WO2023087550A1 - 一种检测新型冠状病毒抗原的试剂盒及检测方法 - Google Patents

一种检测新型冠状病毒抗原的试剂盒及检测方法 Download PDF

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WO2023087550A1
WO2023087550A1 PCT/CN2022/074466 CN2022074466W WO2023087550A1 WO 2023087550 A1 WO2023087550 A1 WO 2023087550A1 CN 2022074466 W CN2022074466 W CN 2022074466W WO 2023087550 A1 WO2023087550 A1 WO 2023087550A1
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sample
pad
antibody
detection
kit
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PCT/CN2022/074466
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English (en)
French (fr)
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蒋析文
齐文闯
刘双
潘秀华
苏松康
徐鸿
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广州达安基因股份有限公司
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Priority to EP22711856.9A priority Critical patent/EP4435430A1/en
Priority to US17/721,382 priority patent/US20230152315A1/en
Publication of WO2023087550A1 publication Critical patent/WO2023087550A1/zh

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • the present application relates to the field of novel coronavirus antigen detection, in particular to a kit for detecting novel coronavirus antigen and its application.
  • new coronavirus Novel coronavirus (Corona Virus Disease 2019, COVID-19), referred to as "new coronavirus”.
  • the clinical manifestations of pneumonia patients infected by the new coronavirus are: fever, fatigue, dry cough as the main manifestations, nasal congestion, runny nose and other upper respiratory symptoms are rare, and hypoxia and hypoxia will occur.
  • the PCR (polymerase chain reaction) method is the earliest applied method.
  • the preparation of viral nucleic acid-specific primer sequences is relatively fast, and the theoretical specificity and sensitivity of nucleic acid detection are high. Therefore, the viral nucleic acid detection kit based on the PCR method has become the earliest kit developed for the detection of the new coronavirus.
  • positive real-time fluorescent RT-PCR is used as one of the confirmed evidences for new coronavirus infection.
  • nucleic acid detection also includes two steps of nucleic acid extraction and PCR amplification.
  • the kits and equipment used include nucleic acid extraction kits, fluorescent probes, and fluorescent RT-PCR instruments.
  • Antibody detection method is based on the human body's immune response to foreign pathogens.
  • the specific antibody IgM against the immunogen usually appears in the serum within 7-10 days, and they are the "vanguard" of the body's anti-infection. If pathogen-specific IgM is detected in the serum, it indicates a recent infection and can be used for early diagnosis of infection. Usually after 20 days, specific IgG against the pathogen can be detected.
  • the antibody detection method has exposed many problems, and it cannot be used as a diagnostic indicator alone for screening, but can only be used as an auxiliary diagnostic method when the nucleic acid test is negative.
  • antibody detection cannot be used alone for the diagnosis of the new coronavirus, and cannot rule out infection or explain the infection status.
  • Antibody tests may also be positive when there are past or current infections with other viruses such as HKU1, NL63, OC43, or 229E.
  • Most antibody detection kits use the principle of capture ELISA to detect specific antibodies. That is, the IgM or IgG in the sample is captured by the anti-IgM/IgG antibody. In the case of pathogen-specific IgM or IgG, it will bind the labeled antigen and thus be visualized in the test line of a dipstick or in the corresponding well of a 96-well plate.
  • the capture antibody anti-IgM/IgG antibody
  • the specificity of this method is only based on the specific recognition and binding of a pair of antigen-antibody (i.e., provided by the antibody recognition kit in the serum). of viral proteins). This leads to false positives due to non-specific binding in complex sample components.
  • the immunocolloidal gold method (ICA) to detect the new coronavirus antigen is to mark the colloidal gold with high electron density as a marker on the antibody or antigen, and when it aggregates at the corresponding ligand, it will produce a color visible to the naked eye. Therefore, it can be used in qualitative or semi-quantitative detection research.
  • ICA immunocolloidal gold method
  • certain modifications can be carried out on the surface of the gold core, mainly sulfur-containing ligands, phosphides, phosphorus oxides, amino and carboxyl ligands. The sensitivity of this method is low, and quantitative detection cannot be carried out, which cannot meet the requirements of clinical diagnosis.
  • the problem to be solved in this application is to provide a rapid, accurate, low-cost and clinically applicable kit for detecting novel coronavirus antigens and its use.
  • a kit for detecting a novel coronavirus antigen comprising a detection card, on which a binding pad and a reaction pad are provided; on the binding pad, a labeled antibody Ab1 and a labeled antibody Ab3 of the novel coronavirus antigen; A quality control line and a detection line are arranged in parallel on the reaction pad, the detection line is provided with coated antibody Ab2, and the quality control line is provided with coated antibody Ab4.
  • the present application also provides a detection method for novel coronavirus antigen, using the detection card in the above kit, comprising the following steps:
  • sampling swab into the extraction bottle pre-added with the sample extraction solution, mix the sample thoroughly, and keep the oropharyngeal mucous membrane fluid in the extraction bottle as much as possible;
  • the present application also provides a detection method for novel coronavirus antigen, using the detection card in the above kit, comprising the following steps:
  • a disposable sampling swab insert it into the nasal cavity perpendicular to the nose, and penetrate at least half the length from the earlobe to the tip of the nose to collect nasal mucous fluid;
  • sampling swab into the extraction bottle that has been pre-added with the sample extraction solution, and mix the sample thoroughly so that the nasal mucous membrane fluid remains in the extraction bottle as much as possible;
  • the present application also provides a detection method for novel coronavirus antigen, using the detection card in the above kit, comprising the following steps:
  • sampling swab into the single-ear tube that has been pre-added with the sample extraction solution, and mix the sample thoroughly so that the nasal mucosal fluid or oropharyngeal mucosal fluid remains in the single-ear tube as much as possible;
  • the kit for detecting the novel coronavirus antigen provided by this application, the binding pad in the built-in detection card is provided with the labeled antibody Ab1 and the labeled antibody Ab3 of the novel coronavirus antigen, and the reaction pad is provided with a quality control line and a detection line in parallel at intervals, and Coating antibody Ab2 is provided on the detection line, and coating antibody Ab4 is provided on the quality control line.
  • AIE fluorescent immunochromatography technology can be used, AIE fluorescent microspheres can be used as markers, and fluorescent flashlights can be used to directly interpret the test results of the test card, or use a fluorescence detector to read the test results.
  • This kit has high sensitivity. , strong specificity, simple and fast operation, etc.
  • Fig. 1 is the structural representation of the test card in the test kit provided by the present application.
  • FIG. 2 is a schematic diagram of the structure of the clamping case provided by the detection card provided by the present application.
  • AIE fluorescence immunochromatography technology is a dry quantitative new detection technology developed and innovated based on the combination of AIE fluorescence technology and traditional immunochromatography technology.
  • this technology also achieves accurate detection results through aggregation-induced fluorescent material trace enhancement technology. Compared with the performance of traditional fluorescence rapid detection technology, this technology has higher sensitivity, wider detection range, low price, no background, and no quenching.
  • This application uses the double-antibody sandwich method in the AIE fluorescence immunochromatography technique to quantitatively detect the content of novel coronavirus antigens in the oropharynx or nasopharynx.
  • An effective fluorescent immunochromatographic detection kit for novel coronavirus (COVID-19) antigen was established by optimizing process conditions, performance evaluation, and clinical application.
  • a kit for detecting novel coronavirus antigens includes a detection card, a disposable sampling swab, an extraction bottle, and a sample extraction liquid placed in a sample extraction liquid bottle; Pad and reaction pad; The labeled antibody Ab1 and the labeled antibody Ab3 of the new coronavirus antigen are provided on the binding pad; quality control lines and detection lines are arranged in parallel on the reaction pad, and a bag is provided on the detection line. It is coated with antibody Ab2, and the quality control line is equipped with coated antibody Ab4.
  • the above-mentioned kit also includes a sample extraction solution (phosphate buffered saline) for diluting the sample, an extraction bottle, and a disposable sampling swab; the disposable sampling swab is used to collect human oropharyngeal mucus or nasal cavity mucus, and will collect
  • the mucus is placed in the sample extraction solution added to the extraction bottle in advance, and the sample solution to be tested is mixed; then, the sample solution to be tested is dropped into the test card, and the test result is interpreted through the test line and quality control line.
  • the main components of the sample extract include 0.01M BB, 1% PVP, 1% sodium chloride and 1% Tween.
  • the biogenicity of the labeled antibody of the new coronavirus antigen in the above test card is shown in Ab1
  • the biological origin of the coated antibody of the new coronavirus antigen in the reaction pad is shown in Ab2.
  • the biological origin of the labeled antibody of the quality control line is shown in Ab3
  • the biological origin of the coated antibody of the quality control line in the reaction pad is shown in Ab4; details are shown in Table 1.
  • labeled antibody Ab1 contains AIE fluorescent microspheres (ie, AIE fluorescently labeled antibody, or AIE fluorescently labeled)
  • labeled antibody Ab3 contains AIE fluorescent microspheres (ie, AIE fluorescently labeled antibody or AIE fluorescently labeled).
  • the final concentration of the labeled antibody Ab1 on the detection card is 0.3 mg/mL
  • the final concentration of the coated antibody Ab2 on the detection card is 1 mg/mL
  • the final concentration of the labeled antibody Ab3 of the quality control line on the detection card is 0.3 mg/mL.
  • the final concentration of the coated antibody Ab4 of the quality control line on the test card is 0.5 mg/mL.
  • the novel coronavirus antigen in the sample combines with the AIE fluorescently labeled antibody on the test card and diffuses to the test area, and the novel coronavirus monoclonal antibody coated by the test line Capture and form an "antibody-antigen-fluorescent antibody" complex; at the same time, the concentration of the new coronavirus antigen in the sample is proportional to the fluorescence intensity of the complex, and the fluorescent flashlight can be used to directly judge the negative and positive.
  • the above-mentioned test card 10 also includes a sample pad 11, a water-absorbing pad 14 and a base plate 15;
  • the reaction pad 13 and the water-absorbing pad 14 are arranged in sequence and fixedly pasted on one surface of the bottom plate 15 .
  • the two ends of the reaction pad 13 are overlapped with one end of the water-absorbing pad 14 and one end of the binding pad 12 respectively, and the other end of the binding pad 12 is overlapped with one end of the sample pad 11, and the sample pad 11 and the binding pad 14 are respectively located on the bottom plate 15
  • the quality control line (C line) 16 and the detection line (T line) 17 are set on the reaction pad 13 .
  • a casing 20 is also fitted on the outer surface of the detection card 10 , which includes an observation window 22 and a sample injection hole 21 .
  • the shell 20 is made of hard material, such as hard paper, PVC and so on.
  • test card 10 with the card case 20 and the desiccant are sealed and packaged together in an aluminum foil bag.
  • each part is composed as follows:
  • the sample pad 11 is mainly composed of glass cellulose film.
  • the binding pad 12 is mainly composed of glass cellulose membrane, and the binding pad 12 is provided with AIE fluorescently labeled labeled antibody Ab1 and AIE fluorescently labeled labeled antibody Ab3; wherein, the labeled antibody Ab1 is the new coronavirus antibody Ab1, and the labeled antibody Ab3 It is chicken IgY antibody Ab3.
  • Reaction pad 13 is made of nitrocellulose membrane (NC membrane), and its detection line is coated with antibody Ab2, that is, coated with novel coronavirus monoclonal antibody Ab2; the quality control line is coated with antibody Ab4, that is, coated with goat anti-chicken IgY antibody.
  • N membrane nitrocellulose membrane
  • Absorbent pad 14 is H-1 absorbent paper.
  • Base plate 15 is a PVC sheet.
  • the present application also provides a sampling method for the novel coronavirus antigen, which uses the above-mentioned kit for detection. Including oropharyngeal swab and nasopharyngeal swab sampling test.
  • Oropharyngeal swab Insert the sample collection swab from the mouth into the throat, slowly rotate and wipe the bilateral pharyngeal tonsils and the posterior pharyngeal wall, avoiding touching the tongue.
  • Nasopharyngeal swab Use the sample collection swab to measure the distance from the tip of the nose to the earlobe and mark it with your finger. Insert the swab into the nasal cavity in a direction perpendicular to the nose (face). The depth of the swab should be at least half the length from the earlobe to the tip of the nose. After encountering resistance, it reaches the posterior nasopharynx, so that the collected swab stays in the nasopharynx for 15-30 seconds, gently rotates 3-5 times, and then slowly takes it out.
  • Sample storage The collected samples should be tested immediately. If the sample collection swab is not used immediately after sampling, it should be stored immediately in a dry, sterile and sealed plastic tube.
  • the sample After the sample is collected, it should be processed with the sample extraction solution provided by this kit as soon as possible.
  • the processed samples can be stored at room temperature for 2 hours, at 2-8°C for 3 hours, at -20 ⁇ 5°C for 2 months. Note: The samples after cryopreservation need to return to room temperature before testing.
  • AIE fluorescence activation Add 100uLAIE fluorescent microspheres into a centrifuge tube of 800uL MES (2-morpholineethanesulfonic acid), and sonicate for 3min. Measure 30uL of EDC (1-ethyl-carbodiimide hydrochloride) and NHS (N-hydroxysuccinimide) (1:1) activation solution and mix it in a centrifuge tube, sonicate until uniformly dispersed , equilibrate at room temperature for a certain period of time, centrifuge at 12000r/min, 4°C for 15min, and remove the supernatant.
  • EDC 1-ethyl-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antibody coupling add 1mL MES to the above centrifuge tube, vortex, sonicate for 3min, add 0.3mg of the new coronavirus antibody, spin coupling reaction for 2h, after resting for 5min, add 0.3mg of chicken IgY antibody.
  • Blocking and preservation add 100 uL of 10% BSA (bovine serum albumin) solution to the above centrifuge tube for blocking for 30 min, centrifuge at 12000 r/min, 4° C. for 15 min. Remove the supernatant, add 1mL Tris buffer (trishydroxymethylaminomethane), sonicate for 3min, then centrifuge at 12000r/min, 4°C for 15min, repeat this step once. Remove the supernatant and add 1mL microsphere reconstituted solution, sonicate for 3min, centrifuge at 2000r/min for 3min, remove the precipitate and keep at 4°C in the dark.
  • BSA bovine serum albumin
  • Conjugation pad Spray the prepared fluorescent particle solution evenly on the 1.5-1.8cm glass fiber at a speed of 6uL/cm, and dry it at 55°C for 3-4h.
  • treatment solution 0.1M BB, 1% sucrose, 1% trehalose, 1% PVPK3O (polyvinylpyrrolidone).
  • Sample pad treatment Apply 3-4mL of the above treatment solution (width: 17-18, length: 30cm) evenly on the 0.8-1.0cm sample pad, and dry it at 55°C for 6-8h.
  • quality control line goat anti-chicken IgY coating antibody Ab4 concentration is 0.5mg/mL
  • Step 4 Assembling the test card: Paste the treated sample pad, bonding pad, and water-absorbing pad on the PVC board in sequence, and cut the assembled PVC board into 3.85mm wide immunochromatographic test cards by a strip cutter.
  • sample extract 0.01M BB, 1% PVP (polyvinylpyrrolidone), 1% sodium chloride, 1% Tween.
  • PVP polyvinylpyrrolidone
  • composition, packaging and quantity (25 servings/box) of a novel coronavirus antigen detection kit in human oropharyngeal or nasopharyngeal samples are shown in Table 2.
  • the extraction bottle in the above-mentioned kit for detecting the novel coronavirus antigen of the present application can be replaced with a single ear tube.
  • composition, packaging and quantity of the kit are shown in Table 3:
  • a method for detecting novel coronavirus antigens using the above kit comprising the following steps:
  • sampling swab into the single-ear tube that has been pre-added with the sample extraction solution, and mix the sample thoroughly so that the nasal mucosal fluid or oropharyngeal mucosal fluid remains in the single-ear tube as much as possible;
  • the specific sampling steps include:

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Abstract

检测新型冠状病毒抗原的试剂盒及其检测方法；试剂盒包括检测卡（10），在检测卡（10）上设有结合垫（12）和反应垫（13）；在结合垫（12）上设有新型冠状病毒抗原的标记抗体Ab1和标记抗体Ab3；在反应垫（13）上间隔平行设置有质控线（16）和检测线（17），在检测线（17）上有包被抗体Ab2，质控线（16）上有包被抗体Ab4。该试剂盒采用AIE荧光免疫层析技术，用AIE荧光微球作为标记物，使用荧光手电直接判读检测卡（10）的检测结果，或使用荧光检测仪读出检测结果，这种试剂盒具有灵敏度高、特异性强、操作简单快速等优点。

Description

一种检测新型冠状病毒抗原的试剂盒及检测方法
本申请要求于2021年11月16日提交中国专利局、申请号为202111357502.6，发明名称为“一种检测新型冠状病毒抗原的试剂盒及检测方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本申请涉及新型冠状病毒抗原检测领域，尤其涉及一种检测新型冠状病毒抗原的试剂盒及其用途。
背景技术
新型冠状病毒(Corona Virus Disease 2019，COVID-19)，简称“新冠病毒”。新型冠状病毒感染的肺炎患者的临床表现为：以发热、乏力、干咳为主要表现，鼻塞、流涕等上呼吸道症状少见，会出现缺氧低氧状态。约半数患者多在一周后出现呼吸困难，严重者快速进展为急性呼吸窘迫综合征、脓毒症休克、难以纠正的代谢性酸中毒和出凝血功能障碍。值得注意的是，重症、危重症患者病程中可为中低热，甚至无明显发热。部分患者起病症状轻微，可无发热，多在1周后恢复。多数患者预后良好，少数患者病情危重，甚至死亡。
[根据细则26改正07.07.2022]　
面对来势汹汹的新冠病毒传染病的大规模流行，大量人群需要被筛查、确诊并做相应的处理，临床对诊断试剂的需求极为迫切。除口罩、防护服等医疗用品外，在新冠疫情早期以及现在的意大利、西班牙等国都出现了检测试剂的巨大缺口。
对于新型冠状病毒感染的检测，目前有多种新型冠状病毒的检测方法研究，根据检测的原理以及方法的不同主要可以分为病原检测、抗体检测及免疫检测。具体如下：
1、对于病原体的检测，如，PCR(聚合酶链式反应)方法是最早被应用的方法。制备病毒核酸特异性引物序列相对较快，核酸检测的理论特异性和灵敏度都很高，因此基于PCR方法的病毒核酸检测试剂盒成为此次针对新冠病毒检测最早被开发的试剂盒。在中国的《新型冠状病毒肺炎诊疗方案(试行第七版)》中，实时荧光RT-PCR阳性被作为新冠病毒感染的确诊证据之一。
然而，发明人意识到在实际诊断过程中，核酸法却暴露出很多问题：第一、假阴性问题；第二、设备或检测平台要求较高；第三，检测时间较长。首先，假阴性意味着漏检，不仅会导致临床中对疑似患者不能快速确诊，而且会使漏检者成为潜在的病毒传染源。其次，据报道，甚至出现了多次核酸检测阴性而出院的患者又再次复发的情况，也很可能属于假阴性引起的误诊。在采样后，核酸检测还包括核酸提取和PCR扩增两个步骤，所采用的试剂盒和设备包括核酸提取试剂盒、荧光探针、以及荧光RT-PCR仪等。高灵敏度的RT-PCR仪价格不菲。另外，为避免空气中各种微生物可能带有的核酸酶的影响，核酸检测对实验室的洁净度要求也较高。最后，核酸检测是一项精密的实验，对操作人员的要求也较高，需要经过专门的操作培训。这使得在大规模疫情爆发时，各大医院具有核酸检测资质的实验室很快被饱和，大量样本堆积无法及时获得检测。完成一个RT-PCR的检测通常需要4-6个小时，然而考虑到样本运输、大量样本积压的情况，通常最快24小时才可以报告结果。
2、抗体检测法，是基于人体对于外源病原体的免疫反应。当感染病原体后，人通常在7-10天在血清中出现针对该免疫原的特异性抗体IgM，它们是机体抗感染的“先头部队”。如果血清中检出病原体特异性IgM，提示新近发生感染，可用于感染的早期诊断。通常在20天后，可以检测到针对病原体的特异性IgG。然而在实际诊断过程中，抗体检测法却暴露出多种问题，不能单独作为诊断指标用于筛查，只能作为当核酸检测为阴性时的辅助诊断方法。在FDA的SARS-COV-2检测指导中明确指出，抗体检测不能单独用于新冠病毒诊断，不能排除感染或说明感染状态。有其它病毒如HKU1、NL63、OC43或229E等的既往或现时感染时抗体检测也可能呈阳性。大部分抗体检测试剂盒采用捕获法ELISA原理实现对特异性抗体的检测。即通过抗IgM/IgG的抗体，捕获样本中的IgM或IgG。如果是病原体特异性的IgM或IgG，它将结合带有标记的抗原，从而在试纸条的检测线或96孔板的相应孔中显现出来。由于捕获抗体(抗IgM/IgG抗体)会结合血清中的任何IgM或IgG，因此该方法的特异性仅基于一对抗原-抗体的特异性识别和结合(即血清中的抗体识别试剂盒所提供的病毒蛋白)。这造成了在复杂的样本成分中，由于非特异性结合而引起的假阳性。
3、免疫胶体金方法(ICA)检测新型冠状病毒抗原是将高电子密度的胶体金作为标记物标记在抗体或者抗原上，当其在相应的配体处发生聚集时会产生肉眼可见的颜色，因而可以 用以定性或者半定量的检测研究中。随着胶体金研究的发展，在金核的表面可以进行一定的修饰，主要为含硫的配体、含磷化物、磷氧化物、氨基和羧基的配体。该方法灵敏度较低，不能进行定量的检测达不到临床诊断的要求。
发明内容
基于上述问题，本申请所要解决的问题在于提供一种可以快速、准确、低成本且可适用临床应用的检测新型冠状病毒抗原的试剂盒及其用途。
本申请的技术方案如下：
一种检测新型冠状病毒抗原的试剂盒，包括检测卡，在所述检测卡上设有结合垫和反应垫；在所述结合垫上设有新型冠状病毒抗原的标记抗体Ab1和标记抗体Ab3；在所述反应垫上间隔平行设置有质控线和检测线，在所述检测线上设有包被抗体Ab2，质控线上设有包被抗体Ab4。
本申请还提供一种新型冠状病毒抗原的检测方法，使用上述试剂盒中的检测卡，包括如下步骤：
采用一次性采样拭子，从人体口腔完全插入咽喉中，缓慢旋转擦拭双侧咽扁桃体及咽后壁，采集口咽粘膜液；
将采样拭子放入预先加入了样本提取液的抽提瓶中，充分混匀样本，使口咽粘膜液尽可能留在抽提瓶中；
将采集的口咽粘膜液样本滴入试剂盒中的检测中进行检测，并根据检测线和质控线判断检测结果。
本申请还提供一种新型冠状病毒抗原的检测方法，使用上述试剂盒中的检测卡，包括如下步骤：
采用一次性采样拭子，以垂直鼻子方向插入鼻腔，深入距离最少应达耳垂部位到鼻尖长度的一半，采集鼻腔粘膜液；
将采样拭子放入预先加入了样本提取液的抽提瓶中，充分混匀样本，使鼻腔粘膜液尽可能留在抽提瓶中；
将采集的鼻腔粘膜液样本滴入试剂盒中的检测中进行检测，并根据检测线和质控线判断检测结果。
本申请还提供一种新型冠状病毒抗原的检测方法，使用上述试剂盒中的检测卡，包括如下步骤：
采用一次性采样拭子，采集鼻腔粘膜液或口咽粘膜液；
将采样拭子放入预先加入了样本提取液的单耳管中，充分混匀样本，使鼻腔粘膜液或口咽粘膜液尽可能留在单耳管中；
将采集的鼻腔粘膜液样本或或口咽粘膜液样本滴入试剂盒中的检测中进行检测，并根据检测线和质控线判断检测结果。
本申请提供的检测新型冠状病毒抗原的试剂盒，其内置检测卡中的结合垫设有新型冠状病毒抗原的标记抗体Ab1和标记抗体Ab3，反应垫上间隔平行设置有质控线和检测线，且检测线上设有包被抗体Ab2，质控线上设有包被抗体Ab4。这样就可以采用AIE荧光免疫层析技术，用AI E荧光微球作为标记物，使用荧光手电直接判读检测卡的检测结果，或使用荧光检测仪读出检测结果，这种试剂盒，具有灵敏度高、特异性强、操作简单快速等优点。
附图说明
图1为本申请提供的试剂盒中检测卡的结构示意图；
图2为本申请提供的检测卡配套的卡壳结构示意图。
具体实施方式
下面结合附图，对本申请的较佳实施例作进一步详细说明。
AIE荧光免疫层析技术，是基于AIE荧光技术和传统免疫层析技术相结合发展创新的一种干式定量新型检测技术。该技术在保留胶体金免疫层析技术操作简便、检测快速、便携性强的优点外，还通过聚集诱导荧光材料示踪增强技术实现了检测结果的精准。与传统荧光快速检测技术性能比较，该技术灵敏度更高、检测范围更宽、价格低廉、无背景、不淬灭。
本申请使用AIE荧光免疫层析技术中双抗夹心的方法定量检测口咽或鼻咽中新型冠状病 毒抗原的含量。通过优化工艺条件、性能评估、临床应用等手段建立了一个有效的新型冠状病毒(COVID-19)抗原荧光免疫层析检测试剂盒。
本申请提供的一种检测新型冠状病毒抗原的试剂盒，包括检测卡、一次性采样拭子、抽提瓶以及置于样本提取液瓶中的样本提取液；在所述检测卡上设有结合垫和反应垫；在所述结合垫上设有新型冠状病毒抗原的标记抗体Ab1和标记抗体Ab3；在所述反应垫上间隔平行设置有质控线和检测线，在所述检测线上设有包被抗体Ab2，质控线上设有包被抗体Ab4。
上述试剂盒还包括用于稀释样本的样本提取液(磷酸盐缓冲液)、抽提瓶、及一次性采样拭子；一次性采样拭子用于采集人体口咽粘液或鼻腔粘液，并将采集的粘液置于预先加入到抽提瓶的样本提取液中，混匀待检样本液；随后，将待检样本液滴入检测卡中，通过检测线和质控线来判读检测结果。
所述样本提取液的主要成分包括0.01M的BB、1％的PVP、1％的氯化钠以及1％吐温。
上述检测卡，其结合垫中设有新型冠状病毒抗原的标记抗体生物源性如Ab1所示，反应垫中的新型冠状病毒抗原的包被抗体生物源性如Ab2所示，结合垫中用于质控线的标记抗体生物源性如Ab3所示，反应垫中质控线的包被抗体生物源性如Ab4所示；具体如表1所示。
表1检测新型冠状病毒抗原的抗体和抗原的生物源性
主要原材料 生物源性
新型冠状病毒抗原的标记抗体Ab1 鼠源单克隆IgG
新型冠状病毒抗原的包被抗体Ab2 鼠源单克隆IgG
鸡IgY标记抗体Ab3 鸡源抗体
羊抗鸡IgY包被抗体Ab4 羊源抗体
进一步，标记抗体Ab1含有AIE荧光微球(即AIE荧光标记抗体、或AIE荧光标记)，标记抗体Ab3含有AIE荧光微球(即AIE荧光标记抗体或AIE荧光标记)。
优选地，标记抗体Ab1在检测卡上的终浓度为0.3mg/mL，包被抗体Ab2在检测卡上的终浓度1mg/mL，质控线的标记抗体Ab3在检测卡上的终浓度为0.3mg/mL，质控线的包被抗体Ab4在检测卡上的终浓度为0.5mg/mL。
使用检测卡时，在毛细效应的层析作用下，样本中的新型冠状病毒抗原与检测卡上的AIE 荧光标记抗体结合，并扩散至测试区，被检测线包被的新型冠状病毒单克隆抗体捕获，并形成“抗体-抗原-荧光抗体”复合物；同时，样本中的新型冠状病毒抗原浓度与复合物荧光强度成正比，可用荧光手电直接判读阴阳性。
如图1所示，上述检测卡10中还包括样品垫11、吸水垫14以及底板15；底板15外形构造为长条形；该检测卡10的组成结构为：样品垫11、结合垫12、反应垫13、吸水垫14依次排列固定粘贴在底板15的一个表面。反应垫13的两端分别与吸水垫14的一端及结合垫12的一端层叠搭接，结合垫12的另一端与样品垫11的一端层叠搭接，样品垫11和结合垫14分别位于底板15的两端；质控线(C线)16和检测线(T线)17则设置在反应垫13上。
进一步地，如图2所示，在检测卡10的外表面还适配套设一外壳20，其包括观察窗22和加样孔21。外壳20由硬质材质制得，如，硬质纸、PVC等。检测卡10装入外壳20后，样品垫11则对应设置在加样孔21位置处，而反应垫13则对应设置在观察窗22位置处。由于检测卡10比较柔软，检测操作不方便，外壳20的作用主要是方便检测卡10的检测，强化检测卡10的硬度，确保检测卡10在检测时的直挺性，避免检测卡10弯曲，影响检测。
进一步地，生产制造制作过程中，为避免检测卡10受潮或污染，一般都会将装有卡壳20的检测卡10及干燥剂一同用铝箔袋密封包装。
上述检测卡10中，各部分组成如下：
样品垫11主要为玻璃纤维素膜构成。
结合垫12主要为玻璃纤维素膜构成，且在结合垫12上设有AIE荧光标记的标记抗体Ab1和AIE荧光标记的标记抗体Ab3；其中，标记抗体Ab1为新型冠状病毒抗体Ab1，标记抗体Ab3为鸡IgY抗体Ab3。
反应垫13为硝酸纤维素膜(NC膜)构成，其检测线包被抗体Ab2，即包被新型冠状病毒单克隆抗体Ab2；质控线包被抗体Ab4，即包被羊抗鸡IgY抗体。
吸水垫14为H-1吸水纸。
底板15为PVC片。
本申请还提供了一种针对新冠病毒抗原的采样方法，该方法采用上述试剂盒进行采用检测。包括口咽拭子和鼻咽拭子采样检测。
口咽拭子：将样本采集拭子从口腔完全插入咽喉中，缓慢旋转擦拭双侧咽扁桃体及咽后壁，避免触及舌部。
鼻咽拭子：以样本采集拭子测量鼻尖到耳垂的距离并用手指做标记，将拭子以垂直鼻子(面部)方向插入鼻腔，拭子深入距离最少应达耳垂部位到鼻尖长度的一半。遇到阻力后即抵达后鼻咽，使采集拭子在鼻咽停留15～30s，轻轻旋转3～5次后缓慢取出。
样本保存：所采集样本应立即进行检测。如果样本采集拭子在取样后不立即使用，应立即置于干燥、消毒并密封的塑料管内储存。
样本采集后应尽快用本试剂盒提供的样本提取液进行处理。经过处理的样本，常温下保存2小时，2～8℃下可保存3小时，-20±5℃保存2个月。注意：经冷冻保存后的样本需恢复室温后进行检测。
具体采样步骤如下：
1.打开滴瓶，垂直滴加11滴(约400微升)样本提取液至抽提瓶中；
2.样本采集；口咽拭子或鼻咽拭子采集；
3.将采样后的一次性采样拭子放入含样本提取液的抽提瓶中，将拭子浸在样本提取液中挤压旋转10次，充分混匀样本，再将拭子置于液面上挤压拭子，使拭子上的液体尽可能留在管内；
4.将一次性滴头接到含样本提取液的抽提瓶上；
5.打开铝箔袋，取出测试卡，放置在一个干燥、干净的平面；
6.将抽提瓶中的液体垂直滴入3滴至检测卡的加样孔中，因为操作的差异，如果3滴样本提取液滴入后2min发现上面窗口还没有液体层析出，则往该窗口下面对应的加样孔再滴1滴样本提取液；
7.在加样后10～15分钟内用荧光手电照射窗口处检测线、质控线线的位置判读结果，在30分钟后显示的结果无临床意义。
实施例1试剂盒的制备
1、检测卡的制备
步骤一、结合垫的制备
AIE荧光活化：取100uLAIE荧光微球加入800uL MES(2-吗啉乙磺酸)的离心管中，超声3min。量取30uL的EDC(1-乙基-碳酰二亚胺盐盐酸盐)和NHS(N-羟基琥珀酰亚胺)(1:1)活化液与离心管中的混合，超声至分散均匀，在室温下平衡一定时间，12000r/min，4℃，离心15min，去上清。
抗体偶联：向上述离心管加入1mL MES，旋涡后，超声3min，加入0.3mg的新型冠状病毒抗体，旋转偶联反应2h，静止5min后，加入0.3mg鸡IgY抗体。
封闭与保存：向上述离心管中加入100uL10％BSA(牛血清蛋白)溶液封闭30min，12000r/min，4℃，离心15min。去上清，加入1mL Tris缓冲液(三羟甲基氨基甲烷)，超声3min，随后12000r/min，4℃，离心15min，重复一次此步骤。去上清加入1mL微球复溶液，超声3min，2000r/min离心3min，去沉淀4℃避光保持。
结合垫：将制备的荧光微粒溶液以6uL/cm的速度均匀喷在1.5-1.8cm的玻纤上，在55℃烘干3-4h，即可。
步骤二、样品垫的制备及处理
处理液的配制：0.1M BB，1％蔗糖，1％海藻糖，1％PVPK3O(聚乙烯吡咯烷酮)。
样品垫处理：将上述处理液按3-4mL/条(宽：17-18，长：30cm)均匀涂抹在0.8-1.0cm的样品垫上，在在55℃烘干6-8h，即可。
步骤三、反应垫的制备
包被液配制：0.05M Tris(三羟甲基氨基甲烷)，1％蔗糖。划线处理：
1)、检测线，将反应垫(即硝酸纤维素膜，NC膜)贴在底板(PVC板)上，用上述包被液配制新型冠状病毒包被抗体Ab2浓度为1mg/mL，使用定量喷膜仪以1.2μL/cm均匀喷涂于硝酸纤维素膜上，于50℃鼓风干燥箱中烘干24小时，制得含检测线的反应垫；
2)、质控线，羊抗鸡IgY的包被抗体Ab4浓度为0.5mg/mL，使用定量喷膜仪以1.0μL/cm均匀喷涂于含检测线的反应垫，于50℃鼓风干燥箱中烘干24小时，制得含检测线和质控线的反应垫。
步骤四、检测卡的组装：将处理好的样品垫、结合垫、吸水垫依次贴在PVC板上，经切条机将组装好的PVC板切割成3.85mm宽度的免疫层析检测卡。
2、样品检验及结果判定
样本提取液配制：0.01M BB，1％PVP(聚乙烯吡咯烷酮)，1％氯化钠，1％吐温。
样品检验：
1.打开滴瓶，垂直滴加11滴(约400微升)样本提取液至抽提瓶中；
2.样本采集；口咽拭子或鼻咽拭子采集；
3.将采样后的一次性采样拭子放入含样本提取液的抽提瓶中，将拭子浸在样本提取液中挤压旋转10次，充分混匀样本，再将拭子置于液面上挤压拭子，使拭子上的液体尽可能留在管内；
4.将一次性滴头接到含样本提取液的抽提瓶上；
5.打开铝箔袋，取出测试卡，放置在一个干燥、干净的平面；
6.将抽提瓶中的液体垂直滴入3滴至检测卡的加样孔中，因为操作的差异，如果3滴样本提取液滴入后2min发现上面窗口还没有液体层析出，则往该窗口下面对应的加样孔再滴1滴样本提取液，15分钟后用荧光手电进行判读。荧光检测时，无论阴性还是阳性，质控线上都要显示出荧光条带，才能证明检测结果是有效的。
3、试剂盒的包装
一种人体口咽或鼻咽样本中新型冠状病毒抗原检测试剂盒的组成成分、包装及数量(25人份/盒)，如表2所示。
表2试剂盒的组成成分、包装及数量
Figure PCTCN2022074466-appb-000001
Figure PCTCN2022074466-appb-000002
此外，本申请还提供另一实施例：
本申请的上述检测新型冠状病毒抗原的试剂盒中的抽提瓶，可以替换为单耳管。
试剂盒的组成成分、包装及数量(25人份/盒)，如表3所示：
表3试剂盒的组成成分、包装及数量
Figure PCTCN2022074466-appb-000003
一种使用上述试剂盒进行新型冠状病毒抗原的检测方法，包括步骤如下：
采用一次性采样拭子，采集鼻腔粘膜液或口咽粘膜液；
将采样拭子放入预先加入了样本提取液的单耳管中，充分混匀样本，使鼻腔粘膜液或口咽粘膜液尽可能留在单耳管中；
将采集的鼻腔粘膜液样本或或口咽粘膜液样本滴入试剂盒中的检测中进行检测，并根据 检测线和质控线判断检测结果。
具体采样步骤包括：
1、样本采集；口咽拭子或鼻咽拭子采集；
2、将采样后的一次性采样拭子放入含样本提取液的单耳管中，将拭子浸在样本提取液中挤压旋转10次，充分混匀样本，再将拭子置于液面上挤压拭子，使拭子上的液体尽可能留在管内；
3、将一次性滴头接到含样本提取液的单耳管上；
4、打开铝箔袋，取出测试卡，放置在一个干燥、干净的平面；
5、将单耳管中的液体垂直滴入4滴至检测卡的加样孔中，因为操作的差异，如果3滴样本提取液滴入后2min发现上面窗口还没有液体层析出，则往该窗口下面对应的加样孔再滴1滴样本提取液；
6、在加样后10～15分钟内用荧光手电照射窗口处检测线、质控线线的位置判读结果，在30分钟后显示的结果无临床意义。荧光检测时，无论阴性还是阳性，质控线上都要显示出荧光条带，才能证明检测结果是有效的。
应当理解的是，上述针对本申请较佳实施例的表述较为详细，并不能因此而认为是对本申请专利保护范围的限制，本申请的专利保护范围应以所附权利要求为准。

Claims (20)

  1. 一种检测新型冠状病毒抗原的试剂盒，该试剂盒包括检测卡，在所述检测卡上设有结合垫和反应垫；在所述结合垫上设有新型冠状病毒抗原的标记抗体Ab1和标记抗体Ab3；在所述反应垫上间隔平行设置有质控线和检测线，在所述检测线上设有包被抗体Ab2，质控线上设有包被抗体Ab4。
  2. 根据权利要求1所述的试剂盒，其中，所述标记抗体Ab1的生物源性为鼠源单克隆IgG。
  3. 根据权利要求1所述的试剂盒，其中，所述包被抗体Ab2的生物源性为鼠源单克隆IgG。
  4. 根据权利要求1所述的试剂盒，其中，所述标记抗体Ab3的生物源性为鸡源抗体。
  5. 根据权利要求1所述的试剂盒，其中，所述包被抗体Ab4的生物源性为羊源抗体。
  6. 根据权利要求1所述的试剂盒，其中，所述标记抗体Ab1和所述标记抗体Ab3均有AIE荧光微球。
  7. 根据权利要求1所述的试剂盒，其中，所述标记抗体Ab1的终浓度为0.3mg/mL；所述包被抗体Ab2的终浓度为1mg/mL。
  8. 根据权利要求1所述的试剂盒，其中，所述标记抗体Ab3的终浓度为0.3mg/mL；所述包被抗体Ab4的终浓度为0.5mg/mL。
  9. 根据权利要求1所述的试剂盒，其中，所述试剂盒还包括用于稀释样本的样本提取液、提瓶及一次性采样拭子。
  10. 根据权利要求9所述的试剂盒，其中，所述样本提取液包括0.01M的BB、1％的PVP、1％的氯化钠以及1％吐温。
  11. 根据权利要求1所述的试剂盒，其中，所述标记抗体Ab1含有AIE荧光微球，所述标记抗体Ab3含有AIE荧光微球。
  12. 根据权利要求1所述的试剂盒，其中，所述标记抗体Ab1在检测卡上的终浓度为0.3mg/mL，所述包被抗体Ab2在检测卡上的终浓度1mg/mL，质控线的标记抗体Ab3 在检测卡上的终浓度为0.3mg/mL，质控线的包被抗体Ab4在检测卡上的终浓度为0.5mg/mL。
  13. 根据权利要求1所述的试剂盒，其中，所述检测卡还包括样品垫、吸水垫以及底板，其中，所述检测卡的组成结构为：样品垫、结合垫、反应垫、吸水垫依次排列固定粘贴在底板的一个表面，反应垫的两端分别与吸水垫的一端及结合垫的一端层叠搭接，结合垫的另一端与样品垫的一端层叠搭接，样品垫和结合垫分别位于底板的两端，所述质控线和所述检测线设置在反应垫上。
  14. 根据权利要求13所述的试剂盒，其中，所述样品垫为玻璃纤维素膜构成。
  15. 根据权利要求13所述的试剂盒，其中，所述吸水垫为H-1吸水纸。
  16. 根据权利要求13所述的试剂盒，其中，所述底板为PVC片。
  17. 根据权利要求1所述的试剂盒，其中，所述检测卡的外表面还适配套设一外壳，所述外壳包括观察窗和加样孔。
  18. 一种使用权利要求1至17任一所述试剂盒进行新型冠状病毒抗原的检测方法，包括步骤如下：
    采用一次性采样拭子，从人体口腔完全插入咽喉中，缓慢旋转擦拭双侧咽扁桃体及咽后壁，采集口咽粘膜液；
    将采样拭子放入含预先加入了样本提取液的抽提瓶中，充分混匀样本，使口咽粘膜液尽可能留在抽提瓶中；
    将采集的口咽粘膜液样本滴入试剂盒中的检测中进行检测，并根据检测线和质控线判断检测结果。
  19. 一种使用权利要求1至17任一所述试剂盒进行新型冠状病毒抗原的检测方法，包括步骤如下：
    采用一次性采样拭子，以垂直鼻子方向插入鼻腔，深入距离最少应达耳垂部位到鼻尖长度的一半，采集鼻腔粘膜液；
    将采样拭子放入预先加入了样本提取液的抽提瓶中，充分混匀样本，使鼻腔粘膜液尽可能留在抽提瓶中；
    将采集的鼻腔粘膜液样本滴入试剂盒中的检测中进行检测，并根据检测线和质控线判断检测结果。
  20. 一种使用权利要求1至17任一所述试剂盒进行新型冠状病毒抗原的检测方法，包括步骤如下：
    采用一次性采样拭子，采集鼻腔粘膜液或口咽粘膜液；
    将采样拭子放入预先加入了样本提取液的单耳管中，充分混匀样本，使鼻腔粘膜液或口咽粘膜液尽可能留在单耳管中；
    将采集的鼻腔粘膜液样本或或口咽粘膜液样本滴入试剂盒中的检测中进行检测，并根据检测线和质控线判断检测结果。
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