WO2021185357A1 - Protein chip for detecting human immunodeficiency virus hiv1/2 antibody and preparation method therefor - Google Patents

Protein chip for detecting human immunodeficiency virus hiv1/2 antibody and preparation method therefor Download PDF

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WO2021185357A1
WO2021185357A1 PCT/CN2021/081827 CN2021081827W WO2021185357A1 WO 2021185357 A1 WO2021185357 A1 WO 2021185357A1 CN 2021081827 W CN2021081827 W CN 2021081827W WO 2021185357 A1 WO2021185357 A1 WO 2021185357A1
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hiv
detection
protein chip
immunodeficiency virus
human immunodeficiency
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PCT/CN2021/081827
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French (fr)
Chinese (zh)
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杜卫东
刘胜胜
吴昊
姜淼
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北京良芯生物科技发展有限公司
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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
    • G01N33/50—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
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G01N33/56988—HIV or HTLV
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • This application belongs to the field of immunochemistry, and specifically relates to a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies and a preparation method thereof.
  • HIV Human Immunodeficiency Virus
  • AIDS Abreted Immune Deficiency Syndrome virus
  • retrovirus a type of retrovirus that infects cells of the human immune system.
  • Proteinchip is a high and new technology integrating microelectronics, micromechanics, chemical physics technology, and computer technology. It is considered to be an efficient tool in life science research and is to fix various proteins on a carrier in an orderly manner. Become a chip for detection, and then use the protein or other components labeled with specific fluorescent antibodies to interact with the chip. After rinsing, the components that cannot be complementary to the protein on the chip are washed away, and then fluorescent scanner or laser confocal scanning technology is used , Measure the fluorescence intensity of each point on the chip, and analyze the interaction relationship between protein and protein by fluorescence intensity, thereby achieving the purpose of measuring the function of various proteins. This technology can be used for parallel detection and analysis of multiple proteins at the same time, so that the analysis that requires thousands of times with conventional ELISA technology can be completed only once on the protein chip, and the detected parallel data error is smaller and more precise.
  • Protein chip technology has the advantages of high throughput, miniaturization and rapid parallel analysis, small sample size, high sensitivity and specificity, etc., and has a wide range of applications in the detection of clinical tumors, infectious diseases, autoimmune diseases, and drug development. .
  • the high-throughput protein microarray method can classify HIV monoclonal antibodies and variant antigens.
  • the polyclonal immune response in the plasma samples of HIV-infected persons showed different binding modes.
  • the response to different antigen proteins of the imprinting showed the precise diversity and specificity of antibodies for the humoral response to HIV. Therefore, current researchers have used protein chip technology to coat recombinant HIV antigens to diagnose AIDS.
  • the method research has high specificity (99.97%) and sensitivity (100%), and improves the efficiency and accuracy of diagnosis.
  • HIV chips can be used to monitor the disease process and the antibody response during treatment.
  • HIV laboratory testing mainly includes HIV antibody testing, HIV nucleic acid qualitative and quantitative testing, CD4 + T lymphocyte count, HIV drug resistance testing, etc.
  • HIV1/2 antibody (human immunodeficiency virus 1+2 antibody) test is the gold standard for HIV diagnosis. HIV1/2 antibody testing includes screening tests and supplementary tests. The most commonly used method for clinical HIV antibody screening is immunoenzyme-linked immunosorbent assay (ELISA). HIV ELISA reagents have been developed to the fourth generation so far. The current mainstream international and domestic HIV testing reagents and methods are the third-generation reagents-double antigen sandwich method to detect antibodies in specimens.
  • the detection sensitivity and specificity are significantly higher than those of the first and second-generation reagents, which improves the O group specimens.
  • the reactivity, the window period is shortened to 22 days.
  • this method has lower sensitivity. False positives due to contamination.
  • the fourth-generation reagents have increased the detection of P24 antigen, and the positive rate of detection is increased.
  • the specificity of the fourth-generation reagent is lower than that of the third-generation reagent, because the former coating component contains HIV P24 antibody, which is a mouse antibody, and 1% to 2% of blood donors respond to it or have unknown reasons. The antigen response.
  • WB Western blotting
  • A-J 10 subtypes
  • O group O group
  • N group 7 subtypes
  • A-G 7 subtypes
  • HIV-1 is prevalent in my country, and there are 8 subtypes detected.
  • subtype B is found in almost all regions of my country
  • subtype C is mainly found in southwest and northwest China
  • subtype E is more common in the southeast coast.
  • HIV protein immunoblotting test WB criterion is: HIV-1 antibody positive: at least 2 env bands (gp41 and gp160/gp120), or 1 env band and p24 band; HIV-2 antibody Positive: At least two env bands (gp36 and gp140/gp105) appear and meet the positive standard provided by the kit.
  • WB HIV protein immunoblotting test
  • HIV immunoblotting experiments have a 2% false positive rate, which is mostly seen in patients with a history of vaccination and autoimmune diseases.
  • the detection rate of HIV antibodies can be greatly increased (about 99%), but it will also increase the cost, detection time, and additional burden on patients.
  • WB has strict experimental technology requirements and many influencing factors.
  • the false positive rate of band misjudgment and improper operation exceeds 2%.
  • the initial HIV-positive 4%-20% of WB samples The test result is "antibody indeterminate". Uncertain specimens can only be confirmed after 3-6 months of follow-up. Therefore, it is necessary to provide a highly sensitive and highly specific method and product for the diagnosis of AIDS-infected persons.
  • the purpose of this application is to provide a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies.
  • Another purpose of this application is to provide a protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies.
  • Another purpose of this application is to provide a method for preparing a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies.
  • the embodiment of the application provides a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies.
  • the protein chip includes a substrate on which HIV-specific recombinant antigens gp36, gp41, gp120, and gp160 are fixed on the surface. .
  • the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the substrate is an aldehyde modified glass plate.
  • the molecular weight bands of HIV-specific recombinant antigens gp36, gp41, gp120, and gp160 are used for the WB clinical verification test.
  • the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies can be used to target HIV-specific recombinant antigens gp36 and gp41 of AIDS patients by changing different labeled fluorescent agents. , Gp120, gp160 antibodies (IgG antibody and IgM antibody) are tested simultaneously;
  • the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies wherein the HIV-specific recombinant antigens gp36, gp41, gp120, and gp160 diluent use 2 ⁇ SCC (sodium citrate ) Buffer dilution is beneficial to the preservation of antigen activity.
  • SCC sodium citrate
  • the coating concentration of HIV-specific recombinant antigen gp36 is 3.125ug/mL; the coating concentration of gp41 is 12.5ug/mL, the coating concentration of gp120 is 200ug/mL, and the coating concentration of gp160 is 12.5ug/mL.
  • the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the control area has a negative result of immobilization of 10% bovine serum albumin (Bovine Serum Album, BSA) Contrast.
  • BSA Bovine Serum Album
  • the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies wherein the surface of the substrate includes a plurality of detection areas and control areas, and any detection area includes at least one set of HIV-specific Recombinant antigens gp36, gp41, gp120 and gp160 detection points, the same antigen concentration in the same detection area is the same.
  • the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies wherein the detection chip includes ten detection areas, and each detection area includes 10 detection points, fixed with The diameter of the HIV recombinant antigen detection spots is 50 ⁇ m, the spot spacing is 100 ⁇ m, and the detection spots form an array of 2.5 mm ⁇ 2.5 mm.
  • the embodiment of the application provides a protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies, wherein the detection kit includes a protein chip and a detection reagent, and the protein chip includes a substrate, a substrate
  • the HIV-specific recombinant antigens gp36, gp41, gp120 and gp160 are fixed on the surface.
  • the protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the detection reagent is an enzyme-labeled antibody, more preferably horseradish peroxidase Labeled antibodies.
  • the embodiment of the application provides a method for preparing a protein chip for detecting human immunodeficiency virus HIV1/2 antibody, which includes the following steps:
  • the HIV recombinant antigens are fixed on the substrate of the protein chip, and the recombinant antigens include HIV antigens gp36, gp41, gp120 and gp160.
  • the method for preparing a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies includes the step of treating the surface of the substrate of the protein chip with aldehyde groups.
  • Fig. 1 is a diagram showing the arrangement of antigens in a glass chip block prepared according to Example 1 of the present application, in which 10 servings per chip;
  • FIG. 2 is a scanning entity diagram of the quality control experimental chip for the aldehyde-based chip protein IgG coated detection line during the preparation process according to Example 1 of the present application;
  • Example 3 is a chemiluminescence scan diagram of the concentration gradient of the coating antigen and the plasma dilution gradient during the preparation process according to Example 1 of the present application;
  • Figure 4 is a chemiluminescence scan of avidin HRP-labeled secondary antibody concentration gradient experiment.
  • Figure 5 shows the fluorescence intensity scan of the results of HIV protein chip detection of patients' plasma, including 8 HIV patients, 1 normal control and 1 blank control.
  • the protein chip includes a substrate, and the surface of the substrate is replaceable detection paper.
  • the surface of the detection paper includes one or more detections. Area and a control area, the detection area includes one or more detection points, the detection points are fixed with HIV recombinant antigens, and the HIV recombinant antigens include HIV-specific recombinant antigens gp36, gp41, gp120, and gp160, each of which is Corresponds to a plasma or serum sample.
  • the substrate of the protein chip can be a glass or silicon wafer, and the active functional group aldehyde group is introduced into the surface of the substrate, which can effectively and firmly fix the protein.
  • Adhesive tape is adhered to the surface of the substrate to physically separate the chip into ten independent detection areas, which can be used interchangeably.
  • the 1-10nL recombinant antigen solution and the negative control solution can be spotted on the substrate with a high-speed automatic spotting instrument.
  • the spotting conditions are: temperature 25°C and dry environment. Under such spotting conditions, the morphology of spotting can be improved, and the distribution of antibodies on the slide is more even.
  • the protein is connected to the aldehyde-treated substrate through a chemical bond to form a Schiff base to be immobilized. After standing overnight at 4°C, vacuum drying for 1-4 hours.
  • Any detection point is fixed with one of HIV-specific recombinant antigens gp36, gp41, gp120 or gp160, and the detection area includes at least one gp36, gp41, gp120 and gp160 detection point.
  • the size of the spot coating of the antigen and the control can be changed according to the size of the spot and the change of the spot spacing; the arrangement and distribution number of the array can be changed according to the number of samples to be tested.
  • the substrate has a plurality of the detection areas, each detection area includes 4 detection points arranged in 4 rows, and the control area includes 1 4 control points in a row; the test points and control points are arranged in 5 parallel rows.
  • the detection area is obtained by separating the detachable adhesive separation tape. Before spotting, a specific frame structure is adopted to optimize the chip lattice, so that the samples can be tested in batches, the operation is simple, the sample amount is small, and there is no cross-contamination.
  • the diameter of the antigen spot and the control spot is 50 ⁇ m, and the spot spacing is 100 ⁇ m, the size of the antigen array is 2.5mm ⁇ 2.5mm, and 10 detection areas can be set on a glass substrate at the same time, and each chip detects 10 samples .
  • the method of using the protein chip to detect AIDS antibody includes the following steps:
  • the detection reagent may be an enzyme-labeled antibody commonly used in a chemiluminescence reaction method, such as a horseradish peroxidase-labeled antibody.
  • the concentration of the enzyme-labeled antibody is related to the detection effect.
  • the concentration of the enzyme-labeled antibody is preferably ⁇ 0.4 ug/mL.
  • sample spotter NanoPlotter NP 2.1/2GeSim ultra-micro sample spotter from Germany
  • chemiluminescence scanner Bio-rad ChemiDoc MP chemiluminescence imaging system
  • centrifuge Thermo Scientific Sorvall Legend Micro 17&21 series centrifugation machine.
  • Scanning with a chemiluminescence imaging system is the quality control experiment of the aldehyde-modified chip protein IgG coated detection line. The results are shown in Figure 2. Within a certain range, the intensity of the luminescence signal generated varies with the concentration of human IgG. The signal intensity is significantly different from that of the blank control. At the same time, as the concentration of the HRP-labeled secondary antibody decreases, the signal intensity also changes. Therefore, the aldehyde modified chip can bind well to protein biomolecules.
  • the indirect ELISA checkerboard titration method was used to determine the optimal coating concentration and plasma dilution of HIV-1/2 recombinant antigen.
  • PBST-BSA coating solution to dilute 4 kinds of HIV recombinant antigens at 50 ⁇ g/mL, 12.5 ⁇ g/mL, 3.125 ⁇ g/mL, 0.78 ⁇ g/mL, 0.195 ⁇ g/mL, 0 ⁇ g/mL 6 concentrations, Spot the 4 HIV recombinant antigens with different concentrations on the detection area on the substrate, each with 6 multiple spots, and incubate at 4°C for 12 hours;
  • the experimental results of the gradient concentration of different plasma concentrations corresponding to different HIV recombinant antigens can be obtained.
  • the results are shown in Figure 3.
  • the corresponding plasma concentration in the detection area of the chip 1-9 is: stock solution, 1:10 , 1: 102, 1: 103, 1: 104, 10 5 and the detection region is blank, can be obtained from each of the antigens in FIG optimal coating concentration and plasma dilution are shown in Table 2.
  • each detection square use a high-speed automatic spotting instrument to spot HIV recombinant antigens gp36, gp41, gp120 and gp160, and the negative control solution on the chip in an array pattern to form a detection spot and a control spot, 4°C After standing overnight, vacuum drying for 2 hours; further, after vacuum drying, air-sealed with a plastic pouch resistant to water vapor and stored at 4°C.
  • This application realizes the simultaneous dot matrix of different HIV type antigens on one substrate, which can detect the IgG and/or IgM antibodies of multiple human samples on one glass slide at the same time, and the required sample volume is small (15 ⁇ L). Multiple index response results can be obtained through only one response, which improves the detection speed and efficiency, and can provide more indexes for the clinic.
  • the amount of antigen required for the protein chip of this application is reduced. At least 5 chips can be spotted with 1uL. The amount of antigen required to detect a patient's blood sample is much lower than the ELISA method, which is simple and fast, and reduces the cost of detection.
  • the protein chip of the present application has high detection specificity, stable detection results, and high reliability, which is beneficial to clinical diagnosis and treatment.
  • the problem of false positives caused by misjudgment of bands and improper operation of the existing method is avoided, and the problem of "undefined antibody" in the WB test results of 4%-20% of samples that are HIV-positive by the existing detection method is avoided.
  • the use of the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies of the present application for HIV antibody detection has the characteristics of small sample amount, time-saving, labor-saving, and high accuracy.

Abstract

Disclosed are a protein chip for detecting a human immunodeficiency virus HIV1/2 antibody and a preparation method therefor. The protein chip comprises a detection substrate, the surface of which is a replaceable aldehyde group modification, wherein the surface of the detection substrate comprises a detection area formed by detection points, and HIV recombinant antigens gp36, gp41, gp120 and gp160 are fixed to the detection points.

Description

一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片及其制备方法Protein chip for detecting human immunodeficiency virus HIV1/2 antibody and preparation method thereof
本申请要求提交在中国专利局、申请号为202010203740.0,发明名称为“一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片及其制备方法”的中国专利申请的优先权，该申请的全部内容通过引用结合在本申请中。This application requires the priority of a Chinese patent application filed in the Chinese Patent Office with the application number 202010203740.0 and the invention title "A protein chip for detecting human immunodeficiency virus HIV1/2 antibody and its preparation method". The entire content is incorporated into this application by reference.
技术领域Technical field
本申请属于免疫化学领域，具体是一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片及其制备方法。This application belongs to the field of immunochemistry, and specifically relates to a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies and a preparation method thereof.
背景技术Background technique
艾滋病是由人类免疫缺陷病毒感染所导致的传染病。人类免疫缺陷病毒(Human Immunodeficiency Virus；HIV)，即艾滋病(AIDS，获得性免疫缺陷综合征)病毒，它属于逆转录病毒的一种，是一种感染人类免疫***细胞的慢病毒(Lentivirus)。AIDS is an infectious disease caused by human immunodeficiency virus infection. Human Immunodeficiency Virus (HIV), that is, AIDS (Acquired Immune Deficiency Syndrome) virus, is a type of retrovirus, a type of Lentivirus that infects cells of the human immune system.
蛋白芯片(proteinchip)是集微电子、微机械、化学物理技术、计算机技术为一体的一门高新技术，被认为是生命科学研究中的高效工具，是将各种蛋白质有序地固定于载体上成为检测用的芯片，然后用标记了特定荧光抗体的蛋白质或其他成分与芯片作用，经漂洗将未能与芯片上的蛋白质互补结合的成分洗去，再利用荧光扫描仪或激光共聚焦扫描技术，测定芯片上各点的荧光强度，通过荧光强度分析蛋白质与蛋白质之间相互作用的关系，由此达到测定各种蛋白质功能的目的。利用这项技术可同时对多种蛋白质进行平行检测分析，使得用常规ELISA技术需上千次才能完成的分析在蛋白质芯片上仅需一次就可以完成，并且检测到的平行数据误差更小，更准确。Proteinchip is a high and new technology integrating microelectronics, micromechanics, chemical physics technology, and computer technology. It is considered to be an efficient tool in life science research and is to fix various proteins on a carrier in an orderly manner. Become a chip for detection, and then use the protein or other components labeled with specific fluorescent antibodies to interact with the chip. After rinsing, the components that cannot be complementary to the protein on the chip are washed away, and then fluorescent scanner or laser confocal scanning technology is used , Measure the fluorescence intensity of each point on the chip, and analyze the interaction relationship between protein and protein by fluorescence intensity, thereby achieving the purpose of measuring the function of various proteins. This technology can be used for parallel detection and analysis of multiple proteins at the same time, so that the analysis that requires thousands of times with conventional ELISA technology can be completed only once on the protein chip, and the detected parallel data error is smaller and more precise.
蛋白质芯片技术具有高通量，微型化和快速平行分析、样本量少、高敏感性及特异性等优点，在临床肿瘤、感染性疾病、自身免疫性疾病的检测，药物研发等领域有着广泛应用。高通量蛋白质微阵列方法可对HIV单克隆抗体和变异抗原进行分类。Protein chip technology has the advantages of high throughput, miniaturization and rapid parallel analysis, small sample size, high sensitivity and specificity, etc., and has a wide range of applications in the detection of clinical tumors, infectious diseases, autoimmune diseases, and drug development. . The high-throughput protein microarray method can classify HIV monoclonal antibodies and variant antigens.
HIV感染者的血浆样品中的多克隆免疫反应显示出不同的结合模式，对印迹不同抗原蛋白的反应，可以发现对于HIV的体液反应的抗体精准多样性和特异性。因此，目前已有学者使用蛋白质芯片技术包被重组HIV抗原诊断艾滋病，其方法研究具有很高的特异性(99.97％)和敏感性(100％)，提高诊断效率、准确性。此外，HIV芯片可用于监测疾病过程和治疗过程中的抗体反应。The polyclonal immune response in the plasma samples of HIV-infected persons showed different binding modes. The response to different antigen proteins of the imprinting showed the precise diversity and specificity of antibodies for the humoral response to HIV. Therefore, current scholars have used protein chip technology to coat recombinant HIV antigens to diagnose AIDS. The method research has high specificity (99.97%) and sensitivity (100%), and improves the efficiency and accuracy of diagnosis. In addition, HIV chips can be used to monitor the disease process and the antibody response during treatment.
根据《全国艾滋病检测技术规范》(2015年修订版)，HIV实验室检测主要包括HIV 抗体检测、HIV核酸定性和定量检测、CD4 +T淋巴细胞计数、HIV耐药检测等。HIV1/2抗体(人类免疫缺陷病毒1+2型抗体)检测是HIV诊断金标准。HIV1/2抗体检测包括筛查试验和补充试验。临床HIV抗体筛查最常用的方法为免疫酶联免疫吸附法化(ELISA)，HIV ELISA试剂到目前为止已经发展到了第4代。目前国际和国内HIV检测的主流试剂和方法是第3代试剂——双抗原夹心法检测标本中的抗体，其检测的灵敏度和特异性比第1、2代试剂明显提高，改善了O群标本的反应性，窗口期缩短至22天，但是该方法与化学发光和时间分辨荧光免疫分析技术等更先进的分析技术方法相比，其灵敏度较低，用于检测的包被微孔之间会因为携带污染而出现假阳性。第4代试剂比第3代试剂增加了P24抗原的检测，其检测阳性率增高。但有研究表明第4代试剂的特异性低于第3代试剂，因为前者包被的成分含有HIV P24抗体为鼠抗体，在献血人群中有1％～2％的对其有反应或不明原因的抗原反应。 According to the "National AIDS Testing Technical Specifications" (revised in 2015), HIV laboratory testing mainly includes HIV antibody testing, HIV nucleic acid qualitative and quantitative testing, CD4 + T lymphocyte count, HIV drug resistance testing, etc. HIV1/2 antibody (human immunodeficiency virus 1+2 antibody) test is the gold standard for HIV diagnosis. HIV1/2 antibody testing includes screening tests and supplementary tests. The most commonly used method for clinical HIV antibody screening is immunoenzyme-linked immunosorbent assay (ELISA). HIV ELISA reagents have been developed to the fourth generation so far. The current mainstream international and domestic HIV testing reagents and methods are the third-generation reagents-double antigen sandwich method to detect antibodies in specimens. The detection sensitivity and specificity are significantly higher than those of the first and second-generation reagents, which improves the O group specimens. The reactivity, the window period is shortened to 22 days. However, compared with more advanced analytical techniques such as chemiluminescence and time-resolved fluorescence immunoassay techniques, this method has lower sensitivity. False positives due to contamination. Compared with the third-generation reagents, the fourth-generation reagents have increased the detection of P24 antigen, and the positive rate of detection is increased. However, studies have shown that the specificity of the fourth-generation reagent is lower than that of the third-generation reagent, because the former coating component contains HIV P24 antibody, which is a mouse antibody, and 1% to 2% of blood donors respond to it or have unknown reasons. The antigen response.
目前中国《全国艾滋病检测技术规范》中的规定HIV感染唯一的确认方法是免疫印迹实验(WB)，也是证实HIV感染的最特异、最敏感的方法，为抗体检测的金标准。现已发现有HIV-1和HIV-2两型，将HIV-1分为M组，共10个亚型(A-J)、O组和N组，HIV-2也有7个亚型(A-G)。在我国流行的是HIV-1，检测到的亚型有8种之多，其中B亚型几乎见于我国各个地区，而C亚型主要见于中国西南和西北地区，E亚型则多见于东南沿海和西南边境地区，在部分地区发现并证实我国有少数HIV-2型感染者。我国HIV蛋白免疫印迹实验(WB)判定标准是：HIV-1抗体阳性：至少出现2条env条带(gp41和gp160/gp120)，或出现1条env条带和p24条带；HIV-2抗体阳性：至少出现2条env条带(gp36和gp140/gp105)，并符合试剂盒提供的阳性标准。但是HIV免疫印迹实验有2％的假阳性率，大多见于有疫苗接种史和患有自身免疫性疾病的患者。若将酶联免疫实验和免疫印迹实验结合起来检测HIV抗体，可大大提高HIV抗体的检出率(约99％)，但也会增加成本、检测时间，额外增加患者负担。而且WB与ELISA及其他检测方法相比，实验技术要求严格、影响因素较多，条带误判及操作不当的假阳性率超过2％，初检HIV阳性的4％-20％的样品WB的检测结果为“抗体不确定”，不确定的标本需3-6个月随访后方能确定。因此，有必要提供一种高灵敏度、高特异性的用于艾滋病感染者诊断的方法和产品。At present, China's "National AIDS Testing Technical Specifications" stipulates that the only method for confirming HIV infection is Western blotting (WB), which is also the most specific and sensitive method for confirming HIV infection, and is the gold standard for antibody testing. It has been found that there are two types of HIV-1 and HIV-2. HIV-1 is divided into M group, a total of 10 subtypes (A-J), O group and N group, HIV-2 also has 7 subtypes (A-G). HIV-1 is prevalent in my country, and there are 8 subtypes detected. Among them, subtype B is found in almost all regions of my country, subtype C is mainly found in southwest and northwest China, and subtype E is more common in the southeast coast. In the border area with Southwest my country, a small number of people with HIV-2 infection have been found and confirmed in some areas. my country's HIV protein immunoblotting test (WB) criterion is: HIV-1 antibody positive: at least 2 env bands (gp41 and gp160/gp120), or 1 env band and p24 band; HIV-2 antibody Positive: At least two env bands (gp36 and gp140/gp105) appear and meet the positive standard provided by the kit. However, HIV immunoblotting experiments have a 2% false positive rate, which is mostly seen in patients with a history of vaccination and autoimmune diseases. If the enzyme-linked immunosorbent assay and immunoblotting experiment are combined to detect HIV antibodies, the detection rate of HIV antibodies can be greatly increased (about 99%), but it will also increase the cost, detection time, and additional burden on patients. Moreover, compared with ELISA and other detection methods, WB has strict experimental technology requirements and many influencing factors. The false positive rate of band misjudgment and improper operation exceeds 2%. The initial HIV-positive 4%-20% of WB samples The test result is "antibody indeterminate". Uncertain specimens can only be confirmed after 3-6 months of follow-up. Therefore, it is necessary to provide a highly sensitive and highly specific method and product for the diagnosis of AIDS-infected persons.
发明内容Summary of the invention
为了克服HIV第三代试剂假阳性率高及免疫印迹实验检测的局限性的问题，提出并完成了本申请。In order to overcome the high false positive rate of HIV third-generation reagents and the limitations of Western blotting experiments, this application is proposed and completed.
本申请的目的是提供一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片。The purpose of this application is to provide a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies.
本申请的再一目的是提供一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片检测试剂盒。Another purpose of this application is to provide a protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies.
本申请的再一目的是提供了一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片的制备方法。Another purpose of this application is to provide a method for preparing a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies.
本申请实施例提供了一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，所述蛋白芯片包括基片，基片表面固定有HIV特异性重组抗原gp36、gp41、gp120和gp160检测点。The embodiment of the application provides a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies. The protein chip includes a substrate on which HIV-specific recombinant antigens gp36, gp41, gp120, and gp160 are fixed on the surface. .
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，所述的基片为醛基修饰玻璃片。In one of the embodiments, the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the substrate is an aldehyde modified glass plate.
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，将WB临床验证检测HIV特异性重组抗原gp36、gp41、gp120、gp160分子量条带以芯片点的形式显现；In one of the embodiments, according to the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, the molecular weight bands of HIV-specific recombinant antigens gp36, gp41, gp120, and gp160 are used for the WB clinical verification test. The form of dots appears;
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，若通过更换不同标记荧光剂可以实现蛋白芯片针对艾滋病患者HIV特异性重组抗原gp36、gp41、gp120、gp160抗体(IgG抗体和IgM抗体)实行同步检测；In one of the embodiments, according to the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, the protein chip can be used to target HIV-specific recombinant antigens gp36 and gp41 of AIDS patients by changing different labeled fluorescent agents. , Gp120, gp160 antibodies (IgG antibody and IgM antibody) are tested simultaneously;
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，HIV特异性重组抗原gp36、gp41、gp120、gp160稀释液用2×SCC(柠檬酸钠)缓冲液稀释，有利于抗原活性的保存。In one of the embodiments, the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the HIV-specific recombinant antigens gp36, gp41, gp120, and gp160 diluent use 2×SCC (sodium citrate ) Buffer dilution is beneficial to the preservation of antigen activity.
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，HIV特异性重组抗原gp36的包被浓度为3.125ug/mL；gp41的包被浓度为12.5ug/mL，gp120的包被浓度为200ug/mL，gp160的包被浓度为12.5ug/mL。In one of the embodiments, according to the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, the coating concentration of HIV-specific recombinant antigen gp36 is 3.125ug/mL; the coating concentration of gp41 is 12.5ug/mL, the coating concentration of gp120 is 200ug/mL, and the coating concentration of gp160 is 12.5ug/mL.
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，所述对照区域有固定10％牛血清白蛋白(Bovine Serum Albumin，BSA)形成的阴性对照。In one of the embodiments, the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the control area has a negative result of immobilization of 10% bovine serum albumin (Bovine Serum Album, BSA) Contrast.
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，基片表面包括多个检测区域和对照区域，任意检测区域至少包括一组HIV特异性重组抗原gp36、gp41、gp120和gp160检测点，同一个检测区域内的同一种抗原的浓度相同。In one of the embodiments, the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the surface of the substrate includes a plurality of detection areas and control areas, and any detection area includes at least one set of HIV-specific Recombinant antigens gp36, gp41, gp120 and gp160 detection points, the same antigen concentration in the same detection area is the same.
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，所述检测芯片包括十个检测区域，每个检测区域包括10个检测点，固定有HIV 重组抗原的检测点的直径为50μm，点间距为100μm，检测点形成2.5mm×2.5mm的阵列。In one of the embodiments, the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the detection chip includes ten detection areas, and each detection area includes 10 detection points, fixed with The diameter of the HIV recombinant antigen detection spots is 50 μm, the spot spacing is 100 μm, and the detection spots form an array of 2.5 mm×2.5 mm.
本申请实施例提供了一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片检测试剂盒，其中，所述检测试剂盒包括蛋白芯片和检测试剂，所述蛋白芯片包括基片，基片表面固定有HIV特异性重组抗原gp36、gp41、gp120和gp160检测点。The embodiment of the application provides a protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies, wherein the detection kit includes a protein chip and a detection reagent, and the protein chip includes a substrate, a substrate The HIV-specific recombinant antigens gp36, gp41, gp120 and gp160 are fixed on the surface.
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片检测试剂盒，其中，所述检测试剂为酶标记的抗体，更优选为辣根过氧化物酶标记的抗体。In one of the embodiments, the protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application, wherein the detection reagent is an enzyme-labeled antibody, more preferably horseradish peroxidase Labeled antibodies.
本申请实施例提供了一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片的制备方法，包括以下步骤：The embodiment of the application provides a method for preparing a protein chip for detecting human immunodeficiency virus HIV1/2 antibody, which includes the following steps:
将HIV重组抗原固定于蛋白芯片的基片上，所述重组抗原包括HIV抗原gp36、gp41、gp120和gp160。The HIV recombinant antigens are fixed on the substrate of the protein chip, and the recombinant antigens include HIV antigens gp36, gp41, gp120 and gp160.
在其中一个实施例中，根据本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片的制备方法，包括将所述蛋白芯片的基片表面醛基处理的步骤。In one of the embodiments, the method for preparing a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to the present application includes the step of treating the surface of the substrate of the protein chip with aldehyde groups.
本申请本申请本申请本申请本申请 附图说明 This application of the present application of the present application BRIEF DESCRIPTION The present application of the present application
图1为根据本申请的实施例1制备的玻璃芯片区块抗原排布图，其中，10人份/每张芯片；Fig. 1 is a diagram showing the arrangement of antigens in a glass chip block prepared according to Example 1 of the present application, in which 10 servings per chip;
图2为根据本申请实施例1的制备过程中醛基芯片蛋白质IgG包被检测线质量控制实验芯片扫描实体图；2 is a scanning entity diagram of the quality control experimental chip for the aldehyde-based chip protein IgG coated detection line during the preparation process according to Example 1 of the present application;
图3为根据本申请实施例1的制备过程中包被抗原浓度梯度及血浆稀释梯度化学发光扫描图；3 is a chemiluminescence scan diagram of the concentration gradient of the coating antigen and the plasma dilution gradient during the preparation process according to Example 1 of the present application;
图4为亲和素HRP标记二抗浓度浓度梯度实验化学发光扫描图。Figure 4 is a chemiluminescence scan of avidin HRP-labeled secondary antibody concentration gradient experiment.
图5显示HIV蛋白质芯片检测患者血浆结果的荧光强度扫描图，包括8例HIV患者和1例正常对照及1例空白对照。Figure 5 shows the fluorescence intensity scan of the results of HIV protein chip detection of patients' plasma, including 8 HIV patients, 1 normal control and 1 blank control.
具体实施方式Detailed ways
为了使本申请的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本申请进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本申请，并不用于限定本申请。In order to make the purpose, technical solutions, and advantages of this application clearer and clearer, the following further describes the application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present application, and are not used to limit the present application.
以下结合附图，详细说明本申请各实施例提供的技术方案。The technical solutions provided by the embodiments of the present application will be described in detail below with reference to the accompanying drawings.
本申请提供了一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，所述蛋白芯片包括基片，基片表面为可替换的检测纸，所述检测纸表面包括一个或多个检测区域以及对照区域，所述检测区域包括一个或多个检测点，所述检测点固定有HIV重组抗原，所述HIV重组抗原包括HIV特异性重组抗原gp36、gp41、gp120和gp160，每个检测点对应一份血浆或血清样品。This application provides a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies. The protein chip includes a substrate, and the surface of the substrate is replaceable detection paper. The surface of the detection paper includes one or more detections. Area and a control area, the detection area includes one or more detection points, the detection points are fixed with HIV recombinant antigens, and the HIV recombinant antigens include HIV-specific recombinant antigens gp36, gp41, gp120, and gp160, each of which is Corresponds to a plasma or serum sample.
根据本申请的技术方案，蛋白芯片所述的基片可以为玻璃片或硅片，将活性官能基团醛基引入基片表面，能够有效牢固固定蛋白质。According to the technical solution of the present application, the substrate of the protein chip can be a glass or silicon wafer, and the active functional group aldehyde group is introduced into the surface of the substrate, which can effectively and firmly fix the protein.
基片表面粘附胶纸，将芯片分物理分隔成十个独立的检测区，可替换使用。Adhesive tape is adhered to the surface of the substrate to physically separate the chip into ten independent detection areas, which can be used interchangeably.
可以利用高速全自动点样仪将1-10nL重组抗原的溶液及阴性对照溶液点样于基片，点样的条件为：温度25℃、干燥环境。这样的点样条件下，可以改善点样的形态，使抗体在玻片上的分布更加均匀。蛋白质通过化学键连接于醛基处理的基片上，形成Schiff碱而被固定。4℃下，静置过夜后，真空干燥1-4小时。The 1-10nL recombinant antigen solution and the negative control solution can be spotted on the substrate with a high-speed automatic spotting instrument. The spotting conditions are: temperature 25°C and dry environment. Under such spotting conditions, the morphology of spotting can be improved, and the distribution of antibodies on the slide is more even. The protein is connected to the aldehyde-treated substrate through a chemical bond to form a Schiff base to be immobilized. After standing overnight at 4°C, vacuum drying for 1-4 hours.
任意检测点固定有HIV特异性重组抗原gp36、gp41、gp120或gp160之一，检测区域至少包括一个gp36、gp41、gp120和gp160检测点。Any detection point is fixed with one of HIV-specific recombinant antigens gp36, gp41, gp120 or gp160, and the detection area includes at least one gp36, gp41, gp120 and gp160 detection point.
抗原与对照的点涂层的大小，可以根据点的大小，点间距的变化而变化；阵列的排列和分布数量可以根据待检样品的数目而变化。如图1所示的玻璃芯片区块抗原排布图，基片上具有多个所述检测区，所述每个检测区域包括排列成4排的4个检测点，所述对照区域包括排列成1排的4个对照点；所述检测点和对照点排成平行的5列。所述检测区由可拆卸型粘贴分隔胶纸分离得到。点样前采用特定的框架结构，优化了芯片点阵，使得样本可批量检测，操作简便，样本用量小，互不交叉污染。抗原点与对照点的直径为50μm，点间距为100μm时，抗原阵列的大小为2.5mm×2.5mm，且可同时在一张玻璃基片上设置10个检测区，则每张芯片检测10份样品。The size of the spot coating of the antigen and the control can be changed according to the size of the spot and the change of the spot spacing; the arrangement and distribution number of the array can be changed according to the number of samples to be tested. As shown in FIG. 1, the glass chip block antigen arrangement diagram, the substrate has a plurality of the detection areas, each detection area includes 4 detection points arranged in 4 rows, and the control area includes 1 4 control points in a row; the test points and control points are arranged in 5 parallel rows. The detection area is obtained by separating the detachable adhesive separation tape. Before spotting, a specific frame structure is adopted to optimize the chip lattice, so that the samples can be tested in batches, the operation is simple, the sample amount is small, and there is no cross-contamination. The diameter of the antigen spot and the control spot is 50μm, and the spot spacing is 100μm, the size of the antigen array is 2.5mm×2.5mm, and 10 detection areas can be set on a glass substrate at the same time, and each chip detects 10 samples .
使用上述蛋白芯片检测艾滋病抗体的方法包括以下步骤：The method of using the protein chip to detect AIDS antibody includes the following steps:
(1)用10％胎牛血清的封闭液，封闭基片表面非特异性位点，37℃孵育1～1.5小时；所述10％胎牛血清的封闭液是由PBST稀释胎牛血清获得。(1) Use a blocking solution of 10% fetal bovine serum to block non-specific sites on the surface of the substrate, and incubate at 37° C. for 1 to 1.5 hours; the blocking solution of 10% fetal bovine serum is obtained by diluting fetal bovine serum with PBST.
(2)用PBST液冲洗3-4次，每次10秒钟，用PBS冲洗，离心甩干，去除多余封闭液；(2) Rinse 3-4 times with PBST solution, 10 seconds each time, rinse with PBS, centrifuge to dry, and remove excess blocking solution;
(3)滴加病人血浆或血清样品15uL于芯片上，置于湿盒内37℃孵育30分钟，使抗原与抗体充分反应；(3) Add 15uL of the patient's plasma or serum sample onto the chip and place it in a wet box and incubate at 37°C for 30 minutes to make the antigen and antibody fully react;
(4)用PBST洗涤3～4次，每次10秒钟，用PBS冲洗，离心甩干，去除多余样品；(4) Wash with PBST 3 to 4 times, 10 seconds each time, rinse with PBS, centrifuge to dry, and remove excess samples;
(5)滴加PBS稀释过的HRP标记抗人二抗(HRP-Anti-Human-IgG)，浓度为0.4ug/ml的 混合液，37℃避光孵育30分钟；(5) Add dropwise a mixture of HRP-Anti-Human-IgG diluted with PBS at a concentration of 0.4ug/ml, and incubate for 30 minutes at 37°C in the dark;
(6)用PBST洗涤3次，每次10秒钟，用PBS冲洗，离心甩干；(6) Wash 3 times with PBST, 10 seconds each time, rinse with PBS, and centrifuge to dry;
(7)用化学发光扫描仪扫描并收集信号，依据扫描显示，分析检测结果。(7) Scan and collect signals with a chemiluminescence scanner, and analyze the detection results according to the scan display.
显然地，额外的检测试剂是使用上述蛋白芯片检测艾滋病抗体所必须的。所述检测试剂可以为化学发光反应方法中常用的酶标记抗体，例如辣根过氧化物酶标记的抗体。Obviously, additional testing reagents are necessary to use the above-mentioned protein chip to detect AIDS antibodies. The detection reagent may be an enzyme-labeled antibody commonly used in a chemiluminescence reaction method, such as a horseradish peroxidase-labeled antibody.
酶标记抗体的浓度与检测效果相关，本申请的实施例优选酶标记抗体的浓度≥0.4ug/mL。The concentration of the enzyme-labeled antibody is related to the detection effect. In the embodiments of the present application, the concentration of the enzyme-labeled antibody is preferably ≥0.4 ug/mL.
以下实施例中所使用的设备及试剂为：The equipment and reagents used in the following embodiments are:
1、主要仪器设备：点样仪，德国NanoPlotter NP 2.1/2GeSim超微量样品点量仪；化学发光扫描仪，Bio-rad伯乐ChemiDoc MP化学发光成像***；离心机，Thermo Scientific Sorvall Legend Micro 17&21系列离心机。1. Main instruments and equipment: sample spotter, NanoPlotter NP 2.1/2GeSim ultra-micro sample spotter from Germany; chemiluminescence scanner, Bio-rad ChemiDoc MP chemiluminescence imaging system; centrifuge, Thermo Scientific Sorvall Legend Micro 17&21 series centrifugation machine.
2、主要试剂及其来源：醛基芯片购自上海百傲公司；人IgG购自英国abcam公司；HRP标记的驴抗人IgG抗体购自上海生工生物工程股份有限公司；Immobilon Western HRP底物购自Merckmillipore公司；PBS粉末、Tween20及胎牛血清(BSA)粉末均购自Sigma公司；PBST缓冲液：将商品化PBS粉末溶解在去离子水中，形成浓度0.01M、pH＝7.4的PBS缓冲液；然后加入Tween-20，混匀，获得PBST溶液，在PBST溶液中Tween20的体积浓度为0.1％。2. The main reagents and their sources: aldehyde-based chips were purchased from Shanghai Baiao Company; human IgG was purchased from abcam company in the UK; HRP-labeled donkey anti-human IgG antibody was purchased from Shanghai Shenggong Bioengineering Co., Ltd.; Immobilon Western HRP substrate Purchased from Merckmillipore; PBS powder, Tween20 and fetal bovine serum (BSA) powder were all purchased from Sigma; PBST buffer: Dissolve commercial PBS powder in deionized water to form a PBS buffer with a concentration of 0.01M and pH=7.4 ; Then add Tween-20 and mix well to obtain a PBST solution. The volume concentration of Tween20 in the PBST solution is 0.1%.
HIV-1/2重组抗原来源见表1。See Table 1 for the sources of HIV-1/2 recombinant antigens.
表1 HIV1+2型重组抗原来源及货号Table 1 Sources and article numbers of HIV1+2 recombinant antigens
Figure PCTCN2021081827-appb-000001
Figure PCTCN2021081827-appb-000001
实施例1Example 1
一、质控实验1. Quality control experiment
1.醛基芯片蛋白质IgG包被检测线质量控制实验1. Quality control experiment of aldehyde-based chip protein IgG coating detection line
1)将梯度稀释200μg/mL、100μg/mL、50μg/mL、25μg/mL、12.5μg/mL、6.25μg/mL、 3.13μg/mL、1.56μg/mL、0.78μg/mL、0.39μg/mL、0.19μg/mL的人IgG溶液及PBS空白对照溶液，分别点样于基片上的检测区，每种浓度IgG点3个复点，4℃孵育12h；1) Dilute gradients 200μg/mL, 100μg/mL, 50μg/mL, 25μg/mL, 12.5μg/mL, 6.25μg/mL, 3.13μg/mL, 1.56μg/mL, 0.78μg/mL, 0.39μg/mL , 0.19μg/mL human IgG solution and PBS blank control solution, respectively spot on the detection area on the substrate, each concentration of IgG 3 spots, 4 ℃ incubate for 12 hours;
2)芯片用10％胎牛血清白蛋白封闭100min；2) Block the chip with 10% fetal bovine serum albumin for 100 minutes;
3)用PBST清洗芯片3次，每次2min；3) Wash the chip 3 times with PBST, 2 min each time;
4)芯片上1-9检测区加入不同浓度的已知浓度梯度的HRP标记二抗15μL,HRP标记二抗浓度梯度为1.6μg/mL、0.8μg/mL、0.4μg/mL、0.2μg/mL、0.1μg/mL、0.05μg/mL、0.025μg/mL、0.0125μg/mL、0.006μg/mL，第10检测区加入空白对照PBS 15μL，37℃孵育30min；4) Add 15μL of HRP-labeled secondary antibodies with known concentration gradients of different concentrations to the detection areas 1-9 on the chip, and the concentration gradients of HRP-labeled secondary antibodies are 1.6μg/mL, 0.8μg/mL, 0.4μg/mL, 0.2μg/mL , 0.1μg/mL, 0.05μg/mL, 0.025μg/mL, 0.0125μg/mL, 0.006μg/mL, add 15μL of blank control PBS to the 10th detection area, and incubate at 37°C for 30min;
5)PBST清洗3次，每次2min；5) Wash with PBST 3 times, 2 min each time;
6)每个检测区加入15μL的HRP发光底物混合液，室温孵育2min。6) Add 15 μL of HRP luminescent substrate mixture to each detection area, and incubate at room temperature for 2 minutes.
用化学发光成像***扫描，即为醛基修饰芯片蛋白质IgG包被检测线的质控实验，结果如图2所示，在一定范围内，随人IgG浓度变化，所产生的发光信号强度也随之变化，并与空白对照产生的信号强度有明显的差异，同时随HRP标记二抗浓度降低，信号强度也随之变化。故醛基修饰芯片能与蛋白质生物分子良好的结合。Scanning with a chemiluminescence imaging system is the quality control experiment of the aldehyde-modified chip protein IgG coated detection line. The results are shown in Figure 2. Within a certain range, the intensity of the luminescence signal generated varies with the concentration of human IgG. The signal intensity is significantly different from that of the blank control. At the same time, as the concentration of the HRP-labeled secondary antibody decreases, the signal intensity also changes. Therefore, the aldehyde modified chip can bind well to protein biomolecules.
2.HIV-1/2重组抗原包被浓度及血浆稀释度实验2. HIV-1/2 recombinant antigen coating concentration and plasma dilution experiment
间接ELISA棋盘滴定法确定HIV-1/2重组抗原最佳包被浓度及血浆稀释度，所用10份HIV-I阳性血清，均经WB确认，含有表达的HIV-I重组抗原对应的抗体，10份HIV-I阴性血清。The indirect ELISA checkerboard titration method was used to determine the optimal coating concentration and plasma dilution of HIV-1/2 recombinant antigen. An HIV-I negative serum.
1)用0.1％PBST-BSA包被液将4种HIV重组抗原梯度稀释50μg/mL、12.5μg/mL、3.125μg/mL、0.78μg/mL、0.195μg/mL、0μg/mL 6个浓度，将不同浓度的4种HIV重组抗原分别点样于基片上的检测区，每种抗原6个复点，4℃孵育12h；1) Use 0.1% PBST-BSA coating solution to dilute 4 kinds of HIV recombinant antigens at 50μg/mL, 12.5μg/mL, 3.125μg/mL, 0.78μg/mL, 0.195μg/mL, 0μg/mL 6 concentrations, Spot the 4 HIV recombinant antigens with different concentrations on the detection area on the substrate, each with 6 multiple spots, and incubate at 4°C for 12 hours;
2)芯片用10％胎牛血清白蛋白封闭100min；2) Block the chip with 10% fetal bovine serum albumin for 100 minutes;
3)用PBST清洗芯片3次，每次2min；3) Wash the chip 3 times with PBST, 2 min each time;
4)芯片上1-9检测区加入不同浓度的已知阳性血浆15μL，第10检测区加入空白对照PBS 15μL，37℃孵育30min；4) Add 15μL of known positive plasma of different concentrations to the detection zone 1-9 on the chip, add 15μL of blank control PBS to the detection zone 10, and incubate at 37°C for 30min;
5)PBST清洗3次，每次2min；5) Wash with PBST 3 times, 2 min each time;
6)每个检测区加入15μL的亲和素HRP标记的二抗，37℃孵育30min；6) Add 15μL of avidin HRP-labeled secondary antibody to each detection area, and incubate at 37°C for 30min;
7)PBST清洗后，去除非特异结合，加入HRP发光底物混合液，室温孵育2min，化学发光扫描仪进行扫描。7) After washing with PBST, remove non-specific binding, add HRP luminescent substrate mixture, incubate at room temperature for 2 minutes, and scan with chemiluminescence scanner.
使用化学发光扫描仪对芯片扫描，可得不同血浆浓度对应不同HIV重组抗原的梯度浓 度实验结果，结果如图3所示，其中，芯片1-9检测区血浆对应浓度为：原液，1：10，1：10 2，1：10 3，1：10 4，第5和10个检测区为空白对照，从图中可得每种抗原最佳包被浓度及血浆稀释浓度见表2。 Using a chemiluminescence scanner to scan the chip, the experimental results of the gradient concentration of different plasma concentrations corresponding to different HIV recombinant antigens can be obtained. The results are shown in Figure 3. The corresponding plasma concentration in the detection area of the chip 1-9 is: stock solution, 1:10 , 1: 102, 1: 103, 1: 104, 10 5 and the detection region is blank, can be obtained from each of the antigens in FIG optimal coating concentration and plasma dilution are shown in Table 2.
表2各种抗原包被浓度及血浆稀释度Table 2 Coating concentration of various antigens and plasma dilution
Figure PCTCN2021081827-appb-000002
Figure PCTCN2021081827-appb-000002
二、蛋白质芯片制备2. Preparation of protein chip
每个检测方格内，利用高速全自动点样仪将将HIV重组抗原gp36、gp41、gp120和gp160，阴性对照溶液按阵图排列方式依次点在芯片上，形成检测斑和对照斑，4℃下，静置过夜后，真空干燥2小时；进一步地，真空抽气干燥后，用抗水蒸气的塑料小袋抽气密封并于4℃保存。In each detection square, use a high-speed automatic spotting instrument to spot HIV recombinant antigens gp36, gp41, gp120 and gp160, and the negative control solution on the chip in an array pattern to form a detection spot and a control spot, 4℃ After standing overnight, vacuum drying for 2 hours; further, after vacuum drying, air-sealed with a plastic pouch resistant to water vapor and stored at 4°C.
三、芯片检测HRP-抗体浓度梯度实验3. Chip detection HRP-antibody concentration gradient experiment
上述制备的蛋白质芯片室温复温30min后，10％胎牛血清白蛋白封闭100min，PBST清洗3次，每次2min，离心甩干；将上述实验浓度的阳性血浆加入1-10检测区，37℃孵育30min，PBST清洗3次，每次2min，离心甩干；将梯度稀释1.6μg/mL、0.8μg/mL、0.4μg/mL、0.2μg/mL、0.1μg/mL、0.05μg/mL、0.025μg/mL、0.0125μg/mL、0.06μg/mL的HRP标记的抗人IgG二抗溶液，点样于芯片上1-9检测区，第10区点样10％BSA作为空白对照，置于湿盒内，37℃下孵育30min后，用PBST缓冲液清洗、离心甩干。After the protein chip prepared above was rewarmed at room temperature for 30 minutes, 10% fetal bovine serum albumin was blocked for 100 minutes, washed with PBST 3 times, 2 minutes each time, and centrifuged to dry; the positive plasma of the above experimental concentration was added to the 1-10 detection zone at 37°C Incubate for 30 min, wash with PBST 3 times, 2 min each time, and centrifuge to dry; Dilute the gradient 1.6μg/mL, 0.8μg/mL, 0.4μg/mL, 0.2μg/mL, 0.1μg/mL, 0.05μg/mL, 0.025 μg/mL, 0.0125μg/mL, 0.06μg/mL HRP-labeled anti-human IgG secondary antibody solution, spot on the chip 1-9 detection zone, spot 10% BSA as a blank control in the 10th zone, placed in a wet In the box, after incubating for 30 min at 37°C, it was washed with PBST buffer and centrifuged to dry.
使用化学发光扫描仪对芯片进行扫描，即为HRP标记检测抗体浓度梯度确定实验，结果如图4所示，在抗原及血浆浓度条件不变的情况下，当HRP标记二抗孵育浓度大于0.4μg/mL时，所产生的信号强度与阴性对照产生的荧光信号强度有明显的差异。故显示孵育HRP标记山羊抗人IgG抗体的最优浓度应在0.4μg/ml以上。在一定范围内，随人HRP标记抗人二抗浓度变化，所产生的荧光信号强度也随之变化，并与空白对照产生的荧光信号强度有明显的差异。Use a chemiluminescence scanner to scan the chip, which is the HRP-labeled detection antibody concentration gradient determination experiment. The results are shown in Figure 4. When the antigen and plasma concentration conditions remain unchanged, when the HRP-labeled secondary antibody incubation concentration is greater than 0.4μg /mL, the intensity of the signal generated is significantly different from the intensity of the fluorescent signal generated by the negative control. Therefore, the optimal concentration of HRP-labeled goat anti-human IgG antibody should be above 0.4μg/ml. Within a certain range, as the concentration of the human HRP-labeled anti-human secondary antibody changes, the intensity of the fluorescent signal generated also changes, and there is a significant difference between the intensity of the fluorescent signal generated by the blank control.
实施例2样品检测检验Example 2 Sample detection and inspection
血清样本：Serum sample:
50份已知HIV阳性血浆，来源于首都医科大学附属佑安医院标本库；50 known HIV-positive plasmas were sourced from the specimen bank of You'an Hospital Affiliated to Capital Medical University;
5份已知正常健康人血浆；5 parts of plasma from known normal healthy persons;
5份空白对照(空白对照为1×PBS)。5 blank controls (blank control is 1×PBS).
蛋白芯片操作流程：Protein chip operation process:
(1)将上述实施例1制备好的蛋白质芯片室温放置30min；(1) Place the protein chip prepared in Example 1 above at room temperature for 30 minutes;
(2)用10％胎牛血清的封闭液，封闭基片表面非特异性位点，37℃孵育1～1.5小时；(2) Use a blocking solution of 10% fetal bovine serum to block non-specific sites on the surface of the substrate, and incubate at 37°C for 1 to 1.5 hours;
(3)用PBST液冲洗3-4次，每次10秒钟，用PBS冲洗，离心甩干；(3) Rinse 3-4 times with PBST solution, 10 seconds each time, rinse with PBS, and centrifuge to dry;
(4)滴加患者血浆或血清样品于芯片上，置于湿盒内37℃孵育30分钟，使抗原与抗体充分反应；(4) Drop the patient's plasma or serum sample on the chip, and incubate it in a wet box at 37°C for 30 minutes to make the antigen and antibody fully react;
(5)用PBST洗涤3～4次，每次10秒钟，用PBS冲洗，离心甩干，去除多余样品；(5) Wash with PBST 3 to 4 times, 10 seconds each time, rinse with PBS, centrifuge and spin dry to remove excess samples;
(6)滴加PBS稀释过的HRP-抗人IgG，浓度为0.4μg/mL的混合液，37℃避光温育30分钟；(6) Add dropwise a mixture of HRP-anti-human IgG diluted in PBS with a concentration of 0.4 μg/mL, and incubate at 37°C for 30 minutes in the dark;
(7)用PBST洗涤3次，每次10秒钟，用PBS冲洗，离心甩干；(7) Wash with PBST 3 times, 10 seconds each time, rinse with PBS, and centrifuge to dry;
(8)滴加HRP发光底物混合液，室温孵育2min；(8) Add HRP luminescent substrate mixture dropwise, and incubate at room temperature for 2 minutes;
(9)用化学发光扫描仪扫描并收集信号，依据扫描显示结果如图5所示，分析检测结果如下表3。(9) Scan and collect the signals with a chemiluminescence scanner. The displayed results according to the scan are shown in Figure 5, and the analysis and detection results are shown in Table 3 below.
表3 50例临床样本检测结果Table 3 Test results of 50 clinical samples
Figure PCTCN2021081827-appb-000003
Figure PCTCN2021081827-appb-000003
本芯片的检测结果：Test results of this chip:
空白对照5份没有检测到阳性指标：说明本实验采用的芯片有效。已知健康血浆5份，没有检测到阳性指标；说明本申请提供的芯片及方法检测的假阳性为0。No positive indicators were detected in 5 blank controls: it indicates that the chip used in this experiment is effective. It is known that 5 samples of healthy plasma have no positive indicators detected; indicating that the false positives detected by the chip and method provided in this application are 0.
50份已知HIV阳性患者血浆中50份检测到AFP(100％)，其中内阴性对照均为阴性。证明：本申请的芯片和方法具有50/50＝100％检出灵敏性，100％的特异性，具有可靠的临床应用价值。上述数据说明，本申请的芯片和方法稳定性，准确性和可靠性良好。AFP (100%) was detected in 50 plasmas of 50 known HIV-positive patients, and the internal negative controls were all negative. It is proved that the chip and method of this application have 50/50=100% detection sensitivity, 100% specificity, and reliable clinical application value. The above data shows that the chip and method of this application are stable, accurate and reliable.
与现有技术相比，本申请的有益效果为：Compared with the prior art, the beneficial effects of this application are:
1)本申请实现了把不同的HIV类型抗原同时点阵于一张基片上，可同时在一张玻片上检测多人份样品的IgG和/或IgM抗体，所需样本量少(15μL)，仅通过一次反应就可得到多指标的反应结果，提高了检测速度和效率，可为临床提供更多的指标。1) This application realizes the simultaneous dot matrix of different HIV type antigens on one substrate, which can detect the IgG and/or IgM antibodies of multiple human samples on one glass slide at the same time, and the required sample volume is small (15μL). Multiple index response results can be obtained through only one response, which improves the detection speed and efficiency, and can provide more indexes for the clinic.
2)本申请蛋白质芯片所需抗原量减少，1uL可以点样至少5张芯片，检测1例患者血样所需抗原量远远低于ELISA方法，简便快速，又降低了检测费用。2) The amount of antigen required for the protein chip of this application is reduced. At least 5 chips can be spotted with 1uL. The amount of antigen required to detect a patient's blood sample is much lower than the ELISA method, which is simple and fast, and reduces the cost of detection.
3)本申请蛋白质芯片的检测特异性高，检测结果稳定，可靠性高，有利于临床的诊断治疗。避免了现有方法条带误判及操作不当导致的假阳性，以及现有检测方法初检HIV阳性的4％-20％的样品WB的检测结果为“抗体不确定”问题。3) The protein chip of the present application has high detection specificity, stable detection results, and high reliability, which is beneficial to clinical diagnosis and treatment. The problem of false positives caused by misjudgment of bands and improper operation of the existing method is avoided, and the problem of "undefined antibody" in the WB test results of 4%-20% of samples that are HIV-positive by the existing detection method is avoided.
4)制作工艺简单，操作安全，可自动化，实用性强。4) The production process is simple, the operation is safe, it can be automated, and the practicability is strong.
综上，使用本申请的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片进行HIV抗体的检测，具有样品用量少、省时、省力、准确度高的特点。In summary, the use of the protein chip for detecting human immunodeficiency virus HIV1/2 antibodies of the present application for HIV antibody detection has the characteristics of small sample amount, time-saving, labor-saving, and high accuracy.
以上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。The technical features of the above embodiments can be combined arbitrarily. In order to make the description concise, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction in the combination of these technical features, they should be It is considered as the range described in this specification.
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本申请专利范围的限制。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。The above-mentioned embodiments only express several implementation manners of the present application, and their description is relatively specific and detailed, but they should not be understood as a limitation to the patent scope of the present application. It should be pointed out that for those of ordinary skill in the art, without departing from the concept of this application, several modifications and improvements can be made, and these all fall within the protection scope of this application. Therefore, the scope of protection of the patent of this application shall be subject to the appended claims.

Claims (10)

  1. 一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，所述蛋白芯片包括基片，基片表面固定有HIV特异性重组抗原gp36、gp41、gp120和gp160检测点。A protein chip for detecting human immunodeficiency virus HIV1/2 antibodies, wherein the protein chip comprises a substrate, and HIV-specific recombinant antigens gp36, gp41, gp120 and gp160 are fixed on the surface of the substrate.
  2. 根据权利要求1所述的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，所述基片为醛基修饰玻璃片。The protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to claim 1, wherein the substrate is an aldehyde modified glass plate.
  3. 根据权利要求1所述的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，所述HIV特异性重组抗原gp36的包被浓度为3.125μg/mL；HIV特异性重组抗原gp41的包被浓度为12.5μg/mL，HIV特异性重组抗原gp120的包被浓度为200μg/mL，HIV特异性重组抗原gp160的包被浓度为12.5μg/mL。The protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to claim 1, wherein the coating concentration of the HIV-specific recombinant antigen gp36 is 3.125 μg/mL; the HIV-specific recombinant antigen gp41 is coated at a concentration of 3.125 μg/mL; The coating concentration of HIV-specific recombinant antigen gp120 was 12.5μg/mL, the coating concentration of HIV-specific recombinant antigen gp120 was 200μg/mL, and the coating concentration of HIV-specific recombinant antigen gp160 was 12.5μg/mL.
  4. 根据权利要求1所述的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，基片表面包括多个检测区域和对照区域，任意检测区域至少包括一组HIV特异性重组抗原gp36、gp41、gp120和gp160检测点，同一个检测区域内的同一种抗原的浓度相同。The protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to claim 1, wherein the surface of the substrate includes multiple detection areas and control areas, and any detection area includes at least a group of HIV-specific recombinant antigens gp36, The gp41, gp120 and gp160 detection points have the same concentration of the same antigen in the same detection area.
  5. 根据权利要求1所述的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片，其中，固定有HIV重组抗原的检测点的直径为50μm，点间距为100μm，检测点形成2.5mm×2.5mm的阵列。The protein chip for detecting human immunodeficiency virus HIV1/2 antibodies according to claim 1, wherein the diameter of the detection spot on which the HIV recombinant antigen is immobilized is 50 μm, the spot spacing is 100 μm, and the detection spot forms 2.5mm×2.5mm的Array.
  6. 一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片检测试剂盒，其中，所述检测试剂盒包括蛋白芯片和检测试剂，所述蛋白芯片包括基片，基片表面固定有HIV特异性重组抗原gp36、gp41、gp120和gp160检测点。A protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies, wherein the detection kit includes a protein chip and a detection reagent, the protein chip includes a substrate, and the surface of the substrate is fixed with HIV specificity Detection points for recombinant antigens gp36, gp41, gp120 and gp160.
  7. 根据权利要求6所述的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片检测试剂盒，其中，所述检测试剂为酶标记的抗体。The protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies according to claim 6, wherein the detection reagent is an enzyme-labeled antibody.
  8. 根据权利要求7所述的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片检测试剂盒，其中，所述酶标记的抗体的浓度≥0.4μg/mL。The protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies according to claim 7, wherein the concentration of the enzyme-labeled antibody is ≥ 0.4 μg/mL.
  9. 根据权利要求7所述的用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片检测试剂盒，其中，所述检测试剂为辣根过氧化物酶标记的抗体。The protein chip detection kit for detecting human immunodeficiency virus HIV1/2 antibodies according to claim 7, wherein the detection reagent is an antibody labeled with horseradish peroxidase.
  10. 一种用于检测人类免疫缺陷病毒HIV1/2抗体的蛋白芯片的制备方法，其中，所述方法包括以下步骤：将HIV重组抗原固定于蛋白芯片的基片上，所述重组抗原包括HIV抗原gp36、gp41、gp120和gp160。A method for preparing a protein chip for detecting human immunodeficiency virus HIV1/2 antibodies, wherein the method comprises the following steps: immobilizing HIV recombinant antigens on a substrate of the protein chip, and the recombinant antigens include HIV antigen gp36, gp41, gp120 and gp160.
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