibody is 8-12:1; And / or, the purification in step (2) and step (3) is performed independently by dialysis, desalting column elution or ultrafiltration using a PBS buffer having a pH value of 7.0 to 7.8; And / or, in step (4), the streptavidin magnetic particles and the biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides are coupled in a lyophilization buffer, and then lyophilized to obtain the sample treatment agent, the lyophilization buffer has a pH value of 7.2-7.6, contains 10-50 mM Tris-HCl, 0.8-1.0 wt% NaCl, 10-20 wt% mannitol and 0.04-0.06 wt% ProClin 300, the amount of the streptavidin magnetic particles in the coupling reaction system is 50-100 mg / mL, and the amount of the biotinylated anti-human IgG antibody connected with multiple CCD glycopeptides in the coupling reaction system is 20-50 μg / mg.

7. The preparation method according to claim 5, characterized in that: The preparation method of the CCD glycopeptide comprises the following steps: (1) using a non-specific protease to hydrolyze the CCD glycopeptide in a buffer having a pH value of 7.5 to 8.0 to obtain a first digestion mixture from the glycoprotein; (2) using a dextran gel column to separate and collect glycopeptides in the digested mixture; (3) hydrolyzing the glycopeptide with the non-specific protease again to obtain a second digestion mixture; (4) Purifying the second digestion mixture using an anion exchange chromatography column to obtain the CCD glycopeptide.

8. Use of the sample treatment agent for reducing interference in allergen-specific IgE antibody detection according to any one of claims 1 to 4 in the processing of samples for allergen-specific IgG antibody detection.

9. An allergen-specific IgG antibody detection kit, characterized in that: The allergen-specific IgG antibody detection kit comprises the sample processing agent for reducing interference in allergen-specific IgE antibody detection according to any one of claims 1 to 4.

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