WO2021185357A1 - Puce à protéines pour détecter un anticorps anti-vih 1/2 du virus de l'immunodéficience humaine et son procédé de préparation - Google Patents

Puce à protéines pour détecter un anticorps anti-vih 1/2 du virus de l'immunodéficience humaine et son procédé de préparation 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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English (en)
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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Publication of WO2021185357A1 publication Critical patent/WO2021185357A1/fr

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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

L'invention concerne une puce à protéines permettant de détecter un anticorps HIV1/2 du virus de l'immunodéficience humaine et son procédé de préparation. La puce à protéines comprend un substrat de détection, dont la surface est une modification de groupe aldéhyde remplaçable, la surface du substrat de détection comprenant une zone de détection formée par des points de détection, et des antigènes de recombinaison du VIH gp36, gp41, gp120 et gp160 étant fixés aux points de détection.
PCT/CN2021/081827 2020-03-20 2021-03-19 Puce à protéines pour détecter un anticorps anti-vih 1/2 du virus de l'immunodéficience humaine et son procédé de préparation WO2021185357A1 (fr)

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