WO2017008179A1 - Réactif pour une détection combinée à rendement élevé d'un antigène et d'un anticorps du virus de l'hépatite c - Google Patents

Réactif pour une détection combinée à rendement élevé d'un antigène et d'un anticorps du virus de l'hépatite c Download PDF

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WO2017008179A1
WO2017008179A1 PCT/CN2015/000701 CN2015000701W WO2017008179A1 WO 2017008179 A1 WO2017008179 A1 WO 2017008179A1 CN 2015000701 W CN2015000701 W CN 2015000701W WO 2017008179 A1 WO2017008179 A1 WO 2017008179A1
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hepatitis
antibody
microspheres
fitc
add
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PCT/CN2015/000701
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English (en)
Chinese (zh)
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甘宜梧
王进
谭柏清
李静
王绮
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山东博科生物产业有限公司
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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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • 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
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
    • 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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
    • G01N33/545—Synthetic resin
    • 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
    • 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
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention belongs to the technical field of hepatitis C detection, and particularly relates to a reagent for high-throughput combined detection of hepatitis C virus antigen-antibody.
  • Hepatitis C is a global infectious disease caused by hepatitis C virus (HCV) infection. It is estimated that there are currently 170 million people infected with hepatitis C virus in the world. The hepatitis C infection rate in China is about 30%, and there are at least 40-40 million hepatitis C patients. Among them, 80-85% of infected people will develop chronic hepatitis C, and 20% of them can develop liver fibrosis. Finally, 4 to 5% of patients with liver fibrosis develop hepatocellular carcinoma, and the damage is very serious.
  • HCV hepatitis C virus
  • the existing clinical detection methods of HCV mainly include PCR detection, hepatitis C virus antibody detection and hepatitis C virus core antigen detection kit.
  • the PCR detection method has high sensitivity and accurate results, but the method operation is cumbersome.
  • the required personnel have strong technical ability and are not easy to promote in various hospitals;
  • the hepatitis C virus antibody detection reagent is the most common hepatitis C detection method in clinical practice, but there is a “window period” in the detection, that is, infection in hepatitis C.
  • the hepatitis C virus core antigen detection reagent has gradually been accepted by hospitals in recent years, and the market share has also increased. However, for the follow-up detection of hepatitis C patients, there is no good tracking effect.
  • the present invention provides a reagent for high-throughput detection of hepatitis C virus antibodies and antigens.
  • the reagent utilizes the principle of flow cytometry to coat the antibody and the antigen in different concentrations of FITC-fluorescein-labeled microspheres, and after sandwiching the test sample, the sandwiched sandwich structure with the PE-labeled paired antibody and antigen constitutes a sandwich structure at 480 nm.
  • different concentrations of fluorescein emit light with different intensity, which can achieve high-throughput simultaneous detection of hepatitis C antigen and antibody.
  • the principle of fluorescence detection is adopted to improve the sensitivity of detection, and the antigen and antibody can be qualitatively detected at the same time, and it is possible to determine whether the detection sample has hepatitis C virus infection, and has high clinical application value.
  • the present invention is achieved by the following measures:
  • a chemiluminescence method combined with detection of hepatitis C virus antibody-antigen reagent the components of which are as follows:
  • the FITC high-brightness microsphere preparation process is: dissolving fluorescein isothiocyanate (FITC) into dimethyl sulfoxide to prepare a 1 mg/mL concentrated dye solution, and the surface is simultaneously aminated and carboxylated polystyrene.
  • FITC low-light microspheres The preparation process of FITC low-light microspheres is: dissolving fluorescein isothiocyanate (FITC) into dimethyl sulfoxide to prepare a concentrated dye solution of 1 mg/mL, and the surface is simultaneously aminated and carboxylated polystyrene micro
  • FITC highlight microspheres coated with hepatitis C core antigen antibody 1 mL of the above FITC highlight microspheres was added, and 1 mL of carbonate buffer containing 0.005 g of N-hydroxysuccinimide and 0.005 g of carbodiimide was added.
  • the PE labeling antibody or protein preparation process is: (1) Preparation of phycoerthrin PE: 600 ⁇ L of 15.5 mg/mL iminothiolane hydrochloride is added to 1.2 mL of 3.6 mg/mL. In PE (phycoerythrin), mix with 1.2 mL of phosphate buffer (0.2 M, pH 6.8), place in a dialysis bag and place in 50 mmol/L pH 6.8 phosphate buffer for dialysis, overnight at 4 ° C. The cells were dialyzed against 50 mmol/L pH 7.5 phosphate buffer for 6 h.
  • (2) PE-labeled antibody or protein Take 30 ⁇ L of 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide (SPDP) 1.1 mg/mL in ethanol, and add 700 ⁇ L of 4.2 mg/mL antibody or The protein was phosphate buffered (50 mmol/L, pH 7.5) and allowed to react at room temperature for 2.5 h. 400 ⁇ L of the above thiolated phycoerythrin was further added, and reacted at room temperature for 12 hours.
  • SPDP 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide
  • the kit of the present invention is used as follows:
  • R1 FITC fluorescent microsphere reagent
  • Negative control mean FL2-H value ⁇ 2.1 (if the negative control average FL2-H value ⁇ 5, calculated as 5; OD > 5, according to the actual measurement negative control average FL2-H value ⁇ 2.1).
  • the FL2-H value of the test specimen is less than the critical value and is HCV negative.
  • the FL2-H value in the test specimen is equal to or greater than the critical value and is HCV positive.
  • a combined detection kit for simultaneously detecting a hepatitis C virus antigen-antibody can be obtained, which is coated by binding antibodies and antigens at different concentrations of FITC-fluorescein-labeled microspheres. After detecting the sample, the PE-labeled paired antibody and antigen form a sandwich structure. Under the action of 480 nm excitation light, different concentrations of fluorescein emit different light intensity, which can achieve high-throughput simultaneous detection of hepatitis C antigen and antibody.
  • the sensitivity of the reaction is enhanced by the fluorescent color development effect, and the hepatitis C virus antibody and the core antigen can be simultaneously detected by using FITC-labeled microspheres to simultaneously detect antigens and antibodies.
  • the high-throughput detection kit can simultaneously monitor the hepatitis C core antigen and hepatitis C antibody on the flow cytometer, and more intuitively identify the infection period of hepatitis C, and has high application value for clinical hepatitis C detection.
  • FIG. 2 The result of detecting the positive result of the kit of the present invention is shown in FIG. 2 .
  • the innovation of the kit for detecting hepatitis C virus antigen-antibody of the present invention is:
  • the immune reaction can be used in the sample.
  • the antibodies to hepatitis C core antigen and various fragment antigens can be adsorbed to the surface of the microspheres by an immune reaction, thereby avoiding missed detection.
  • the PE label in the kit is a PE enzyme label for the corresponding antibody and antigen paired with the coated antibody and the antigen, and can be paired with the coated antibody and the antigen to ensure the specificity of the kit detection and accurate detection. Hepatitis C virus.
  • the kit of the invention adopts the principle of flow cytometry, and uses the fluorescence color rendering technology to calculate the result, thereby effectively ensuring the sensitivity of the kit, and can be detected even in a trace state, and the effect of early detection is achieved.
  • the component of the kit of the invention is a liquid component, which is free from the influence of the solid phase carrier by the enzyme-linked immunosorbent assay, is not restricted by the test sample, and the detection result is more intuitive.
  • the combined detection of hepatitis C core antigen-hepatitis C antibody by enzyme-linked immunosorbent assay has resulted in waste of slats due to the number of samples, and it is not possible to visually express the content of hepatitis C core antigen or antibody.
  • the kit of the present invention uses different FITC contents.
  • the labeled microspheres can distinguish different detection items, and can achieve the purpose of simultaneously detecting hepatitis C core antigen and hepatitis C antibody, and have good clinical application value for detection and treatment tracking of hepatitis C.
  • Figure 1 is a schematic diagram of the reaction principle
  • Figure 2 is a graph showing positive results
  • Figure 3 is a graph showing the results of different infection days in Example 1, wherein a, the test results of 1-6 days of infection (both core antigen and antibody are negative); b, 7-38 days of infection (core antigen positive, Antibody negative); c, test results after 39 days of infection (both core antigen and antibody are positive).
  • composition and concentration of the detection reagent are:
  • the kit of the present invention is used as follows:
  • R1 FITC fluorescent microsphere reagent
  • composition and concentration of the detection reagent are:
  • the washing solution, positive control, negative control and detection method were as in Example 1.
  • composition and concentration of the detection reagent are:
  • the washing solution, positive control, negative control and detection method were as in Example 1.
  • Example 2 Third party verification Serial number Example 1 Example 2 Third party verification Serial number Example 1 Example 2 Third party verification 1 - - twenty one - - 2 + + twenty two + + 3 - - twenty three - - 4 - - twenty four + - + 5 + + 25 + + 6 + + 26 + + 7 - - 27 - - 8 + - + 28 - - 9 + + 29 - - 10 + + 30 + + 11 + + 31 - - 12 + + + 32 + + + 13 - - 33 + + + 14 - - 34 - - 15 - - 35 + + 16 - - 36 + + 17 - - 37 + + 18 + + 38 - - 19 + + 39 + + 20 - - 40 - - -
  • Example 1 The results of the detection of the present invention are more accurate, and the two samples which are abnormal are mainly autoimmune antibodies, and the concentration of the hepatitis C virus core antigen is relatively low, resulting in missed detection. According to the detection result, the detection result of the kit of the present invention is more accurate, and the occurrence of the miss detection is prevented.
  • the sensitivity of the kit was verified by dilution of the positive sample.
  • the specific implementation is as follows:
  • a hepatitis C virus-positive sample was subjected to gradient dilution using a normal negative sample, and each of Example 1, Example 2, Example 3, and the orthovirus hepatitis C virus core antigen detection kit (ELISA) was used.
  • the positive samples of the dilution gradient were tested, and the test results are shown in Table 2.
  • the sensitivity of the ortho company is 1/8, and the sensitivity of the embodiment 1, the embodiment 2, and the embodiment 3 can reach 1/32. This result indicates that the kit of the present invention has higher sensitivity. Even if the concentration of hepatitis C virus in the sample is very low, it can be well detected, and the effect of early detection and early prevention can be achieved.
  • Example 1 The results of Example 1 for different infection days are shown in Figure 3.
  • Example 1 of the present invention can detect hepatitis C infection earlier than the hepatitis C virus core antigen detection kit of Ortho Division (enzyme-linked immunosorbent assay), while utilizing high-brightness microspheres and low-light micro-micro
  • the ball distinguishes different detection items, and can directly obtain the positive status of hepatitis C core antigen and hepatitis C antibody, which has guiding significance for different infection cycles.
  • the traditional hepatitis C virus detection kit is detected by enzyme-linked immunosorbent assay, which has poor mobility, low sensitivity and unacceptable accuracy.
  • the present invention utilizes the principle of flow cytometry by passing microspheres labeled with FITC at different concentrations. Corresponding coated antibody and antigen, after adding the test sample, and sandwiching the PE-labeled paired antibody and antigen to form a sandwich structure (as shown in Fig. 1), under the excitation light of 480 nm, different concentrations of fluorescein emit light intensity is different, can be high The flux achieves the simultaneous detection of hepatitis C antigens and antibodies.
  • the reagent kit is a liquid reagent, which is free from the limitation of the slats of the 96-well plate and can be more maneuverable.
  • the sensitivity of PE fluorescence is much higher than that of the enzyme, which enhances the sensitivity of the reaction, and uses micro-labels with different FITC content.
  • the ball can distinguish different detection items, and can achieve the purpose of simultaneously detecting hepatitis C core antigen and hepatitis C antibody. The detection of hepatitis C is more accurate and will not be missed, and the effect of early detection is achieved, which has high application value for the prevention of hepatitis C.

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Abstract

La présente invention concerne un réactif pour la détection combinée à rendement élevé d'un antigène et d'un anticorps du virus de l'hépatite C. En utilisant le principe de cytométrie de flux, différentes concentrations de microsphères marquées à l'isothiocyanate de fluorescéine (FITC) correspondent à des antigènes et à des anticorps revêtus ; après l'ajout d'un échantillon de détection, les microsphères, l'échantillon de détection et un anticorps et un antigène appairés marqués à la PE forment une structure en sandwich ; et sous l'action d'une lumière d'excitation à 480 nm, les effets de détection simultanée d'un antigène et d'un anticorps du virus de l'hépatite C peuvent être obtenus d'une manière à rendement élevé en fonction de différentes intensités lumineuses émises des fluorescéines à différentes concentrations. Au moyen de l'utilisation de l'effet de coloration fluorescente, la sensibilité de la réaction est améliorée, et un anticorps et un antigène du noyau du virus de l'hépatite C peuvent être détectés simultanément au moyen de l'utilisation de microsphères marquées à la FITC pour la détection simultanée de l'antigène et de l'anticorps.
PCT/CN2015/000701 2015-07-06 2015-10-19 Réactif pour une détection combinée à rendement élevé d'un antigène et d'un anticorps du virus de l'hépatite c WO2017008179A1 (fr)

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CN201510389933.9A CN105004862B (zh) 2015-07-06 2015-07-06 一种高通量联合检测丙肝病毒抗原抗体的试剂

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CN110646616A (zh) * 2019-09-05 2020-01-03 桂林理工大学 一种检测血清中人cTnI的超敏荧光猝灭免疫传感器及检测方法
CN112540174A (zh) * 2020-12-02 2021-03-23 泰州泽成生物技术有限公司 一种新型冠状病毒IgG检测试剂盒及其制备方法
CN114032281A (zh) * 2021-09-15 2022-02-11 陈翠英 一种丙肝肝癌检测试剂及其在丙肝肝癌检测中的应用
CN114354935A (zh) * 2022-01-05 2022-04-15 厦门大学 一种高灵敏免标记的肾癌血清检测生物试剂
CN117434269A (zh) * 2022-09-14 2024-01-23 杭州赛基生物科技有限公司 中枢神经退行性疾病相关标志物试剂盒及检测方法

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CN105004862B (zh) * 2015-07-06 2017-01-25 山东博科生物产业有限公司 一种高通量联合检测丙肝病毒抗原抗体的试剂
CN107782891A (zh) * 2017-10-19 2018-03-09 天津医科大学 一种多功能上转换纳米平台的构建方法
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WO2021134302A1 (fr) * 2019-12-30 2021-07-08 深圳迈瑞生物医疗电子股份有限公司 Instrument de dosage immunologique et méthode de détection du virus de l'hépatite c, ainsi que kit
CN112763709A (zh) * 2020-12-29 2021-05-07 中国科学院苏州生物医学工程技术研究所 基于不同荧光强度微球的联合检测试剂盒及方法
CN115060772A (zh) * 2022-08-16 2022-09-16 天津市协和医药科技集团有限公司 一种基于功能化核孔膜的ca50抗原电化学检测方法

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