Hydrophobic interferents, methods of making and using the same

Abstract

The application relates to a hydrophobic interferent and a preparation method and application thereof. The application provides a preparation method of a hydrophobic interferent, which comprises the following steps: adding a lysis agent into a whole blood sample, fully lysing blood cells in the whole blood sample and releasing contents, centrifuging to obtain supernatant; adding an oxidizing agent into the supernatant, oxidizing the contents to improve hydrophobicity, centrifuging to obtain supernatant, and obtaining the hydrophobic interferent. The hydrophobic interferent can be used for detecting the interference performance of an anti-hydrophobic interferent in an immunodiagnostic reagent, such as a POCT, ELISA, CLIA and the like. In clinical detection, potential interference can be identified and optimized in advance to improve the reagent, and false positive or false negative results in clinical sample detection can be prevented in advance.

Description

Technical Field

[0001] This application relates to the field of in vitro diagnostic technology, specifically to a hydrophobic interfering agent, its preparation method, and its application. Background Technology

[0002] Common interferences in immune responses include endogenous and exogenous interferences. Common endogenous interferences include hemolysis, lipemia, total protein, analogues, high IgG or IgM levels, rheumatoid factor, heterophile antibodies, and biotin interference. Common exogenous interferences include drug interference and bacterial or viral infections. One of the fundamental causes of interference in immune responses is hydrophobic interaction. Interfering substances use hydrophobic interactions to non-specifically bridge the coated conjugate and labeled conjugate, or to non-specifically bridge the reaction cup (reaction well) with the labeled conjugate, producing false positive signals. Protein molecules are composed of amino acids, and different amino acids have different levels of hydrophilicity and hydrophobicity. Therefore, different proteins exhibit significant differences in overall hydrophilicity and hydrophobicity due to variations in the amount of hydrophilic and hydrophobic amino acids they carry. Serum and plasma contain various proteins, each exhibiting different levels of hydrophobicity, potentially interfering with detection reagents based on immune response principles. Furthermore, the protein content varies among individuals, resulting in varying degrees of interference with the immune response.

[0003] Currently, methods for assessing anti-interference capabilities, such as common endogenous and exogenous substances, cannot effectively reflect the anti-interference capability of the reaction system against hydrophobic interfering substances. Summary of the Invention

[0004] Therefore, it is necessary to provide a method for preparing a hydrophobic interfering agent and the prepared hydrophobic interfering agent, which can be used to evaluate the ability of the immune response to resist hydrophobic forces.

[0005] The specific technical solution is as follows:

[0006] A first aspect of this application provides a method for preparing a hydrophobic interfering substance, comprising the following steps:

[0007] Add a lysis agent to the whole blood sample to fully lyse the blood cells and release their contents, then centrifuge and collect the supernatant.

[0008] An oxidant is added to the supernatant to oxidize the contents and improve hydrophobicity. The supernatant is then collected by centrifugation to obtain the hydrophobic interfering substance.

[0009] In one embodiment, the source of the whole blood sample includes peripheral blood from a human or animal.

[0010] In one embodiment, the lysis buffer comprises Triton X-100.

[0011] In one embodiment, the oxidant includes one or more of H2O2, urea peroxide, and potassium permanganate.

[0012] In one embodiment, the final concentration of the oxidant in the reaction system is 0.5% (v / v) to 8% (v / v); the final concentration of the pyrolysis agent in the reaction system is 0.05% (v / v) to 4% (v / v).

[0013] A second aspect of this application provides a hydrophobic interfering substance prepared by the method described in any of the preceding claims.

[0014] A third aspect of this application provides a kit comprising the hydrophobic interfering agent described above.

[0015] A fourth aspect of this application provides a method for assessing the resistance to hydrophobic interactions in an immune response, comprising using the hydrophobic interfering agent or the kit to assess the resistance to hydrophobic interactions in an immune response.

[0016] In one embodiment, the method includes the following steps:

[0017] The hydrophobic interfering substance was gradient diluted to prepare multiple hydrophobic interfering substance solutions of different concentrations;

[0018] The hydrophobic interfering solutions of different concentrations were extracted using an extractant, with the organic phase serving as the interfering sample and the aqueous phase serving as the control sample.

[0019] Interferant samples and control samples were added to the reaction vessel along with the immunoassay reagent, and the luminescence values ​​were measured.

[0020] The resistance of the reaction vessel and / or immunoassay reagent in the immunoassay to hydrophobic interactions was analyzed based on the luminescence values ​​of the interfering and control samples.

[0021] Optionally, the immunoassay reagent includes a solid-phase coating and / or a label conjugate.

[0022] Further optionally, the label conjugate includes a label-labeled antigen or antibody, and the solid-phase coating includes a solid-phase coated antigen or antibody.

[0023] In one embodiment, the immunoassay reagent includes a label conjugate.

[0024] Optionally, the immunoassay reagent includes a solid-phase coating and a label conjugate, and the reaction vessel is made of a hydrophobic material.

[0025] A fifth aspect of this application provides the application of the hydrophobic interfering agent or the kit described herein in detecting the interference performance of at least one of an immunodiagnostic reagent, reaction vessel, antigen to be tested, and antibody to be tested in an immune response.

[0026] Compared with the prior art, this application has the following beneficial effects:

[0027] This application provides a method for preparing hydrophobic interfering agents in immune reactions. The prepared hydrophobic interfering agents can be used in immunodiagnostic reagents, such as POCT, ELISA, CLIA, etc., to study the interference performance of anti-hydrophobic interfering agents. During the product development and validation phase, early detection of the interference performance of anti-hydrophobic interfering agents replaces the need for large-scale clinical sample testing, saving development time and costs. Simultaneously, potential interferences can be identified and optimized in advance to improve reagents and prevent false positives or false positive results in clinical sample testing. Attached Figure Description

[0028] Figure 1 A schematic diagram illustrating the false positives caused by the binding of solid-phase coated antibodies and antibody markers mediated by hydrophobic interferences;

[0029] Figure 2 This is a schematic diagram illustrating how hydrophobic interfering substances mediate the binding of solid-phase coated antigens and antigen markers, leading to false positives. Detailed Implementation

[0030] To make the above-mentioned objectives, features, and advantages of this application more apparent and understandable, the specific embodiments of this application are described in detail below with reference to the accompanying drawings. Many specific details are set forth in the following description to provide a thorough understanding of this application. However, this application can be implemented in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of this application. Therefore, this application is not limited to the specific embodiments disclosed below.

[0031] 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 this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0032] Terminology Explanation

[0033] The terms "first aspect," "second aspect," "third aspect," "fourth aspect," and "fifth aspect" 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 aspect," "second aspect," "third aspect," "fourth aspect," or "fifth aspect" may explicitly or implicitly include at least one of those features. In the description of this application, "multiple" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0034] The term “and / or” includes any and all combinations of one or more of the related listed items.

[0035] The term "hydrophobic interaction" refers to the forces that exist between two and / or more hydrophobic substances, enabling them to bond together.

[0036] The abbreviation "CLIA" refers to chemiluminescent immunoassay, which is an immunoassay method that uses chemiluminescent agents to directly label antigens or antibodies.

[0037] The abbreviation "POCT" refers to point-of-care testing, which mainly refers to rapid testing and analysis technologies performed near the patient's bedside. These technologies can be conducted at the bedside, in wards, or in locations other than central laboratories and testing departments. They include, but are not limited to, immunoturbidimetry and immunochromatography.

[0038] The term "ELISA" refers to enzyme-linked immunosorbent assay, which is a qualitative and quantitative detection method that uses the specific binding of antigens and antibodies to bind soluble antigens or antibodies onto a solid-phase carrier to perform an immune reaction.

[0039] One embodiment of this application provides a method for preparing a hydrophobic interfering agent, which can be used to study the anti-hydrophobic interference performance of diagnostic reagents based on the principle of immune reaction, such as immunochromatography, ELISA, CLIA, and other immunodiagnostic reagents, in order to prevent false positive results in large-scale clinical sample testing. Specifically, the method includes the following steps a to b:

[0040] Step a: Add lysis agent to the whole blood sample to fully lyse the blood cells and release their contents, then centrifuge and collect the supernatant.

[0041] In a specific example, Triton X-100 is preferred as the lysis agent, but other cell lysis solutions such as RIPA can also be used.

[0042] In one specific example, the final concentration of the lysing agent in the reaction system is 0.05% (v / v) to 4% (v / v). Optionally, the final concentration of the lysing agent in the reaction system can be any of the following values: 0.05%, 0.1%, 0.5%, 1%, 2%, 3%, or 4%. Preferably, 1% is used to ensure more complete cell lysis and an appropriate concentration.

[0043] Specifically, a lysing agent is added to the whole blood sample to thoroughly mix the lysing agent with the blood cells in the whole blood sample, and then incubated to allow the blood cells in the whole blood sample to fully lyse and release their contents.

[0044] Specifically, there are no restrictions on the mixing method; you just need to mix the two components evenly. Optionally, you can invert the mixture.

[0045] Specifically, incubation is performed at room temperature for 20-40 minutes, preferably 30 minutes. Centrifugation is performed at 2-8°C and 5000g for 10-30 minutes, preferably 20 minutes.

[0046] Step b: Add an oxidant to the supernatant to oxidize the contents and improve hydrophobicity. Centrifuge to collect the supernatant and obtain the hydrophobic interfering substance.

[0047] In one specific example, the oxidant includes one or more of H2O2, urea peroxide, and potassium permanganate.

[0048] In one specific example, the final concentration of the oxidant in the reaction system is 0.5% (v / v) to 8% (v / v). Optionally, the final concentration of the oxidant in the reaction system can be 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, or 8%. Hydrophobic interferants prepared within the above ranges all exhibit strong hydrophobicity, preferably 4%, with even stronger hydrophobicity.

[0049] Specifically, the oxidation conditions are 37°C for 10-20 minutes. Centrifugation is performed at 2-8°C and 5000g for 10-30 minutes, preferably 20 minutes.

[0050] An embodiment of this application also provides a hydrophobic interfering substance prepared by the above method and a kit comprising the above hydrophobic interfering substance.

[0051] An embodiment of this application also provides a method for evaluating the ability of an immune response to resist hydrophobic interactions, including using the above-mentioned hydrophobic interfering agent or the above-mentioned kit to evaluate the ability of an immune response to resist hydrophobic interactions.

[0052] In one specific example, the method includes the following steps a to c:

[0053] Step a: Gradually dilute the above hydrophobic interfering substances to prepare multiple hydrophobic interfering substance solutions of different concentrations.

[0054] Specifically, physiological saline is used to dilute the hydrophobic interfering substance, and the dilution ratio can be 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, etc.

[0055] Step b: Use an extractant to extract hydrophobic interference solutions of different concentrations. The organic phase is used as the interference sample, and the aqueous phase is used as the control sample.

[0056] Specifically, the volume ratio of the extractant to the hydrophobic interfering substance can be 2:1, 3:1, etc., as long as the hydrophobic interfering substance can be extracted.

[0057] Alternatively, the extractant may include organic solvents such as chloroform, diethyl ether, and toluene.

[0058] Step c: Add the interfering sample and control sample to the reaction vessel along with the immunoassay reagent and measure the luminescence value.

[0059] Step d: Analyze the resistance of the reaction vessel and / or immunoassay reagent in the immunoassay to hydrophobic interactions based on the luminescence values ​​of the interfering sample and the control sample.

[0060] Optionally, the immunoassay reagent includes a solid-phase coating and / or a label conjugate.

[0061] Specifically, the label conjugate includes antigen labels or antibody labels. Further, the labels include, but are not limited to, acridine esters, ruthenium tripyridine, biotin, etc.

[0062] Specifically, solid-phase coatings include solid-phase coated antigens or solid-phase coated antibodies. Further, the solid phase includes magnetic beads, cellulose, glass, silicone rubber, polyacrylamide, polystyrene, etc.

[0063] In a specific example, the aforementioned hydrophobic interfering agent is used to evaluate the resistance of the reaction vessel in the immunoassay to hydrophobic interactions. Specifically, the evaluation is performed using steps a through d above. The immunoassay reagent includes a labeled conjugate and physiological saline, used to replace the solid-phase coating. Optionally, the specific steps and parameters for detecting the interfering sample and control sample and measuring the luminescence value using the immunoassay reagent should refer to the instructions for use of the immunoassay reagent or immunoassay kit.

[0064] In another specific example, the aforementioned hydrophobic interfering agent is used to evaluate the resistance of the immunoassay reagent to hydrophobic interactions during the immunoassay reaction. Specifically, the evaluation is performed using steps a through d above. The immunoassay reagent includes a solid-phase coating and a labeled conjugate. Interfering samples and control samples are detected separately, and luminescence values ​​are measured. The reaction vessel used in the detection process is made of a hydrophobic material such as glass. Other specific detection and luminescence value measurement steps and parameters can be found in the instructions for the immunoassay reagent or immunoassay kit used.

[0065] The above-mentioned hydrophobic interfering agents or kits are used to detect the interference performance of at least one of immunodiagnostic reagents, reaction vessels, test antigens, and test antibodies against hydrophobic interactions. Optionally, the immunodiagnostic reagents include, but are not limited to, POCT, ELISA, CLIA, and other immunodiagnostic reagents.

[0066] The hydrophobic interfering agent of this application can directly mediate the bridging of the label conjugate to the hydrophobic reaction vessel, generating a false positive signal. Furthermore, the hydrophobic interfering agent can also conjugate antibody or antigen labels with the solid-phase coating, generating a false positive signal. Specifically, the antibody label can bind to the solid-phase coated antibody via the hydrophobic interfering agent prepared in this application, leading to a false positive signal. For the reaction principle, please refer to [link to relevant documentation]. Figure 1 The antigen marker can bind to the solid-phase coated antigen via the hydrophobic interfering agent prepared in this application, resulting in a false positive signal. For the reaction principle, please refer to [link to relevant documentation]. Figure 2 . Specific Implementation

[0068] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0069] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0070] The Triton X-100 and H2O2 used in the following examples were both purchased from Aladdin.

[0071] Example 1

[0072] A method for preparing a hydrophobic interfering substance:

[0073] The interfering agent was prepared by processing fully coagulated whole blood samples from red-capped blood collection tubes. Triton X-100 was added to the blood collection tube at a final concentration (v / v) of 1%, the tube was capped, and the tube was inverted to thoroughly mix the Triton X-100 and blood cells. The mixture was incubated at room temperature for 30 min. The tube was then centrifuged at 5000g for 20 min at 2-8°C, and the supernatant was collected. H₂O₂ at a final concentration (v / v) of 4% was added to the supernatant, and the mixture was treated at 37°C for 20 min. The oxidized supernatant was then centrifuged at 5000g for 20 min at 2-8°C, and the supernatant was collected; this was the interfering agent of this application.

[0074] Final concentration refers to the percentage of the concentration of the cleavage reagent or oxidant in the reaction system.

[0075] Example 2

[0076] The anti-interference ability of the Col IV (type IV collagen) chemiluminescence detection kit was studied using the hydrophobic interfering agent prepared in Example 1.

[0077] The specific steps of the detection method are as follows:

[0078] 1. Prepare solution A with the following composition: 50 mM PB, 150 mM NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.0.

[0079] 2. Prepare solution B with the following formula: 50 mM PB, 150 mM NaCl, 0.5% BSA, 2% Tween-20, 0.1% ProClin300, pH 7.0.

[0080] 3. Acridinium ester was labeled onto Col IV antibody to prepare antibody-labeled conjugates.

[0081] 4. Prepare acridine working solution A by diluting the antibody-labeled conjugate with solution A; prepare acridine working solution B by diluting the antibody-labeled conjugate with solution B.

[0082] 5. The magnetic bead working solution is replaced with physiological saline. When combined with acridine working solution A, it forms reagent A. When combined with acridine working solution B, it forms reagent B.

[0083] 6. Interferants were prepared by diluting them with physiological saline at different gradients of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128; physiological saline was used as control sample 1.

[0084] 7. Mix chloroform and the interfering substance at a volume ratio of 2:1, roll on a horizontal roller for 30 minutes, centrifuge at 5000g for 15 minutes at room temperature, and transfer the supernatant to an EP tube. Based on the principle of "like dissolves like," the strongly hydrophobic interfering substance can be extracted using chloroform solution. The lower layer consists of chloroform and the extracted interfering substance, while the upper layer is a solution without the interfering substance. Use the upper layer as control sample 2.

[0085] 8. Referring to the reaction parameters of the Col IV (Type IV Collagen) Chemiluminescence Detection Kit (Catalog No. C86047, YHLO), manually prepare different gradients of interference samples, control sample 1, and control sample 2 using reagent A and diluted at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Add these samples to PP (polypropylene) reaction vessels, incubate for 10 min, then perform magnetic separation, add excitation and pre-excitation solutions, and detect the luminescence value. Similarly, prepare different gradients of interference samples, control sample 1, and control sample 2 using reagent B and diluted at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Add these samples to PP reaction vessels, incubate for 10 min, then perform magnetic separation, add excitation and pre-excitation solutions, and detect the luminescence value.

[0086] 9. Referring to the reaction parameters of the Col IV (Type IV Collagen) chemiluminescence detection kit, manually prepare different gradients of interferon samples, control sample 1, and control sample 2 by diluting reagent A at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Add these samples to glass reaction vessels, incubate for 10 min, then perform magnetic separation, add excitation and pre-excitation solutions, and detect the luminescence value. Similarly, prepare different gradients of interferon samples, control 1, and control 2 by diluting reagent B at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Add these samples to glass reaction vessels, incubate for 10 min, then perform magnetic separation, add excitation and pre-excitation solutions, and detect the luminescence value.

[0087] Experimental results:

[0088] Comparing the results of the interference sample and control sample 2 monitored using reagent A in a PP reaction vessel, the interference sample showed a strong signal, while control sample 2 showed almost no signal. Control sample 2 was the interference sample, which had its interfering substances removed through chloroform extraction. The fact that the interfering substances could be extracted with chloroform indicates that they are highly hydrophobic.

[0089] Because PP material is hydrophobic while glass material is highly hydrophilic, hydrophobic interfering substances can bind to the PP reaction vessel but not to the glass reaction vessel. In this embodiment, reagent A was used to detect the data of interfering samples and control samples 1 and 2, comparing PP and glass reaction vessels. The interfering substances could directly mediate the acridine conjugate bridging to the PP reaction vessel, resulting in false positives, while the glass reaction vessel yielded normal results.

[0090] PP material is the most commonly used material for reaction cups in fully automated immunoassay instruments in the medical device industry. Abbott Architech and Alility series instruments, as well as Beckman DXI series instruments, all use PP reaction cups. There is a potential for false positive results due to hydrophobic interfering substances in the sample mediating the binding of the marker to the reaction cup.

[0091] Tween-20 is a commonly added surfactant in immunoassay reagents. It increases the surface tension of the solution and reduces hydrophobic adsorption. The difference between reagent A and reagent B is the concentration of Tween-20 added. Reagent A contains 0.05% Tween-20, which is a commonly used working concentration in commercial immunoassay reagents. Reagent B contains 2% Tween-20, which is the highest working concentration in immunoassay reagents. For reactions using PP reaction cups, the signal of reagent B is significantly lower than that of A, but B cannot completely eliminate this hydrophobic interference. Therefore, adding commonly used Tween-20 to immunoassay reagents cannot completely improve false positive results caused by hydrophobic interference in the sample.

[0092] The above are examples of Col IV antibody acridine labels binding to PP reaction cups via hydrophobic interfering agents. In further research projects, antibody acridine labels and antigen acridine labels from multiple different diagnostic kits were able to bind to the reaction cups via the hydrophobic interfering agents prepared in this application, resulting in false positive signals. The hydrophobic interfering agents prepared in this application can bind not only labeled acridine esters but also multiple antibodies and antigens. In clinical samples, the interference behavior of high-concentration IgM samples and samples with cardiovascular diseases is consistent with the interference behavior of the hydrophobic interfering agents in this application.

[0093] Table 1

[0094]

[0095]

[0096] Example 3

[0097] The anti-interference ability of the Anti-TP (Treponema pallidum antibody) chemiluminescent detection kit was studied using the hydrophobic interfering agent prepared in Example 1.

[0098] The specific steps of the detection method are as follows:

[0099] 1. Prepare solution A with the following composition: 50 mM PB, 150 mM NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.0.

[0100] 2. Prepare solution B with the following formula: 50 mM PB, 150 mM NaCl, 0.5% BSA, 2% Tween-20, 0.1% ProClin300, pH 7.0.

[0101] 3. The purified Treponema pallidum antigen was coated onto superparamagnetic microparticles to prepare magnetic bead coatings. Acridinium ester was labeled onto the purified Treponema pallidum antigen to prepare labeled conjugates.

[0102] 4. Prepare a magnetic bead working solution by diluting the magnetic bead coating with solution A, prepare an acridine working solution by diluting the acridine label with solution A, and assemble the magnetic bead working solution and the acridine working solution into reagent A.

[0103] 5. Prepare a magnetic bead working solution by diluting the magnetic bead coating with solution B, prepare an acridine working solution by diluting the acridine label with solution B, and assemble the magnetic bead working solution and the acridine working solution into reagent B.

[0104] 6. Interferants were prepared by serial dilution with physiological saline at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, and 1:128 to prepare interferants with different gradients; physiological saline was used as control sample 1.

[0105] 7. Mix chloroform and the interfering substance at a volume ratio of 2:1, roll on a horizontal roller for 30 minutes, centrifuge at 5000g for 15 minutes at room temperature, and transfer the supernatant to an EP tube. Based on the principle of "like dissolves like," the strongly hydrophobic interfering substance can be extracted using chloroform solution. The lower layer consists of chloroform and the extracted interfering substance, while the upper layer is a solution without the interfering substance. Use the upper layer as control sample 2.

[0106] 8. Following the reaction parameters of the Anti-TP (Treponema pallidum antibody) chemiluminescence detection kit (catalog number C88049, YHLO), perform manual operation using glass reaction cups. Prepare interfering samples, control sample 1, and control sample 2 with different serial dilutions of reagent A at 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Incubate for 10 min, then perform magnetic separation, add excitation and pre-excitation solutions, and detect the luminescence value.

[0107] 9. Following the reaction parameters of the Anti-TP (Treponema pallidum antibody) chemiluminescence detection kit, perform manual operation using glass reaction cups. Prepare interfering samples, control sample 1, and control sample 2 with different serial dilutions of reagent B at ratios of 1:2, 1:4, 1:8, 1:16, 1:32, and 1:64. Incubate for 10 min, then perform magnetic separation, add excitation and pre-excitation solutions, and detect the luminescence value.

[0108] Experimental results:

[0109] In reagent A, due to the relatively low concentration of Tween-20, the hydrophobic interfering agent can bridge the magnetic bead coating and acridine label, producing false positive signals. Reagent B increased the concentration of Tween-20 in the reaction system, significantly reducing false positive signals, but still could not completely eliminate the false positive results caused by the interference of hydrophobic interfering agents in the sample.

[0110] Tween-20 is a commonly added surfactant in immunoassay reagents. It increases the surface tension of the solution and reduces hydrophobic adsorption. The difference between reagent A and reagent B is the concentration of Tween-20 added. Reagent A contains 0.05% Tween-20, which is a commonly used working concentration in immunoassay reagents, while reagent B contains 2% Tween-20, which is the highest working concentration in immunoassay reagents. Glass reaction vessels were used in this experiment because they are highly hydrophilic, thus eliminating the possibility of hydrophobic interference bridging the reaction vessels and the signal generated by the acridine label.

[0111] The above are examples of how acridine-labeled anti-TP drugs generate false positive signals through the binding of hydrophobic interferants with magnetic bead coatings. Due to significant inconsistencies in the hydrophilicity and hydrophobicity of different manufacturers and types of magnetic beads—for example, the reaction principle of Tosyl (toluenesulfonyl) magnetic beads involves physical adsorption followed by cross-linking—the surfaces of magnetic beads from different manufacturers generally exhibit strong hydrophobicity. Therefore, antigens or antibodies coated with this type of magnetic bead are more prone to generating false positive signals due to non-specific adsorption caused by hydrophobic interactions. Further research revealed that when using reagents from Abbott and Roche for different projects to detect the hydrophobic interferants prepared in this invention, some projects exhibited a certain degree of false positives.

[0112] Table 2

[0113]

[0114] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0115] The embodiments described above are merely illustrative of several implementation methods of this application, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the protection scope of this application. Therefore, the protection scope of this patent application should be determined by the appended claims, and the specification and drawings can be used to interpret the content of the claims.

Claims

1. A method for preparing a hydrophobic interfering substance, characterized in that, Includes the following steps: Add a lysis agent to the whole blood sample to fully lyse the blood cells and release their contents; centrifuge and collect the supernatant; and An oxidant is added to the supernatant to oxidize the contents and improve hydrophobicity. The supernatant is then collected by centrifugation to obtain the hydrophobic interfering substance. The pyrolysis agent is 1% Triton X-100, and the oxidant is 4% H2O2.

2. The preparation method according to claim 1, characterized in that, The source of the whole blood sample includes peripheral blood from humans or animals.

3. The preparation method according to claim 1, characterized in that, The final concentration of the oxidant in the reaction system is 4% (v / v); the final concentration of the pyrolysis agent in the reaction system is 1% (v / v).

4. The preparation method according to any one of claims 1 to 3, characterized in that, Includes the following steps: Add the lysing agent to the whole blood sample, mix the lysing agent thoroughly with the blood cells in the whole blood sample, incubate to allow the blood cells in the whole blood sample to fully lyse and release the contents, and centrifuge to collect the supernatant; wherein, the centrifugation is performed at 2-8℃ and 5000g centrifugal force for 10-30 minutes. Then, the oxidant is added to the supernatant to oxidize the contents and improve hydrophobicity. The supernatant is then collected by centrifugation to obtain the hydrophobic interfering substance. The oxidation conditions are 37°C for 10-20 minutes and centrifugation is performed at 2-8°C and 5000g for 10-30 minutes.

5. A hydrophobic interfering substance prepared by the method according to any one of claims 1 to 4.

6. A reagent kit, characterized in that, The kit includes the hydrophobic interfering agent as described in claim 5.

7. A method for assessing the resistance to hydrophobic interactions in an immune response, characterized in that, This includes using the hydrophobic interfering agent of claim 5 or the kit of claim 6 to evaluate the resistance of the immune response to the hydrophobic interaction of the reaction vessel, wherein the reaction vessel is made of a hydrophobic material, and the hydrophobic material is polypropylene.

8. The method according to claim 7, characterized in that, The method includes the following steps: The hydrophobic interfering agent was gradient diluted to prepare multiple hydrophobic interfering agent solutions of different concentrations; The hydrophobic interfering solutions of different concentrations were extracted using an extractant, with the organic phase serving as the interfering sample and the aqueous phase serving as the control sample. The interfering sample and the control sample were respectively added to the reaction vessel with the immunoassay reagent to measure the luminescence value; and The resistance of the reaction vessel in the immune response to hydrophobic interactions was analyzed based on the luminescence values ​​of the interfering sample and the control sample.

9. The method according to claim 8, characterized in that, The immunoassay reagent includes a solid-phase coating and / or a label conjugate.

10. The method according to claim 9, characterized in that, The labeled conjugate includes a labeled antigen or antibody, and the solid-phase coating includes a solid-phase coated antigen or antibody.

11. The application of the hydrophobic interfering agent of claim 5 or the kit of claim 6 in detecting the anti-hydrophobic interaction interference performance of reaction vessels in immune reactions; wherein, The reaction vessel is made of a hydrophobic material, specifically polypropylene.

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