WO2024120133A1 - 疏水性干扰物及其制备方法和应用 - Google Patents

疏水性干扰物及其制备方法和应用 Download PDF

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WO2024120133A1
WO2024120133A1 PCT/CN2023/131575 CN2023131575W WO2024120133A1 WO 2024120133 A1 WO2024120133 A1 WO 2024120133A1 CN 2023131575 W CN2023131575 W CN 2023131575W WO 2024120133 A1 WO2024120133 A1 WO 2024120133A1
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hydrophobic
interferor
sample
interference
reagent
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PCT/CN2023/131575
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English (en)
French (fr)
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杨永宏
程方明
王刚
钱纯亘
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深圳市亚辉龙生物科技股份有限公司
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Publication of WO2024120133A1 publication Critical patent/WO2024120133A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present application relates to the field of in vitro diagnostic technology, and in particular to a hydrophobic interferor and a preparation method and application thereof.
  • Common interferences in immune response include endogenous and exogenous interferences.
  • Common endogenous interferences include hemolysis, lipemia, total protein, analogs, high IgG or high IgM, rheumatoid factor, heterophilic antibodies, biotin interference, etc.
  • Common exogenous interferences include drug interference, bacterial or viral infection, etc.
  • hydrophobic force that is, the interfering substance non-specifically bridges the coated conjugate and the labeled conjugate through hydrophobic force, or non-specifically bridges the reaction cup (reaction well) and the labeled conjugate, generating false positive signals.
  • Protein molecules are composed of amino acids, and different amino acids have different strengths of hydrophilicity and hydrophobicity.
  • proteins have different overall hydrophilicity and hydrophobicity due to the different content of hydrophilic and hydrophobic amino acids they carry.
  • proteins in serum and plasma There are many kinds of proteins in serum and plasma. Different proteins show different strengths of hydrophobicity, which may potentially interfere with detection reagents based on the principle of immune response.
  • the protein content of different people will also be different, and the severity of interference with immune response will also be different.
  • hydrophobic interferor can be used to evaluate the anti-interference ability of hydrophobic force in immune response.
  • a method for preparing a hydrophobic interferor comprising the following steps:
  • An oxidant is added to the supernatant to oxidize the contents to increase hydrophobicity, and the supernatant is taken out by centrifugation to obtain the hydrophobic interfering substance.
  • the source of the whole blood sample includes peripheral blood of humans or animals.
  • the lysis solution includes TritonX-100.
  • the oxidant includes one or more of H 2 O 2 , urea peroxide, and potassium permanganate.
  • the final concentration of the oxidant in the reaction system is 0.5% (v/v) to 8% (v/v); the final concentration of the cleavage agent in the reaction system is 0.05% (v/v) to 4% (v/v).
  • hydrophobic interferor prepared by any of the methods described above is provided.
  • kits comprising the hydrophobic interferor described above.
  • the fourth aspect of the present application provides a method for evaluating the anti-interference ability of the immune response to hydrophobic forces, comprising using the hydrophobic interferor or the kit to evaluate the anti-interference ability of the immune response to hydrophobic forces.
  • the method comprises the following steps:
  • the organic phase is used as the interfering substance sample, and the aqueous phase is used as the control sample;
  • the anti-interference ability of the reaction container and/or the immunodetection reagent in the immune reaction to the hydrophobic force is analyzed according to the luminescence values of the interference sample and the control sample.
  • the immunodetection reagent includes a solid phase coating and/or a labeled conjugate.
  • the labeled conjugate includes an antigen or antibody labeled with a marker
  • the solid phase coated object includes an antigen or antibody coated with a solid phase
  • the immunodetection reagent includes a labeled conjugate.
  • the immunoassay reagent includes a solid phase coating and a labeled conjugate, and the reaction container is a hydrophobic material.
  • the fifth aspect of the present application provides the use of the hydrophobic interferor or the kit in detecting the interference performance of at least one of an immunodiagnostic reagent, a reaction container, an antigen to be tested and an antibody to be tested in an immune reaction against hydrophobic forces.
  • the present application provides a method for preparing a hydrophobic interferor in an immune response, and the prepared hydrophobic interferor can be used for immunodiagnostic reagents such as POCT, ELISA, CLIA and other immunodiagnostic reagents to study the interference performance of anti-hydrophobic interferors.
  • immunodiagnostic reagents such as POCT, ELISA, CLIA and other immunodiagnostic reagents to study the interference performance of anti-hydrophobic interferors.
  • the interference performance test of anti-hydrophobic interferors is carried out in advance to replace the use of a large number of clinical samples for amplification and research, saving the time cycle cost and cost cost of development.
  • potential interference can be identified and optimized in advance to improve reagents, and false positive or false positive results can be prevented in advance in clinical sample testing.
  • Figure 1 is an example of a false positive caused by hydrophobic interferences mediating the binding between solid-phase coated antibodies and antibody markers. intention;
  • FIG. 2 is a schematic diagram showing a false positive caused by hydrophobic interferences mediating the binding between solid-phase coated antigen and antigen marker.
  • first aspect means at least two, for example two, three, etc., unless otherwise clearly and specifically defined.
  • hydrophobic interaction refers to the interaction between two and/or more hydrophobic substances, which allows the substances to bind to each other.
  • CLIA chemiluminescent immunoassay
  • chemiluminescent immunoassay which is an immunoassay method in which antigens or antibodies are directly labeled with chemiluminescent agents.
  • POCT point-of-care testing
  • the abbreviation "POCT” refers to point-of-care testing, which mainly refers to rapid testing and analysis technology close to the patient's bedside, and can be carried out at the bedside, in the ward or in other places outside the central laboratory and laboratory. It includes but is not limited to immunoturbidimetry and immunochromatography.
  • ELISA enzyme-linked immunosorbent assay, which is a qualitative and quantitative detection method for immune response by binding soluble antigens or antibodies to a solid carrier and utilizing the specific binding of antigens and antibodies.
  • One embodiment of the present application provides a method for preparing a hydrophobic interfering substance, which can be used for diagnostic reagents based on the principle of immune response, such as immunochromatography, ELISA, CLIA and other immunodiagnostic reagents to study the anti-hydrophobic interference performance, so as to prevent false positive results in clinical large-scale sample testing in advance.
  • the method includes the following steps a to b:
  • Step a Add a lysing agent to the whole blood sample to fully lyse the blood cells in the whole blood sample and release the contents, and then centrifuge to obtain the supernatant.
  • the lysis agent is preferably TritonX-100, and may also be other cell lysis solutions such as RIPA.
  • the final concentration of the lysing agent in the reaction system is 0.05% (v/v) to 4% (v/v).
  • the final concentration of the lysing agent in the reaction system can be in the range of any of the following values: 0.05%, 0.1%, 0.5%, 1%, 2%, 3% or 4%.
  • 1% is used to make the cell lysis more complete and the concentration is appropriate.
  • a lysing agent is added to the whole blood sample, and the lysing agent and the blood cells in the whole blood sample are fully mixed and incubated, so that the blood cells in the whole blood sample are fully lysed and the contents are released.
  • the mixing method is not limited, and the two groups of components only need to be mixed evenly, optionally by turning them upside down.
  • the incubation is at room temperature for 20 min-40 min, preferably, 30 min.
  • the centrifugation is at 2-8°C. Centrifuge at 5000 g for 10 min to 30 min, preferably 20 min.
  • Step b adding an oxidant to the supernatant to oxidize the contents to increase the hydrophobicity, and centrifuging to obtain the supernatant to obtain the hydrophobic interfering substance.
  • the oxidant includes one or more of H 2 O 2 , urea peroxide, and potassium permanganate.
  • the final concentration of the oxidant in the reaction system is 0.5% (v/v) to 8% (v/v).
  • the final concentration of the oxidant in the reaction system can be 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7% or 8%.
  • the hydrophobic interferents prepared within the above range all have strong hydrophobicity, preferably 4%, which is more hydrophobic.
  • the oxidation condition is to treat at 37°C for 10-20 min.
  • the centrifugation is at 2-8°C and 5000 g centrifugal force for 10-30 min, preferably 20 min.
  • One embodiment of the present application also provides a hydrophobic interferor prepared by the above method and a kit comprising the above hydrophobic interferor.
  • One embodiment of the present application also provides a method for evaluating the anti-interference ability of an immune response to hydrophobic forces, comprising using the above-mentioned hydrophobic interferent or the above-mentioned kit to evaluate the anti-interference ability of an immune response to hydrophobic forces.
  • the method includes the following steps a to c:
  • Step a diluting the hydrophobic interferor in a gradient manner to prepare a plurality of hydrophobic interferor solutions of different concentrations.
  • the hydrophobic interferent is diluted with physiological saline, and the dilution ratio may be 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, etc.
  • Step b using an extractant to extract hydrophobic interfering substance solutions of different concentrations, the organic phase is used as the interfering substance sample, and the aqueous phase is used as the control sample.
  • the volume ratio of the extractant to the hydrophobic interferent may be 2:1, 3:1, etc., as long as the hydrophobic interferent can be extracted.
  • the extractant includes an organic solvent such as chloroform, ether, toluene, etc.
  • Step c respectively adding the interference sample, the control sample and the immunoassay reagent into the reaction container to determine the luminescence value.
  • Step d Analyze the anti-interference ability of the reaction container and/or the immunodetection reagent in the immune reaction against the hydrophobic force according to the luminescence values of the interference sample and the control sample.
  • the immunodetection reagent includes a solid phase coating and/or a labeled conjugate.
  • the labeled conjugate includes an antigen marker or an antibody marker.
  • the marker includes but is not limited to acridinium ester, terpyridine ruthenium, biotin, etc.
  • the solid phase coating includes solid phase coated antigen or solid phase coated antibody.
  • the solid phase includes magnetic beads, cellulose, glass, silicone rubber, polyacrylamide, polystyrene and the like.
  • the hydrophobic interferent is used to evaluate the anti-interference ability of the reaction container in the immune reaction to the hydrophobic force.
  • the immunoassay reagent includes a labeled conjugate and physiological saline to replace the solid phase coating.
  • the specific steps and parameters for the immunoassay reagent to detect the interferent sample and the control sample and to determine the luminescence value refer to the instructions of the immunoassay reagent or immunoassay kit used.
  • the above-mentioned hydrophobic interferent is used to evaluate the anti-interference ability of the immunodetection reagent in the immune reaction to the hydrophobic force.
  • the immunodetection reagent includes a solid phase coating and a labeled conjugate, which detects the interferent sample and the control sample respectively, and determines the luminescence value.
  • the reaction container used in the detection process is a hydrophobic material such as a glass material.
  • Other specific detection and determination steps and parameters of the luminescence value can refer to the instructions of the immunodetection reagent or immunodetection kit used.
  • the hydrophobic interfering substance or the kit is used to detect the interference performance of at least one of an immunodiagnostic reagent, a reaction container, an antigen to be tested, and an antibody to be tested against hydrophobic forces.
  • the immunodiagnostic reagent includes but is not limited to POCT, ELISA, CLIA and other immunodiagnostic reagents.
  • the hydrophobic interferor of the present application can directly mediate the bridging of the labeled conjugate to the hydrophobic material reaction container to generate a false positive signal.
  • the hydrophobic interferor can also combine the antibody marker or antigen marker with the solid-phase coated object to generate a false positive signal.
  • the antibody marker can cause a false positive signal by combining the hydrophobic interferor prepared by the present application with the solid-phase coated antibody.
  • Figure 1 for the reaction principle.
  • the antigen marker can cause a false positive signal by combining the hydrophobic interferor prepared by the present application with the solid-phase coated antigen.
  • Figure 2 for the reaction principle.
  • the measured parameters of raw material components may have slight deviations within the range of weighing accuracy unless otherwise specified.
  • acceptable deviations caused by instrument test accuracy or operation accuracy are allowed.
  • TritonX-100 and H 2 O 2 used in the following examples were purchased from Aladdin.
  • a method for preparing a hydrophobic interfering substance :
  • the interfering substance is prepared by treating a whole blood sample that has been fully coagulated in a red-capped blood collection tube. Add TritonX-100 to a final concentration (v/v) of 1% in the blood collection tube, cover the blood collection tube cap, and turn it upside down to allow the TritonX-100 to Mix well with blood cells and incubate at room temperature for 30 minutes. Centrifuge at 2-8°C, 5000g for 20 minutes and take the supernatant. Add H 2 O 2 with a final concentration (v/v) of 4% to the supernatant and treat at 37°C for 20 minutes. Centrifuge the oxidized supernatant at 2-8°C, 5000g for 20 minutes and take the supernatant, which is the interfering substance of the present application.
  • Final concentration refers to the concentration ratio of the cleavage reagent or oxidant in the reaction system.
  • the hydrophobic interferor prepared in Example 1 was used to study the anti-interference ability of the ColIV (type IV collagen) chemiluminescence detection kit.
  • solution A with the formula of 50 mM PB, 150 mM NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.0.
  • the magnetic bead working solution is replaced with physiological saline, and assembled with acridine working solution A to form reagent A, and assembled with acridine working solution B to form reagent B.
  • chloroform and the interfering substance in a volume ratio of 2:1, roll on a horizontal roller for 30 minutes, centrifuge at 5000g, centrifuge at room temperature for 15 minutes, and transfer the upper layer of solution to an EP tube. Based on the principle of like dissolves like, the chloroform solution can extract the strongly hydrophobic interfering substance, and the lower layer is chloroform and the extracted interfering substance.
  • the upper layer is a solution without interferences.
  • the upper layer solution is used as control sample 2.
  • ColIV type IV collagen
  • chemiluminescence detection kit Cat. No. C86047, Yahuilong
  • control sample 1 and control sample 2 prepared by dilution of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 to PP (polypropylene) reaction cups, incubate for 10 minutes, and then perform magnetic separation, add excitation solution and pre-excitation solution to detect luminescence value.
  • control sample 1 and control sample 2 prepared by dilution of 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 to PP reaction cups, incubate for 10 minutes, and then perform magnetic separation, add excitation solution and pre-excitation solution to detect luminescence value.
  • reaction parameters of the ColIV (type IV collagen) chemiluminescence detection kit manually operate, manually add reagent A, and different gradients of interference samples, control sample 1 and control sample 2 prepared by dilution at 1:2, 1:4, 1:8, 1:16, 1:32, 1:64, respectively, to glass reaction cups, incubate for 10 minutes, then perform magnetic separation, add excitation solution and pre-excitation solution to detect luminescence values.
  • the interference sample has a strong signal, while the control sample 2 has almost no signal.
  • the control sample 2 is the interference sample extracted by chloroform to remove the interference.
  • the interference can be extracted by chloroform, indicating that the interference has strong hydrophobicity.
  • PP material is hydrophobic
  • glass material is highly hydrophilic
  • hydrophobic interferences can bind to PP material cuvettes, but cannot bind to glass material interferences.
  • This example compares PP material cuvettes and glass material cuvettes using reagent A to detect interference samples and control samples. According to data 1 and 2, the interference substance can directly mediate the bridging of the acridine conjugate to the PP reaction cup, resulting in a false positive, while the results of the glass reaction cup are normal.
  • PP is the most commonly used material for reaction cups in fully automatic immunoassay instruments in the medical device industry. Abbott's Architech and Alility series instruments, and Beckman's DXI series instruments all use reaction cups made of PP. There is a potential for false positive results due to the binding of hydrophobic interferents in the sample to the marker and the reaction cup.
  • Tween-20 is the most commonly added surfactant in immunoreagents, which can increase the surface tension of the solution and reduce hydrophobic adsorption.
  • the difference between reagent A and reagent B is that the concentration of Tween-20 added is different.
  • the concentration of Tween-20 in reagent A is 0.05%, which is a commonly used working concentration in commercial immunoreagents, and the concentration of Tween-20 in reagent B is 2%, which is the highest working concentration in immunoreagents.
  • the signal of B is significantly lower than that of A, but B cannot completely eliminate this hydrophobic interference. Therefore, in immunoassay reagents, the addition of commonly used Tween-20 cannot completely improve the false positive results caused by hydrophobic interferences in the sample.
  • the above is an example of the ColIV antibody acridine marker binding to the PP material reaction cup through a hydrophobic interfering substance.
  • antibody acridine markers and antigen acridine markers of multiple different diagnostic kits can bind to the reaction cup through the hydrophobic interfering substance prepared in this application, resulting in false positive signals.
  • the hydrophobic interfering substance prepared in this application can not only bind to the labeled acridine ester, but also bind to multiple antibodies and antigens.
  • the interference performance of high-concentration IgM samples and samples of cardiovascular diseases is consistent with the interference performance of the hydrophobic interfering substance of this application.
  • the hydrophobic interferor prepared in Example 1 was used to study the anti-interference ability of the Anti-TP (Treponema pallidum antibody) chemiluminescent detection kit.
  • solution A with the formula of 50 mM PB, 150 mM NaCl, 0.5% BSA, 0.05% Tween-20, 0.1% ProClin300, pH 7.0.
  • the purified Treponema pallidum antigen is coated on superparamagnetic microparticles to prepare magnetic bead coatings.
  • the acridinium ester is labeled on the purified Treponema pallidum antigen to prepare labeled conjugates.
  • chloroform and the interfering substance in a volume ratio of 2:1, roll on a horizontal roller for 30 minutes, centrifuge at 5000g, centrifuge at room temperature for 15 minutes, and transfer the upper layer solution to an EP tube. Based on the principle of like dissolves like, the chloroform solution can extract the strongly hydrophobic interfering substance.
  • the lower layer is chloroform and the extracted interfering substance, and the upper layer is a solution without interfering substance.
  • the upper layer solution is used as control sample 2.
  • Reagent B increases the concentration of Tween-20 in the reaction system, and the false positive signals are significantly reduced, but it still cannot completely improve the false positive results caused by hydrophobic interferences in the sample.
  • Tween-20 is the most commonly added surfactant in immunoreagents, which can increase the surface tension of the solution and reduce hydrophobic adsorption.
  • the difference between reagent A and reagent B is the different concentrations of Tween-20 added.
  • the concentration of Tween-20 in reagent A is 0.05%, which is a commonly used working concentration in immunoreagents, and the concentration of Tween-20 in reagent B is 2%, which is the highest working concentration in immunoreagents.
  • the glass reaction cups used in this experiment are highly hydrophilic, so the signals generated by hydrophobic interferences bridging the reaction cups and acridine markers can be excluded.
  • the above is an example of a false positive signal generated by the acridine marker of the Anti-TP project through the combination of hydrophobic interference and magnetic bead coating.

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Abstract

一种疏水性干扰物及其制备方法和应用，其中疏水性干扰物的制备方法，包括如下步骤：向全血样本中加入裂解剂，使全血样本中的血细胞充分裂解并释放内容物，离心取上清；在上清中加入氧化剂，使内容物被氧化以提高疏水性，离心取上清，获得疏水性干扰物。制备的疏水性干扰物可用于免疫诊断试剂，如POCT，ELISA，CLIA等免疫诊断试剂进行抗疏水性干扰物的干扰性能检测。在临床检测中，可以对于潜在的干扰进行提前识别和优化来改进试剂，提前预防在临床样本检测中产生假阳或假阳的结果。

Description

疏水性干扰物及其制备方法和应用 技术领域
本申请涉及体外诊断技术领域，具体涉及一种疏水性干扰物及其制备方法和应用。
背景技术
免疫反应中常见的干扰包含内源性和外源性干扰，其中常见的内源性的干扰包含溶血、脂血、总蛋白、类似物、高IgG或高IgM、类风湿因子、嗜异性抗体、生物素干扰等，常见的外源性干扰包含药物干扰、细菌或病毒感染等。而免疫反应中产生干扰的本质原因之一是疏水作用力，即干扰物质通过疏水作用力把包被偶联物和标记偶联物非特异桥连起来，或将反应杯(反应孔)与标记偶联物非特异桥连起来，产生假阳性信号。蛋白质分子是通过氨基酸组成的，不同的氨基酸具有不同强度的亲水性和疏水性，因此，不同的蛋白质由于携带的亲水性和疏水性的氨基酸含量的不同，表现出的整体的亲水和疏水性也存在显著的差异。血清和血浆中含有多种蛋白质，不同的蛋白质表现出不同强度的疏水性，潜在会对基于免疫反应原理的检测试剂产生干扰，且不同人蛋白含量也会存在差异，对免疫反应干扰严重程度也不一样。
目前，评估抗干扰能力的方法如常见的内源性物质和外源性物质，均不能有效反映出反应体系对疏水性干扰物的抗干扰能力。
发明内容
基于此，有必要提供一种疏水性干扰物的制备方法及其制备的疏水性干扰 物，该疏水性干扰物可用于评估免疫反应中对疏水作用力的抗干扰能力。
具体技术方案如下：
本申请的第一方面，提供了一种疏水性干扰物的制备方法，包括如下步骤：
向全血样本中加入裂解剂，使所述全血样本中的血细胞充分裂解并释放内容物，离心取上清；
在上清中加入氧化剂，使所述内容物被氧化以提高疏水性，离心取上清，获得所述疏水性干扰物。
在其中一个实施例中，所述全血样本的来源包括人或动物的外周血。
在其中一个实施例中，所述裂解液包括TritonX-100。
在其中一个实施例中，所述氧化剂包括H2O2、过氧化脲、高锰酸钾中的一种或多种。
在其中一个实施例中，所述氧化剂在反应体系中的最终浓度为0.5％(v/v)～8％(v/v)；所述裂解剂在反应体系中的最终浓度为0.05％(v/v)～4％(v/v)。
本申请的第二方面，提供了由上述任一项所述的方法制备的疏水性干扰物。
本申请的第三方面，提供了一种试剂盒，所述试剂盒包括上述所述的疏水性干扰物。
本申请的第四方面，提供了一种评估免疫反应中对疏水作用力的抗干扰能力的方法，包括采用所述的疏水性干扰物或所述的试剂盒来评估免疫反应对疏水作用力的抗干扰能力。
在其中一个实施例中，所述方法包括以下步骤：
将所述疏水性干扰物梯度稀释，制成多个不同浓度的疏水性干扰物溶液；
使用萃取剂分别萃取所述不同浓度的疏水性干扰物溶液，有机相作为干扰物样本，水相作为对照样本；
分别将干扰物样本和对照样本与免疫检测试剂加入到反应容器中测定发光值；及
根据干扰样本和对照样本的发光值分析免疫反应中的反应容器和/或免疫检测试剂对疏水作用力的抗干扰能力。
可选地，所述免疫检测试剂包括固相包被物和/或标记偶联物。
进一步可选地，所述标记偶联物包括标记物标记的抗原或抗体，所述固相包被物包括固相包被的抗原或抗体。
在其中一个实施例中，所述免疫检测试剂包括标记偶联物。
可选地，所述免疫检测试剂包括固相包被物和标记偶联物，所述反应容器为疏水性材料。
本申请的第五方面，提供了所述的疏水性干扰物或所述的试剂盒在对免疫反应中的免疫诊断试剂、反应容器、待测抗原和待测抗体中至少一种进行抗疏水作用力的干扰性能检测中的应用。
相对于现有技术，本申请具备如下有益效果：
本申请提供了一种免疫反应中疏水性干扰物的制备方法，制备的疏水性干扰物可用于免疫诊断试剂，如POCT，ELISA，CLIA等免疫诊断试剂进行抗疏水性干扰物的干扰性能研究。在产品的开发和验证阶段，提前进行抗疏水性干扰物的干扰性能检测，来替代用大量的临床样本放大进行研究，节省了开发的时间周期成本和费用成本。同时，可以对于潜在的干扰进行提前识别和优化来改进试剂，提前预防在临床样本检测中产生假阳或假阳的结果。
附图说明
图1为疏水性干扰物介导固相包被抗体和抗体标记物结合导致假阳性的示 意图；
图2为疏水性干扰物介导固相包被抗原和抗原标记物结合导致假阳性的示意图。
具体实施方式
为使本申请的上述目的、特征和优点能够更加明显易懂，下面结合附图对本申请的具体实施方式做详细的说明。在下面的描述中阐述了很多具体细节以便于充分理解本申请。但是本申请能够以很多不同于在此描述的其它方式来实施，本领域技术人员可以在不违背本申请内涵的情况下做类似改进，因此本申请不受下面公开的具体实施例的限制。
除非另有定义，本文所使用的所有的技术和科学术语与属于本申请的技术领域的技术人员通常理解的含义相同。本文中在本申请的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本申请。
术语解释
术语“第一方面”、“第二方面”、“第三方面”、“第四方面”、“第五方面”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所指示的技术特征的数量。由此，限定有“第一方面”、“第二方面”、“第三方面”、“第四方面”、“第五方面”的特征可以明示或者隐含地包括至少一个该特征。在本申请的描述中，“多种”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
术语“疏水作用力”指两个和/或多个疏水性物质之间存在的作用力，能够让物质之间相互结合。
术语简称“CLIA”指化学发光免疫分析，是用化学发光剂直接标记抗原或抗体的免疫分析方法。
术语简称“POCT”指即时检测，主要指靠近患者床旁进行快速检测分析技术，能够在床旁、病房或中心试验室和检验科之外其它地方开展检测技术。包括但不限于免疫比浊和免疫层析。
术语简称“ELISA”指酶联免疫吸附剂测定，是将可溶性的抗原或抗体结合到固相载体上，利用抗原抗体特异性结合进行免疫反应的定性和定量检测方法。
本申请一实施方式提供了一种疏水性干扰物的制备方法，可用于基于免疫反应原理的诊断试剂，如免疫层析，ELISA，CLIA等免疫诊断试剂进行抗疏水性干扰性能研究，来提前预防在临床大量样本检测中产生假阳的结果。具体地，该方法包括以下步骤a～步骤b：
步骤a：向全血样本中加入裂解剂，使全血样本中的血细胞充分裂解并释放内容物，离心取上清。
在一个具体示例中，裂解剂优选TritonX-100，还可以为其他细胞裂解液如RIPA。
在一个具体示例中，裂解剂在反应体系中的最终浓度为0.05％(v/v)～4％(v/v)。可选地，裂解剂在反应体系中的最终浓度可以是以下任意数值组成的范围：0.05％、0.1％、0.5％、1％、2％、3％或者4％。优选1％，使细胞裂解更充分，且浓度适宜。
具体地，向全血样本中加入裂解剂，使裂解剂与全血样本中的血细胞充分混匀，孵育，使全血样本中的血细胞充分裂解并释放内容物。
具体地，混匀的方式不受限制，只需要将两组成分混合均匀即可，可选，上下颠倒。
具体地，孵育是在室温下孵育20min-40min，优选，30min。离心是在2-8℃， 5000g离心力离心10min-30min，优选20min。
步骤b：在上清中加入氧化剂，使内容物被氧化以提高疏水性，离心取上清，获得所述疏水性干扰物。
在一具体示例中，氧化剂包括H2O2、过氧化脲和高锰酸钾中的一种或多种。
在一具体示例中，氧化剂在反应体系中的最终浓度为0.5％(v/v)～8％(v/v)。可选地，氧化剂在反应体系中的最终浓度可以为0.5％、1％、2％、3％、4％、5％、6％、7％或8％。在上述范围内制备的疏水性干扰物均具有强的疏水性，优选4％，疏水性更强。
具体地，氧化条件是在37℃处理10min-20min。离心是在2-8℃，5000g离心力离心10min-30min，优选20min。
本申请一实施方式还提供了上述方法制备的疏水性干扰物以及包括上述疏水性干扰物的试剂盒。
本申请一实施方式还提供了一种评估免疫反应中对疏水作用力的抗干扰能力的方法，包括采用上述疏水性干扰物或上述试剂盒来评估免疫反应对疏水作用力的抗干扰能力。
在其中一具体示例中，所述方法包括以下步骤a～步骤c：
步骤a:将上述疏水性干扰物梯度稀释，制成多个不同浓度的疏水性干扰物溶液。
具体地，使用生理盐水对疏水性干扰物进行稀释，稀释比例可以是1:2、1:4、1:8、1:16、1:32、1:64等。
步骤b:使用萃取剂分别萃取不同浓度的疏水性干扰物溶液，有机相作为干扰物样本，水相作为对照样本。
具体地，萃取剂与疏水性干扰物的体积比可以为2:1、3:1等，只要能将疏水性干扰物萃取出即可。
可选地，萃取剂包括氯仿、乙醚、甲苯等有机有机溶剂。
步骤c:分别将干扰物样本和对照样本与免疫检测试剂加入到反应容器中测定发光值。
步骤d：根据干扰样本和对照样本的发光值分析免疫反应中的反应容器和/或免疫检测试剂对疏水作用力的抗干扰能力。
可选地，免疫检测试剂包括固相包被物和/或标记偶联物。
具体地，标记偶联物包括抗原标记物或抗体标记物。进一步地，标记物包括但不限于吖啶酯、三联吡啶钌、生物素等。
具体地，固相包被物包括固相包被抗原或固相包被抗体。进一步地，固相包括磁珠、纤维素、玻璃、硅橡胶、聚丙稀酰胺、聚苯乙烯等。
在一具体示例中，上述疏水性干扰物用于评估免疫反应中的反应容器对疏水作用力的抗干扰能力。具体地，采用上述步骤a～步骤d进行评估。免疫检测试剂包括标记偶联物，还包括生理盐水，用于替代固相包被物。可选地，免疫检测试剂检测干扰物样本和对照样本及测定发光值的具体步骤和参数参考所使用的免疫检测试剂或免疫检测试剂盒的说明书。
在另一具体示例中，上述疏水性干扰物用于评估免疫反应中的免疫检测试剂对疏水作用力的抗干扰能力。具体地，采用上述步骤a～步骤d进行评估。免疫检测试剂包括固相包被物和标记偶联物，分别检测干扰物样本和对照样本，测定发光值。在检测过程中使用的反应容器为疏水性材料如玻璃材料。其他具体检测及测定发光值的步骤和参数可参考所使用的免疫检测试剂或免疫检测试剂盒的说明书。
上述的疏水性干扰物或上述的试剂盒在对免疫诊断试剂、反应容器、待测抗原和待测抗体中的至少一种进行抗疏水作用力的干扰性能检测中的应用。可选地，免疫诊断试剂包括但不限于POCT，ELISA，CLIA等免疫诊断试剂。
本申请的疏水性干扰物能够直接介导标记偶联物桥连到疏水性材质反应容器上，产生假阳性信号。此外，疏水性干扰物也能将抗体标记物或抗原标记物与固相包被物侨联产生假阳信号。具体地，抗体标记物能够通过本申请制备的疏水性干扰物与固相包被抗体结合上导致假阳信号，反应原理请参阅图1。抗原标记物能够通过本申请制备的疏水性干扰物与固相包被抗原结合上导致假阳信号，反应原理请参阅图2。
具体实施例
下面将结合实施例对本申请的实施方案进行详细描述。应理解，这些实施例仅用于说明本申请而不用于限制本申请的范围。下列实施例中未注明具体条件的实验方法，优先参考本申请中给出的指引，还可以按照本领域的实验手册或常规条件，还可以按照制造厂商所建议的条件，或者参考本领域已知的实验方法。
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。
以下实施例中使用的TritonX-100和H2O2均采购自阿拉丁。
实施例1
一种疏水性干扰物的制备方法：
干扰物是通过处理红头采血管中已充分凝结的全血样本来制备。在采血管中添加终浓度(v/v)为1％TritonX-100，盖上采血管盖，上下颠倒使TritonX-100 和血细胞充分混匀，室温孵育30min。2-8℃，5000g离心力离心20min，取上清。在上清中加入终浓度(v/v)为4％的H2O2，37℃处理20min。将氧化后的上清2-8℃，5000g离心力离心20min，取上清，即为本申请的干扰物。
终浓度是指反应体系中裂解试剂或氧化剂的浓度占比。
实施例2
使用实施例1制备的疏水性干扰物，研究ColⅣ(四型胶原)化学发光检测试剂盒的抗干扰能力。
检测方法的具体步骤如下：
1.配置溶液A，配方为50mM PB，150mM NaCl，0.5％BSA，0.05％Tween-20，0.1％ProClin300，pH 7.0。
2.配置溶液B，配方为50mM PB，150mM NaCl，0.5％BSA，2％Tween-20，0.1％ProClin300，pH 7.0。
3.将吖啶酯标记到ColⅣ抗体上，制备抗体标记偶联物。
4.用溶液A稀释抗体标记偶联物制备吖啶工作液A；用溶液B稀释抗体标记偶联物制备吖啶工作液B。
5.磁珠工作液用生理盐水替代，与吖啶工作液A组装是试剂A，与吖啶工作液B组装成试剂B。
6.用生理盐水对干扰物进行1:2、1：4、1:8、1:16、1:32、1:64、1:128稀释制备不同梯度的干扰物；以生理盐水作为对照样本1。
7.用氯仿与干扰物按体积比2:1混合，在水平滚轴上滚动30min，5000g离心力，室温离心15min，取上层溶液转到到EP管中。基于相似相溶的原理，通过氯仿溶液，能够把强疏水性的干扰物萃取出来，下层为氯仿及萃取的干扰物， 上层为没有干扰物的溶液。将上层溶液作为对照样本2。
8.参考ColⅣ(四型胶原)化学发光检测试剂盒(货号C86047，亚辉龙)的反应参数，手工操作，将试剂A，以及1:2、1：4、1:8、1:16、1:32、1:64稀释制备不同梯度的干扰物样本、对照样本1和对照样本2，分别加入到PP材质(聚丙烯)的反应杯中，孵育10min，然后进行磁分离，加入激发液和预激发液检测发光值。将试剂B，以及1:2、1:4、1:8、1:16、1:32、1:64稀释制备不同梯度的干扰物样本、对照样本1和对照样本2，分别加入到PP材质的反应杯中，孵育10min，然后进行磁分离，加入激发液和预激发液检测发光值。
9.参考ColⅣ(四型胶原)化学发光检测试剂盒的反应参数，手工操作，手工操作，将试剂A，以及1:2、1：4、1:8、1:16、1:32、1:64稀释制备不同梯度的干扰物样本、对照样本1和对照样本2，分别加入到分别加入到玻璃材质的反应杯中，孵育10min，然后进行磁分离，加入激发液和预激发液检测发光值。将试剂B，以及1:2、1：4、1:8、1:16、1:32、1:64稀释制备不同梯度的干扰物、对照1和对照2，分别加入到玻璃材质的反应杯中，孵育10min，然后进行磁分离，加入激发液和预激发液检测发光值。
试验结果：
对比干扰样本和对照样本2通过试剂A在PP材质反应杯反应监测的结果，干扰样本存在很强的信号，而对照样本2基本没有信号。对照样本2是干扰样本通过氯仿的萃取，去掉了干扰物。干扰物能通过氯仿萃取出来，说明干扰物具有很强的疏水性。
由于PP材质具有疏水性，而玻璃材质具有较强的亲水性，因此，疏水性的干扰物能结合到PP材质反应杯上，而不能结合到玻璃材质的干扰物上。本实施例对比PP材质反应杯和玻璃材质的反应杯使用试剂A检测干扰样本、对照样本 1和2数据，干扰物能够直接介导吖啶偶联物桥连到PP材质反应杯上，导致产生假阳，玻璃材质反应杯结果正常。
PP材质是医疗器械行业全自动免疫检测仪器反应杯最常用的材质，雅培Architech和Alility系列仪器、贝克曼DXI系列仪器均使用PP材质的反应杯，潜在存在由于样本中疏水性干扰物介导标记物与反应杯结合导致假阳结果。
Tween-20是一种免疫试剂中最常添加的表面活性剂，其能够提高溶液表面张力，降低疏水性吸附作用。试剂A和试剂B的区别是添加的Tween-20浓度不一样，试剂A中Tween-20浓度为0.05％，在商业化的免疫试剂中是常用的工作浓度，试剂B中Tween-20浓度为2％，在免疫试剂中是最高的工作浓度。对于使用PP材质反应杯反应的试剂A和试剂B，B的信号显著低于A的信号，但B不能完全消除这种疏水性干扰。因此，在免疫检测试剂中，通过添加常用的Tween-20，无法完全改善样本中疏水性干扰物干扰的假阳结果。
以上是ColⅣ抗体吖啶标记物通过疏水性干扰物与PP材质反应杯结合的实例，在进一步的研究项目中，多个不同诊断试剂盒的抗体吖啶标记物、抗原吖啶标记物均能够通过本申请制备的疏水性干扰物与反应杯结合上导致假阳信号。本申请制备的疏水性干扰物不仅能结合标记的吖啶酯，还能够与多个抗体和抗原结合。在临床样本中，高浓度的IgM样本和心血管疾病等样本的干扰表现与本申请的疏水性干扰物的干扰表现一致。
表1

实施例3
使用实施例1制备的疏水性干扰物，研究Anti-TP(梅毒螺旋体抗体)化学发光检测试剂盒的抗干扰能力。
检测方法的具体步骤如下：
1.配置溶液A，配方为50mM PB，150mM NaCl，0.5％BSA，0.05％Tween-20，0.1％ProClin300，pH 7.0。
2.配置溶液B，配方为50mM PB，150mM NaCl，0.5％BSA，2％Tween-20，0.1％ProClin300，pH 7.0。
3.将纯化的梅毒螺旋体抗原包被到超顺磁微粒上，制备成磁珠包被物。将吖啶酯标记到纯化的梅毒螺旋体抗原标记上，制备成标记偶联物。
4.用溶液A稀释磁珠包被物制备成磁珠工作液，用溶液A稀释吖啶标记物制备成吖啶工作液，将磁珠工作液和吖啶工作液组装成试剂A。
5.用溶液B稀释磁珠包被物制备成磁珠工作液，用溶液B稀释吖啶标记物制备成吖啶工作液，将磁珠工作液和吖啶工作液组装成试剂B。
6.用生理盐水对干扰物进行1:2、1：4、1:8、1:16、1:32、1:64、1:128倍比稀释制备不同梯度的干扰物；以生理盐水作为对照样本1。
7.用氯仿与干扰物按体积比2:1混合，在水平滚轴上滚动30min，5000g离心力，室温离心15min，取上层溶液转到到EP管中。基于相似相溶的原理，通过氯仿溶液，能够把强疏水性的干扰物萃取出来，下层为氯仿及萃取的干扰物，上层为没有干扰物的溶液。将上层溶液作为对照样本2。
8.参考Anti-TP(梅毒螺旋体抗体)化学发光检测试剂盒(货号C88049，亚辉龙)的反应参数，手工操作，使用玻璃材质的反应杯，用试剂A检测1:2、 1:4、1:8、1:16、1:32、1:64倍比稀释制备不同梯度的干扰物样本、对照样本1和对照样本2，孵育10min，然后进行磁分离，加入激发液和预激发液检测发光值。
9.参考Anti-TP(梅毒螺旋体抗体)化学发光检测试剂盒的反应参数，手工操作，使用玻璃材质的反应杯，用试剂B检测1:2、1：4、1:8、1:16、1:32、1:64倍比稀释制备不同梯度的干扰物样本、对照样本1和对照样本2，孵育10min，然后进行磁分离，加入激发液和预激发液检测发光值。
试验结果：
试剂A中，由于Tween-20的添加浓度比较低，疏水性干扰物能桥连磁珠包被物和吖啶标记物，产生假阳性的信号。试剂B提高了反应体系中Tween-20的浓度，假阳信号有显著的降低，但依然无法完全改善样本中疏水性干扰物干扰产生的假阳结果。
Tween-20是一种免疫试剂中最常添加的表面活性剂，其能够提高溶液表面张力，降低疏水性吸附作用。试剂A和试剂B的区别是添加的Tween-20浓度不一样，试剂A中Tween-20浓度为0.05％，在免疫试剂中是常用的工作浓度，试剂B中Tween-20浓度为2％，在免疫试剂中是最高的工作浓度。该实验均用的玻璃材质的反应杯，其具有较强的亲水性，因此，可以排除疏水性干扰物桥连反应杯和吖啶标记物产生的信号。
以上是Anti-TP项目吖啶标记物通过疏水性干扰物与磁珠包被物结合产生假阳信号的实例。由于不同厂家及不同磁珠类型表面的亲疏水性存在很大的不一致，如Tosyl(甲苯磺酰基)磁珠的反应原理为先物理吸附后交联，不同厂家的磁珠表面普遍存在的较强的疏水性。因此，使用该类型的磁珠包被的抗原或 抗体，更容易由于疏水作用力而导致的非特异吸附而产生假阳信号。进一步研究发现，用雅培和罗氏不同项目的试剂检测本发明制备的疏水性干扰物，部分项目存在一定假阳现象。
表2
以上所述实施例的各技术特征可以进行任意的组合，为使描述简洁，未对上述实施例中的各个技术特征所有可能的组合都进行描述，然而，只要这些技术特征的组合不存在矛盾，都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本申请的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本申请构思的前提下，还可以做出若干变形和改进，这些都属于本申请的保护范围。因此，本申请专利的保护范围应以所附权利要求为准，说明书及附图可以用于解释权利要求的内容。

Claims (11)

  1. 一种疏水性干扰物的制备方法，其特征在于，包括如下步骤：
    向全血样本中加入裂解剂，使所述全血样本中的血细胞充分裂解并释放内容物，离心取上清；及
    在上清中加入氧化剂，使所述内容物被氧化以提高疏水性，离心取上清，获得所述疏水性干扰物。
  2. 根据权利要求1所述的制备方法，其特征在于，所述全血样本的来源包括人或动物的外周血。
  3. 根据权利要求1所述的制备方法，其特征在于，所述裂解剂包括TritonX-100。
  4. 根据权利要求1所述的制备方法，其特征在于，所述氧化剂包括H2O2、过氧化脲和高锰酸钾中的一种或多种。
  5. 根据权利要求1～4任一项所述的制备方法，其特征在于，所述氧化剂在反应体系中的最终浓度为0.5％(v/v)～8％(v/v)；所述裂解剂在反应体系中的最终浓度为0.05％(v/v)～4％(v/v)。
  6. 由权利要求1～5任一项所述的方法制备的疏水性干扰物。
  7. 一种试剂盒，其特征在于，所述试剂盒包括权利要求6所述的疏水性干扰物。
  8. 一种评估免疫反应中对疏水作用力的抗干扰能力的方法，其特征在于，包括采用权利要求6所述的疏水性干扰物或权利要求7所述的试剂盒来评估免疫反应对疏作用力的抗干扰能力。
  9. 根据权利要求8所述的方法，其特征在于，所述方法包括以下步骤：
    将所述疏水性干扰物梯度稀释，制成多个不同浓度的疏水性干扰物溶液；
    使用萃取剂分别萃取所述不同浓度的疏水性干扰物溶液，有机相作为干扰物样本，水相作为对照样本；
    分别将干扰物样本和对照样本与免疫检测试剂加入到反应容器中测定发光值；及
    根据干扰样本和对照样本的发光值分析免疫反应中的反应容器和/或免疫检测试剂对疏水作用力的抗干扰能力；
    可选地，所述免疫检测试剂包括固相包被物和/或标记偶联物；
    进一步可选地，所述标记偶联物包括标记物标记的抗原或抗体，所述固相包被物包括固相包被的抗原或抗体。
  10. 根据权利要求9所述的方法，其特征在于，所述免疫检测试剂包括标记偶联物；
    可选地，所述免疫检测试剂包括固相包被物和标记偶联物，所述反应容器为疏水性材料。
  11. 权利要求6所述的疏水性干扰物或权利要求7所述的试剂盒在对免疫反应中的免疫诊断试剂、反应容器、待测抗原和待测抗体中至少一种进行抗疏水作用力的干扰性能检测中的应用。
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