WO2022141394A1 - Non-invasive sampling test kit for testing sars-cov-2 antibody, and test method therefor - Google Patents

Non-invasive sampling test kit for testing sars-cov-2 antibody, and test method therefor Download PDF

Info

Publication number
WO2022141394A1
WO2022141394A1 PCT/CN2020/142092 CN2020142092W WO2022141394A1 WO 2022141394 A1 WO2022141394 A1 WO 2022141394A1 CN 2020142092 W CN2020142092 W CN 2020142092W WO 2022141394 A1 WO2022141394 A1 WO 2022141394A1
Authority
WO
WIPO (PCT)
Prior art keywords
antibody
sample
detection
novel coronavirus
invasive sampling
Prior art date
Application number
PCT/CN2020/142092
Other languages
French (fr)
Chinese (zh)
Inventor
马哲
辛炳川
Original Assignee
马哲
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 马哲 filed Critical 马哲
Priority to PCT/CN2020/142092 priority Critical patent/WO2022141394A1/en
Priority to CN202080107265.5A priority patent/CN116670509A/en
Publication of WO2022141394A1 publication Critical patent/WO2022141394A1/en

Links

Images

Classifications

    • 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

Definitions

  • the invention relates to a non-invasive sampling detection kit for detecting novel coronavirus antibodies and a detection method thereof, belonging to the biological field.
  • Coronavirus is a virus that can infect both humans and animals, and is usually the source of infection in areas where humans and wild animals frequently come into contact.
  • a large number of scientific studies have confirmed that the 2019-nCoV virus originated in nature.
  • the infection mechanism has not been clear so far, including transmission speed, incubation period, human-to-human transmission of highly pathogenic viruses, etc., resulting in the existence of a large number of infected people around the world.
  • the death rate is gradually increasing.
  • the new coronavirus has become a pandemic, which has a huge impact on the stability of human society and the global economy.
  • 2019-nCoV 2019 novel coronavirus disease
  • the gold standard for 2019-nCoV detection is nucleic acid detection, but in clinical work, differences in specimen type, specimen quality, patient condition and other factors may lead to false negative results in nucleic acid detection.
  • nucleic acid testing relies on laboratory testing equipment, which has the problem of long testing time and inability to adapt to large-scale screening at the point of care. Therefore, how to accurately and rapidly identify viral infection is an important challenge.
  • serum-specific antibodies as an important immune response product of the body against viral infection, are rapidly applied to laboratory detection of 2019-nCoV, and combined with nucleic acid detection for rapid diagnosis and screening of COVID-19 patients.
  • Serum assays can be used to supplement the diagnosis of SARS-CoV-2 infection when nasopharyngeal swab collection is inappropriate and molecular assay results are unsatisfactory.
  • Antibody rapid detection can make up for the sensitivity and time window of nucleic acid detection, and provide relevant information for infection control.
  • 2019-nCoV/SARS-CoV-2 antibody tests on the market are based on blood or serum samples.
  • tests based on venous blood or serum are carried out by professional medical institutions such as hospitals, and professional blood collection is required. Therefore, the scope of use of these test products is greatly limited by the place where they are applied.
  • home blood requires fingertip blood collection, and fingertip blood collection and pipettes require matching needles. This invasive method is not acceptable to everyone, so the popularity of this test product has certain limitations. Therefore, it is urgent to invent a simpler and more versatile detection product for the new coronavirus.
  • the purpose of the present invention is to obtain a non-invasive sampling detection kit for detecting novel coronavirus antibodies and a detection method thereof.
  • the technical scheme of the non-invasive sampling detection kit for detecting novel coronavirus antibodies of the present invention is as follows:
  • a non-invasive sampling detection kit for detecting novel coronavirus which is used to detect whether urine contains novel coronavirus IgA antibody to quickly determine whether a subject is infected with novel coronavirus
  • the kit includes a test card, and the test card includes Nitrocellulose membrane (NC membrane), sample pad, colloidal gold connection pad, absorbing film and PVC backboard, the structures in the detection card are in the order of passing the sample to be tested: sample pad, colloidal gold connection pad, nitrocellulose Membrane and absorption membrane, wherein the nitrocellulose membrane includes ⁇ chain body/anti-human IgG antibody, anti-rabbit polyclonal antibody, and the connection pad includes 2019-nCoV recombinant antigen, rabbit IgG antibody, and the recombinant antigen is colloid connected by colloidal gold particles Gold antibodies.
  • NC membrane Nitrocellulose membrane
  • sample pad includes ⁇ chain body/anti-human IgG antibody, anti-rabbit polyclonal antibody
  • the connection pad includes 2019-nCoV recombinant anti
  • the absorption film in the test card has strong water absorption.
  • the sample to be tested can move from the sample pad to the direction of the absorption film through capillary action, and pass through the detection area and the quality control area on the nitrocellulose membrane in turn.
  • the IgA in the sample binds to the recombinant antigen
  • the detection area of the nitrocellulose membrane binds to the anti-human IgG antibody to form a red area visible to the naked eye.
  • the detection card is provided with a sample addition hole, and the sample to be tested can penetrate into the interior of the detection card through the sample addition hole, that is, the sample to be tested can penetrate into the sample pad through the sample addition hole.
  • the recombinant antigen sequence on the sample pad can match at least 90% with the SARS-CoV-2 IgA antibody sequence, and the antigen sequence is shown in SEQ ID NO: 1 in the sequence listing. More preferably, the recombinant antigen and antibody sequences are at least 95% compatible.
  • sequence shown in the sequence SED ID NO: 1 targets the SARS-CoV-2 surface glycoprotein, which is a highly flexible S protein with a mobile domain that provides a mobile target for the immune system.
  • the recombinant antigen also includes the sequences shown in SEQ ID NOs: 2-10 in the sequence listing, or at least 500 consecutive amino acid sequences including the above-mentioned sequences.
  • sequence SED ID NO: 2 shows the ⁇ -CoV S-protein structure, which is based on the analysis of the present invention.
  • Two extra-protein domain antigens designed for the S protein have high immunogenicity and are easier to interact with all receptors. Body binding domain binding, it is easier to obtain binding.
  • the receptor-binding domain protein represented by the sequence SED ID NO:6 has two distinct conformational states, a "down" state, which is shielded by the receptor, and an "up” state, which the receptor can enter.
  • the sequence SED ID NO: 10 shows the structurally conserved subdomain 2, and the S1 subunit is subdivided into an N-terminal domain (NTD), a receptor binding domain (RBD) and two structurally conserved subdomains ( SD1 and SD2). Together, these regions confine the S2 subunit, protecting the conserved fusion machinery.
  • NTD N-terminal domain
  • RBD receptor binding domain
  • SD1 and SD2 structurally conserved subdomains
  • sequence SED ID NO: 11 shows the complete protein expression region sequence of SARS-CoV-2.
  • the recombinant antigen can recognize SARS-CoV-2 surface glycoprotein (coronavirus S protein), SARS-CoV-2 surface glycoprotein S2S1 (coronavirus S2S1 protein), or at least 500 consecutive amino acids of the above proteins Fragment.
  • colloidal gold particles label the antigen can also be replaced by any other label known to those skilled in the art, including latex particles, quantum dots, fluorescent nanoparticles, and the like.
  • the detection zone and the quality control zone can choose the same or different labels, as long as the type of labels can be understood by those skilled in the art.
  • the rabbit IgG antibody is selected from other types of immunoglobulins, including IgA and IgG, preferably non-human IgG, such as mouse IgG and the like.
  • whether a red band appears in the quality control area can be used as an indication of whether the liquid flow in the detection card meets the preset requirements. If there is no red band in the quality control area, there is an obstacle or error in the liquid flow. Should not be used as a reference standard.
  • the sample pad, colloidal gold connection pad, nitrocellulose membrane and absorption membrane in the detection card are all adsorbed on a support pad, and the support pad is made of glass fiber, polyester film or non-woven fabric.
  • Another object of the present invention is to provide a detection method using the above-mentioned non-invasive sampling detection kit for detecting novel coronavirus, the method comprising the following steps:
  • the sample to be tested is placed in the sample filling hole of the sample pad, so that the sample to be tested flows from the sample pad to the colloidal gold connection pad;
  • the sample to be tested flows through the detection area and the quality control area in turn by capillary action.
  • the pairing of the antigen and the antibody to be tested is more than 90%, the antibody to be tested can be captured by the recombinant antigen to form a red marked area;
  • the detection sample in this method is human urine that has not undergone secondary processing.
  • the detection kit and detection method of the present invention can be used in clinics, at home, and in various places and uses that require rapid detection of novel coronavirus.
  • polypeptide and “protein” are used interchangeably and refer to a polymer of amino acid residues and are not limited by length.
  • Polypeptides including antibodies and antibody chains and other polypeptides, such as linkers, may include amino acid residues, including natural and/or unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like.
  • a polypeptide may contain modifications relative to the native or native sequence as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or accidental, such as by host mutation to produce the protein or due to PCR amplification errors.
  • percent (%) amino acid sequence identity and “percent identity” and “identity” when used with a sequence of an amino acid sequence (a reference polypeptide sequence) is defined as the ratio of amino acid residues in a candidate sequence (e.g., protein or fragment) identical to the amino acid residue sequence of the reference polypeptide, the sequence and the gaps introduced after adjustment, if necessary, to achieve the maximum percent sequence identity, and disregard any conservative substitutions as part of the sequence identity.
  • Alignment for the purpose of determining percent amino acid sequence identity can be accomplished by various technical means, eg, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. These skilled artisans can determine appropriate parameters for sequence alignment, including any algorithms needed to achieve maximal alignment over the entire length of the sequences being compared.
  • the kit of this application does not cross-react with the following positive samples: parainfluenza virus antibody, influenza A virus antibody, influenza B virus antibody, Chlamydia pneumoniae antibody, Mycoplasma pneumoniae antibody, adenovirus antibody, RSV antibody, B Hepatitis surface antibody, hepatitis C antibody, tp antibody, HIV antibody, EB virus antibody, measles virus antibody, CMV antibody, enterovirus 71 antibody, mumps virus antibody, varicella-zoster virus antibody.
  • the present invention adopts a non-invasive sampling method for sampling, which can conveniently, quickly and reliably detect whether the subject is infected with the new coronavirus SARS-CoV-2, the non-invasive sampling does not cause any pain, and the source of the sample is non-invasive.
  • the detection is fast and the results are visible, and the results can be quickly obtained within 10 minutes; the detection results can avoid the hook effect or the front zone effect, and the detection result has a high accuracy rate .
  • the negative detection rate of morning urine was more than 91%; the operation method is simple, without any equipment or device assistance, suitable for staff who have not received medical or other training, the use cost of the test kit is low, and the use of There are no restrictions on the environment and place, which has strong promotion significance and is more conducive to the monitoring of high-risk groups.
  • the invention can also prevent asymptomatic individuals from spreading the source of infection, and is an important detection means for saving medical manpower and material resources and consumption.
  • Fig. 1 is the production process block diagram of the kit of the present invention
  • FIG. 2 is a schematic diagram of the operation of the kit of the present invention during and after sampling;
  • FIG. 3 is a schematic diagram of the expected detection result of the kit of the present invention.
  • non-invasive sampling detection kit for detecting novel coronavirus antibodies and the detection method thereof provided by the present invention will be further described in detail and completely below with reference to the examples.
  • the embodiments described below are exemplary, only for explaining the present invention, and should not be construed as limiting the present invention.
  • the detection kit of the present invention adopts the double-antigen sandwich capture method to detect the antibody in the sample.
  • the conjugated gold label contains the recombinant antigen of the new coronavirus, and the membrane of the test strip contains (recombinant antigen and mouse anti-human IgA antibody).
  • the antibody-bearing region binds to the antigen on the gold-labeled conjugate.
  • the rest can be bound to the antigen-antibody field, and on the other hand, the antibodies on the membrane can be identified.
  • the gold marker will be fixed on the corresponding test area, which shows a red indicator.
  • the gold indicator will not be immobilized in the detection area and the area will not display red.
  • the mixture on the gold-labeled conjugate includes the label containing rabbit IgG gold particles, and the control region (C line) of the test strip contains goat anti-rabbit antibody. Regardless of whether the detection area shows color, the color is shown by the immobilized labeling of the rabbit IgG gold particles. If no color appears in this area, the test is invalid.
  • This example uses the principle of immunochromatography to detect the presence of antibodies to the novel coronavirus SARS-CoV-2 (also known as 2019-nCoV) in human urine samples using a capture method.
  • SARS-CoV-2 also known as 2019-nCoV
  • the antibody and the antigen label are coupled and bound, and the secondary antibody (anti- ⁇ chain antibody/anti-human IgG) is combined in the detection zone (T zone).
  • Antibody is coupled again to form a red reaction band, at this time the result will be judged as positive, if no band is produced, it will be judged as negative.
  • the quality control area (area C) will always be colored to indicate that the test is valid. When there is no red line in the quality control area (C area), it indicates that the detection is invalid.
  • the test card of the test paper of the present invention includes glass fiber, polyester film or non-woven fabric treated in solution and dried.
  • the kit of this embodiment includes a test card, a desiccant and a urine cup.
  • the test card is composed of a nitrocellulose membrane (NC membrane), a sample pad, a colloidal gold connection pad, an absorbing film, and a PVC backing plate.
  • the membrane includes anti- ⁇ chain/anti-human IgG antibody, anti-rabbit polyclonal antibody, and the junction pad includes 2019-nCoV recombinant antigen, rabbit IgG antibody.
  • the sample pad, colloidal gold connection pad, nitrocellulose membrane and absorption membrane are all adsorbed on a support pad made of glass fiber.
  • test card 1. The processing process of the test card is as follows:
  • Buffer system Mix one to several buffers into a composite buffer system of 0.01-5M, including but not limited to Tris-hydrochloric acid, boric acid-borax, phosphate buffer, and hepd buffer system;
  • Macromolecular substances 0.1-3% of one to several macromolecular substances, including bovine serum albumin, casein, polyethylene glycol, and gelatin;
  • Salt content 0.1%-5% sodium chloride/magnesium chloride
  • Colloidal protective ingredients 0.1%-5%, PVP;
  • Urine is collected in the morning after the first urination and can be tested directly without processing.
  • Female Before sampling, use soap or liquid containing 0.1% alcohol to wash and disinfect the vulva, open the labia with fingers, continuously urinate and collect urine from the middle section, collect 10-20mL of urine and store it in a sterile container.
  • Testing should be performed within 2 hours of sample collection. If it cannot be detected immediately, it can be stored at 2°C-8°C for 2 days; after 2 days, it must be stored at -20°C for up to 7 days. Before testing, samples must be brought to room temperature to avoid repeated freezing and thawing. Heat inactivated samples are not recommended.
  • test card directly into the urine sample to ensure sufficient sample volume.
  • the detection card of this embodiment is carried out in a clean area of class 100,000 according to the process flow chart of FIG. 1 , wherein the marks marked with ⁇ are the key control points of the working process.
  • test reagents and related test materials used for the test must be equilibrated to room temperature, and the test must be carried out at room temperature.
  • specific detection steps are as follows:
  • test strip can be retracted and placed flat on the table.
  • test when there is no red line in the quality control area (C), the test is invalid. It is recommended to take a new test strip for re-testing. When testing, note that the test strip needs to be inserted deep enough into the urine sample and not exceed the max line. When the detection area (T) and the quality control area (C) have two red lines, it is positive. When there is only one red line in the quality control area (C), it is negative. Other cases are abnormal, which is an invalid test, and it is recommended to re-test.
  • Positive reference product rate The industry positive reference product rate must be 5/5;
  • Negative reference product ratio The industry negative reference product ratio must be 10/10;

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Virology (AREA)
  • Microbiology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

A non-invasive sampling test kit for testing a SARS-CoV-2 antibody. The test kit tests whether there is a SARS-CoV-2 IgA antibody in urine to quickly determine whether a subject has been infected with SARS-CoV-2. The kit comprises a test card; the test card comprises a nitrocellulose membrane, a sample pad, a colloidal gold connection pad, an absorption membrane, and a PVC backboard; the nitrocellulose membrane comprises a μ-chain/anti-human IgG antibody and an anti-rabbit polyclonal antibody; the connection pad comprises a 2019-nCoV recombinant antigen and a rabbit IgG antibody; the recombinant antigen is a colloidal gold antibody linked to a colloidal gold particle. Sampling is performed by means of a non-invasive sampling approach, whether a subject has been infected with SARS-CoV-2 can be tested conveniently, quickly, and reliably, the non-invasive sampling does not cause any pain, and non-invasive sampling eliminates the possibility of secondary infection and reduces the risk of virus propagation; the test is implemented quickly and the result is visible, and the result can be quickly obtained within 10 minutes.

Description

一种用于检测新型冠状病毒抗体的无创取样检测试剂盒及其检测方法A non-invasive sampling detection kit for detecting novel coronavirus antibodies and its detection method 技术领域technical field
本发明涉及一种用于检测新型冠状病毒抗体的无创取样检测试剂盒及其检测方法，属于生物领域。The invention relates to a non-invasive sampling detection kit for detecting novel coronavirus antibodies and a detection method thereof, belonging to the biological field.
背景技术Background technique
[根据细则91更正 07.09.2021]　
[Correction 07.09.2021 under Rule 91]
冠状病毒是一种既能感染人类又能感染动物的病毒，通常是人类与野生动物频繁接触地区的感染源。经过大量科学研究证实，2019-nCoV病毒起源于自然界，然而到目前为止感染机制尚未明确，包括传输速度、潜伏期、高致病性病毒的人际传播等等，导致存在大量的感染者在世界各地的许多国家，且死亡率逐渐增加。目前，新型冠状病毒已成为一种大流行，对人类社会稳定和全球经济造成巨大影响。Coronavirus is a virus that can infect both humans and animals, and is usually the source of infection in areas where humans and wild animals frequently come into contact. A large number of scientific studies have confirmed that the 2019-nCoV virus originated in nature. However, the infection mechanism has not been clear so far, including transmission speed, incubation period, human-to-human transmission of highly pathogenic viruses, etc., resulting in the existence of a large number of infected people around the world. In many countries, the death rate is gradually increasing. At present, the new coronavirus has become a pandemic, which has a huge impact on the stability of human society and the global economy.
面对2019年新型冠状病毒病(COVID-19)在全球蔓延的严重大流行形势，快速识别新冠病毒(2019-nCoV)感染者是防控的重中之重。2019-nCoV检测的金标准是核酸检测，但在临床工作中，标本类型、标本质量、患者病情等因素的差异都可能导致核酸检测出现假阴性结果。此外，核酸检测依赖于实验室检测仪器，存在检测时间长，检测不能适应护理点的大规模筛查的问题。因此，如何准确和快速地识别病毒感染是一个重要的挑战。为此，血清特异性抗体作为机体抵抗病毒感染的重要免疫应答产物，迅速应用于实验室检测2019年新型冠状病毒，并与核酸检测相结合，用于COVID-19患者的快速诊断和筛查。In the face of the severe pandemic situation of the 2019 novel coronavirus disease (COVID-19) spreading around the world, rapid identification of people infected with the new coronavirus (2019-nCoV) is the top priority of prevention and control. The gold standard for 2019-nCoV detection is nucleic acid detection, but in clinical work, differences in specimen type, specimen quality, patient condition and other factors may lead to false negative results in nucleic acid detection. In addition, nucleic acid testing relies on laboratory testing equipment, which has the problem of long testing time and inability to adapt to large-scale screening at the point of care. Therefore, how to accurately and rapidly identify viral infection is an important challenge. To this end, serum-specific antibodies, as an important immune response product of the body against viral infection, are rapidly applied to laboratory detection of 2019-nCoV, and combined with nucleic acid detection for rapid diagnosis and screening of COVID-19 patients.
基于胶体金快速免疫层析的诊断模型日益受到重视，快速抗体检测可以弥补核酸检测的时间窗和敏感性问题，并为控制感染措施提供相关信息。在诊断新的和新出现的人冠状病毒时，抗体检测尤其重要。在这种情况下，特别是在疾病的早期阶段，对感染者的病毒DNA进行阳性检测并不是非常可行，但可以回顾性地证明已经产生了免疫反应。在SARS-CoV-2的鉴定中，特别是在快速抗原试验和/或分子测定不能使用且不稳定的情况下，血清学可作为补充诊断工具。最近的一项研究发现，在发病5天后，39名SARS-CoV-2感染者中的每个人体内都发现了IgM和IgG抗体。当采集鼻咽拭子不合适和分子化验结果不满意时，血清化验可用于补充SARS-CoV-2感染的诊断。抗体快速检测可以弥补核酸检测的敏感性和时间窗问题，为控制感染提供相关信息。Diagnostic models based on colloidal gold rapid immunochromatography have received increasing attention, and rapid antibody detection can compensate for the time window and sensitivity of nucleic acid detection and provide relevant information for infection control measures. Antibody testing is especially important when diagnosing new and emerging human coronaviruses. In this case, especially in the early stages of the disease, a positive test for viral DNA in an infected person is not very feasible, but it is possible to retrospectively demonstrate that an immune response has developed. Serology can be used as a complementary diagnostic tool in the identification of SARS-CoV-2, especially when rapid antigen tests and/or molecular assays are not available and are not stable. A recent study found that IgM and IgG antibodies were found in each of 39 SARS-CoV-2-infected individuals five days after onset. Serum assays can be used to supplement the diagnosis of SARS-CoV-2 infection when nasopharyngeal swab collection is inappropriate and molecular assay results are unsatisfactory. Antibody rapid detection can make up for the sensitivity and time window of nucleic acid detection, and provide relevant information for infection control.
目前，市场上的2019-nCoV/SARS-CoV-2抗体检测是基于血液或血清样本的检测。一般以静脉血或血清为基础的检测依靠医院等专业医疗机构进行，并需要专业人员进行采血。因此，这些测试产品的使用范围受到其应用场所的极大限制。此外，家庭用血需要指尖采血，而指尖采血和移液管需要配套针头，这种侵入性的方法并不是所有人都能接受的，因此这种测试产品的普及有一定的局限性。因此，发明一种更简单、更通用的新型冠状病毒检测产品迫在眉睫。Currently, 2019-nCoV/SARS-CoV-2 antibody tests on the market are based on blood or serum samples. Generally, tests based on venous blood or serum are carried out by professional medical institutions such as hospitals, and professional blood collection is required. Therefore, the scope of use of these test products is greatly limited by the place where they are applied. In addition, home blood requires fingertip blood collection, and fingertip blood collection and pipettes require matching needles. This invasive method is not acceptable to everyone, so the popularity of this test product has certain limitations. Therefore, it is urgent to invent a simpler and more versatile detection product for the new coronavirus.
发明内容SUMMARY OF THE INVENTION
针对现有技术存在的上述问题，本发明的目的是获得一种用于检测新型冠状病毒抗体的无创取样检测试剂盒及其检测方法。In view of the above problems existing in the prior art, the purpose of the present invention is to obtain a non-invasive sampling detection kit for detecting novel coronavirus antibodies and a detection method thereof.
为实现上述发明目的，本发明的用于检测新型冠状病毒抗体的无创取样检测试剂盒的技术方案如下：In order to achieve the above-mentioned purpose of the invention, the technical scheme of the non-invasive sampling detection kit for detecting novel coronavirus antibodies of the present invention is as follows:
一种检测新型冠状病毒的无创取样检测试剂盒，用于检测尿液中是否含有新型冠状病毒IgA抗体以快速判断受试者是否感染了新型冠状病毒，所述试剂盒包括检测卡，检测卡包括硝化纤维素膜(NC膜)、样品垫、胶体金连接垫、吸收膜和PVC背板，检测卡内各结构按照待测样品的通过顺序依次为：样品垫、胶体金连接垫、硝化纤维素膜和吸收膜，其中硝化纤维素膜包括μ链体/抗人IgG抗体、抗兔多克隆抗体，连接垫包括2019-nCoV重组抗原、兔IgG抗体，所述重组抗原为胶体金颗粒连接的胶体金抗体。检测卡中吸收膜有较强的吸水性，待测样本可由样品垫向吸收膜方向通过毛细管作用发生运动，并依次经过硝化纤维素膜上的检测区和质控区，当待测样品的浓度大于检测下限时，样品中的IgA与重组抗原结合，至硝酸纤维素膜的检测区与抗人IgG抗体结合后形成肉眼可见的红色区域。A non-invasive sampling detection kit for detecting novel coronavirus, which is used to detect whether urine contains novel coronavirus IgA antibody to quickly determine whether a subject is infected with novel coronavirus, the kit includes a test card, and the test card includes Nitrocellulose membrane (NC membrane), sample pad, colloidal gold connection pad, absorbing film and PVC backboard, the structures in the detection card are in the order of passing the sample to be tested: sample pad, colloidal gold connection pad, nitrocellulose Membrane and absorption membrane, wherein the nitrocellulose membrane includes μ chain body/anti-human IgG antibody, anti-rabbit polyclonal antibody, and the connection pad includes 2019-nCoV recombinant antigen, rabbit IgG antibody, and the recombinant antigen is colloid connected by colloidal gold particles Gold antibodies. The absorption film in the test card has strong water absorption. The sample to be tested can move from the sample pad to the direction of the absorption film through capillary action, and pass through the detection area and the quality control area on the nitrocellulose membrane in turn. When it is greater than the detection limit, the IgA in the sample binds to the recombinant antigen, and the detection area of the nitrocellulose membrane binds to the anti-human IgG antibody to form a red area visible to the naked eye.
优选的，所述检测卡上设有加样孔，待测样品可由加样孔渗入检测卡内部，也就是说，待测样品可由加样孔深入样品垫。Preferably, the detection card is provided with a sample addition hole, and the sample to be tested can penetrate into the interior of the detection card through the sample addition hole, that is, the sample to be tested can penetrate into the sample pad through the sample addition hole.
优选的，所述样品垫上的重组抗原序列与SARS-CoV-2IgA抗体序列可配对至少90％，抗原序列如序列表SEQ ID NO:1所示。更优选的，所述重组抗原与抗体序列可配对至少95％。Preferably, the recombinant antigen sequence on the sample pad can match at least 90% with the SARS-CoV-2 IgA antibody sequence, and the antigen sequence is shown in SEQ ID NO: 1 in the sequence listing. More preferably, the recombinant antigen and antibody sequences are at least 95% compatible.
序列SED ID NO:1所示序列针对的是新冠病毒表面糖蛋白，其为一种高度灵活的s蛋白，具有可移动的结构域，为免疫***提供一个可移动的目标。The sequence shown in the sequence SED ID NO: 1 targets the SARS-CoV-2 surface glycoprotein, which is a highly flexible S protein with a mobile domain that provides a mobile target for the immune system.
优选的，重组抗原还包括如序列表SEQ ID NO:2～10所示的序列，或包括上述序列的至少500个连续的氨基酸序列。Preferably, the recombinant antigen also includes the sequences shown in SEQ ID NOs: 2-10 in the sequence listing, or at least 500 consecutive amino acid sequences including the above-mentioned sequences.
序列SED ID NO:2所示的为β-CoV S-蛋白结构，是基于本发明的分析，为S蛋白设计的两款蛋白外结构域抗原，具有高免疫原性且更易于与所有的受体结合域结合，获取结合更容易。The sequence SED ID NO: 2 shows the β-CoV S-protein structure, which is based on the analysis of the present invention. Two extra-protein domain antigens designed for the S protein have high immunogenicity and are easier to interact with all receptors. Body binding domain binding, it is easier to obtain binding.
序列SED ID NO:6所示的受体结合域蛋白质具有两种不同的构象状态，一种是“down”状态，被受体屏蔽，另一种是“up”状态，受体可进入。The receptor-binding domain protein represented by the sequence SED ID NO:6 has two distinct conformational states, a "down" state, which is shielded by the receptor, and an "up" state, which the receptor can enter.
序列SED ID NO:10所示的是结构上保守的子域2，S1亚基被细分为n端结构域(NTD)、受体结合域(RBD)和两个结构上保守的子域(SD1和SD2)。这些区域共同限制了S2亚基，保护了保守的聚变机制。在S1中通过RBD与宿主受体结合后，宿主蛋白酶对刺突进行蛋白水解裂解2。s蛋白的巨大构象变化导致S1脱落和S2融合机制暴露。The sequence SED ID NO: 10 shows the structurally conserved subdomain 2, and the S1 subunit is subdivided into an N-terminal domain (NTD), a receptor binding domain (RBD) and two structurally conserved subdomains ( SD1 and SD2). Together, these regions confine the S2 subunit, protecting the conserved fusion machinery. After binding to the host receptor via the RBD in S1, the spike is proteolytically cleaved by host proteases. The huge conformational change of the S protein results in the shedding of S1 and the exposure of the S2 fusion machinery.
序列SED ID NO:11所示的是SARS-CoV-2完整的蛋白表达区序列。The sequence SED ID NO: 11 shows the complete protein expression region sequence of SARS-CoV-2.
优选的，所述的重组抗原可以识别SARS-CoV-2表面糖蛋白(冠状病毒S蛋白)、SARS-CoV-2表面糖蛋白S2S1(冠状病毒S2S1蛋白)、或上述蛋白的至少500个连续氨基酸片段。Preferably, the recombinant antigen can recognize SARS-CoV-2 surface glycoprotein (coronavirus S protein), SARS-CoV-2 surface glycoprotein S2S1 (coronavirus S2S1 protein), or at least 500 consecutive amino acids of the above proteins Fragment.
胶体金颗粒标记抗原的方式也可以由任一本领域技术人员了解的其他标记代替，包括乳胶粒子、量子点、荧光纳米粒子等。检测区和质控区可选择相同或不同的标记，只要标记种类是本领域技术人员可以了解到的即可。The manner in which the colloidal gold particles label the antigen can also be replaced by any other label known to those skilled in the art, including latex particles, quantum dots, fluorescent nanoparticles, and the like. The detection zone and the quality control zone can choose the same or different labels, as long as the type of labels can be understood by those skilled in the art.
优选的，所述兔IgG抗体在免疫球蛋白中选择其他类型，包括IgA和IgG，优选非人类IgG，如小鼠IgG等。Preferably, the rabbit IgG antibody is selected from other types of immunoglobulins, including IgA and IgG, preferably non-human IgG, such as mouse IgG and the like.
优选的，质控区是否出现红色条带可以作为测试检测卡内液体流动是否符合预设要求的表现，如质控区未出现红色条带，则液体流动出现障碍或错误，检测区的检测结果不应作为参考标准。Preferably, whether a red band appears in the quality control area can be used as an indication of whether the liquid flow in the detection card meets the preset requirements. If there is no red band in the quality control area, there is an obstacle or error in the liquid flow. Should not be used as a reference standard.
优选的，所述检测卡中的样品垫、胶体金连接垫、硝化纤维素膜和吸收膜均吸附在一支撑垫上，支撑垫由玻璃纤维、聚酯薄膜或无纺布制成。Preferably, the sample pad, colloidal gold connection pad, nitrocellulose membrane and absorption membrane in the detection card are all adsorbed on a support pad, and the support pad is made of glass fiber, polyester film or non-woven fabric.
本发明的另一个目的是提供一种采用上述检测新型冠状病毒的无创取样检测试剂盒的检测方法，所述方法包括如下步骤：Another object of the present invention is to provide a detection method using the above-mentioned non-invasive sampling detection kit for detecting novel coronavirus, the method comprising the following steps:
a)将待测样本置于样品垫的加样孔内，使待测样本由样品垫向胶体金连接垫 流动；a) the sample to be tested is placed in the sample filling hole of the sample pad, so that the sample to be tested flows from the sample pad to the colloidal gold connection pad;
b)待测样本通过毛细管作用依次流过检测区和质控区，当抗原与待测抗体配对大于90％以上时，待测抗体可被重组抗原捕获，形成红色标记区域；b) The sample to be tested flows through the detection area and the quality control area in turn by capillary action. When the pairing of the antigen and the antibody to be tested is more than 90%, the antibody to be tested can be captured by the recombinant antigen to form a red marked area;
c)肉眼观测检测区和质控区是否出现红色标记区域，以确定待测样本来源是否感染新型冠状病毒。c) Visually observe whether the red marked area appears in the detection area and the quality control area to determine whether the source of the sample to be tested is infected with the new coronavirus.
优选的，本方法中的检测样本为不经过二次加工处理的人体尿液。Preferably, the detection sample in this method is human urine that has not undergone secondary processing.
基于上述特征，本发明的检测试剂盒和检测方法可以用于临床、家庭及各类需要进行新型冠状病毒快检的场所和用途。Based on the above features, the detection kit and detection method of the present invention can be used in clinics, at home, and in various places and uses that require rapid detection of novel coronavirus.
本发明中，“多肽”和“蛋白质”这两个词是可以互换使用的，指的是氨基酸残基的聚合物，并且不受长度的限制。多肽，包括抗体和抗体链和其他多肽，如连接子，可能包括氨基酸残基，包括天然和/或非天然氨基酸残基。这些术语还包括多肽表达后的修饰，如糖基化、唾液化、乙酰化、磷酸化等。在某些方面，只要蛋白质保持所期望的活性，多肽就可能包含与天然或自然序列相关的修饰。这些修饰可能是有意的，如通过位点定向诱变，也可能是偶然的，如通过宿主突变产生蛋白质或由于PCR扩增产生错误。In the present invention, the terms "polypeptide" and "protein" are used interchangeably and refer to a polymer of amino acid residues and are not limited by length. Polypeptides, including antibodies and antibody chains and other polypeptides, such as linkers, may include amino acid residues, including natural and/or unnatural amino acid residues. These terms also include post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In certain aspects, a polypeptide may contain modifications relative to the native or native sequence as long as the protein retains the desired activity. These modifications may be intentional, such as by site-directed mutagenesis, or accidental, such as by host mutation to produce the protein or due to PCR amplification errors.
如本文所述，使用“百分比(％)氨基酸序列身份”和“百分比身份”和“身份”序列使用时对一个氨基酸序列(参考多肽序列)被定义为氨基酸残基的比例在候选序列(例如，蛋白质或碎片)一致，参考多肽的氨基酸残基序列，序列和调整后引入的差距，如果有必要，实现最大百分比序列一致性，并且不考虑任何保守替换作为序列一致性的一部分。以确定氨基酸序列身份百分比为目的的比对可以通过各种技术手段实现，例如使用公开的计算机软件，如BLAST、BLAST-2、ALIGN或Megalign(DNASTAR)软件。这些技术人员可以确定序列对齐的适当参数，包括在被比较序列的整个长度上实现最大对齐所需的任何算法。As described herein, the use of "percent (%) amino acid sequence identity" and "percent identity" and "identity" when used with a sequence of an amino acid sequence (a reference polypeptide sequence) is defined as the ratio of amino acid residues in a candidate sequence (e.g., protein or fragment) identical to the amino acid residue sequence of the reference polypeptide, the sequence and the gaps introduced after adjustment, if necessary, to achieve the maximum percent sequence identity, and disregard any conservative substitutions as part of the sequence identity. Alignment for the purpose of determining percent amino acid sequence identity can be accomplished by various technical means, eg, using publicly available computer software, such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. These skilled artisans can determine appropriate parameters for sequence alignment, including any algorithms needed to achieve maximal alignment over the entire length of the sequences being compared.
经试验，本申请的试剂盒与下列阳性样品不发生交叉反应：副流感病毒抗体、甲型流感病毒抗体、乙型流感病毒抗体、肺炎衣原体抗体、肺炎支原体抗体、腺病毒抗体、RSV抗体、乙型肝炎表面抗体、丙型肝炎抗体、tp抗体、HIV抗体、EB病毒抗体、麻疹病毒抗体、CMV抗体、肠病毒71抗体、流行性腮腺炎病毒抗体、水痘-带状疱疹病毒抗体。盐酸组胺、α-干扰素、扎那米韦、利巴韦林、奥司他韦、帕拉米韦、洛匹那韦、利托那韦、阿比度、左氧氟沙星、阿齐霉素、头孢曲松、美罗培南、妥布霉素等不影响本品检测结果。After testing, the kit of this application does not cross-react with the following positive samples: parainfluenza virus antibody, influenza A virus antibody, influenza B virus antibody, Chlamydia pneumoniae antibody, Mycoplasma pneumoniae antibody, adenovirus antibody, RSV antibody, B Hepatitis surface antibody, hepatitis C antibody, tp antibody, HIV antibody, EB virus antibody, measles virus antibody, CMV antibody, enterovirus 71 antibody, mumps virus antibody, varicella-zoster virus antibody. Histamine hydrochloride, alpha-interferon, zanamivir, ribavirin, oseltamivir, peramivir, lopinavir, ritonavir, azithromycin, levofloxacin, azithromycin, Ceftriaxone, meropenem, tobramycin, etc. do not affect the test results of this product.
与现有技术相比，本发明采用非侵入性的取样方式采样，方便、快速而可靠地检测受试者是否感染了新型冠状病毒SARS-CoV-2，无创取样不引起任何疼痛，样本来源无创即杜绝了二次感染的可能性，降低了病毒的传播风险；检测快速且结果可视，10分钟内即可快速得出结果；检测结果可以避免钩效应或前区效应，检测结果准确率高，在早期的临床检验中，晨尿阴性检出率达91％以上；操作方法简单，无需任何设备或装置辅助，适用于没有受过医疗或其他训练的工作人员，检测试剂盒使用成本低，使用的环境和场所不限，具有极强的推广意义，更有利于高危人群的监测。本发明还可防止无症状个体传播传染源，是节约医疗人力物力消耗和消耗的重要检测手段。Compared with the prior art, the present invention adopts a non-invasive sampling method for sampling, which can conveniently, quickly and reliably detect whether the subject is infected with the new coronavirus SARS-CoV-2, the non-invasive sampling does not cause any pain, and the source of the sample is non-invasive. That is, the possibility of secondary infection is eliminated, and the risk of virus transmission is reduced; the detection is fast and the results are visible, and the results can be quickly obtained within 10 minutes; the detection results can avoid the hook effect or the front zone effect, and the detection result has a high accuracy rate , In the early clinical test, the negative detection rate of morning urine was more than 91%; the operation method is simple, without any equipment or device assistance, suitable for staff who have not received medical or other training, the use cost of the test kit is low, and the use of There are no restrictions on the environment and place, which has strong promotion significance and is more conducive to the monitoring of high-risk groups. The invention can also prevent asymptomatic individuals from spreading the source of infection, and is an important detection means for saving medical manpower and material resources and consumption.
附图说明Description of drawings
图1为本发明试剂盒的生产工艺框图；Fig. 1 is the production process block diagram of the kit of the present invention;
图2为本发明的试剂盒在取样时和取样后的操作示意图；2 is a schematic diagram of the operation of the kit of the present invention during and after sampling;
图3为本发明的试剂盒预计的检测结果示意图。FIG. 3 is a schematic diagram of the expected detection result of the kit of the present invention.
具体实施方式Detailed ways
下面结合实施例对本发明提供的用于检测新型冠状病毒抗体的无创取样检测试剂盒及其检测方法作进一步详细、完整地说明。下面描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。The non-invasive sampling detection kit for detecting novel coronavirus antibodies and the detection method thereof provided by the present invention will be further described in detail and completely below with reference to the examples. The embodiments described below are exemplary, only for explaining the present invention, and should not be construed as limiting the present invention.
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的实验 材料如无特殊说明，均为市场购买得到。The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from the market unless otherwise specified.
尿液中的抗体多为分泌型抗体，以IgA为主。因此，为了提高检测的灵敏度，本发明的检测试剂盒采用了双抗原夹心捕获法检测样品中的抗体。偶联金标记物含有新型冠状病毒的重组抗原，试纸条的膜含有(重组抗原和鼠抗人IgA抗体)。当被测尿液样本有相应的抗体时，带有抗体的区域就会与金标记物偶联物上的抗原结合。当金标记共轭-抗体色谱仪测试区域的测试条，一方面其余可以绑定到抗原抗体领域，另一方面，膜上的抗体可被辨认出来。在使用上述两种机制时，金色标记将固定在相应的测试区域，该区域显示红色指示器。反之，如果样品中不含抗体，或含有抗体密度低于试剂检测下限，则金色指示剂将不固定在检测区域，该区域将不显示红色。金标记物偶联物上的混合物包括含有兔IgG金颗粒的标记物，试纸条控制区(C线)含有羊抗兔抗体。无论检测区域是否显示颜色，均通过兔IgG金颗粒的固定标记显示颜色。如果此区域不显示颜色，则表明测试无效。Most of the antibodies in urine are secretory antibodies, mainly IgA. Therefore, in order to improve the detection sensitivity, the detection kit of the present invention adopts the double-antigen sandwich capture method to detect the antibody in the sample. The conjugated gold label contains the recombinant antigen of the new coronavirus, and the membrane of the test strip contains (recombinant antigen and mouse anti-human IgA antibody). When the urine sample to be tested has the corresponding antibody, the antibody-bearing region binds to the antigen on the gold-labeled conjugate. When gold-labeled conjugated-antibody test strips in the test area of an antibody chromatograph, on the one hand the rest can be bound to the antigen-antibody field, and on the other hand, the antibodies on the membrane can be identified. When using the above two mechanisms, the gold marker will be fixed on the corresponding test area, which shows a red indicator. Conversely, if the sample does not contain antibodies, or contains antibodies at a density below the detection limit of the reagent, the gold indicator will not be immobilized in the detection area and the area will not display red. The mixture on the gold-labeled conjugate includes the label containing rabbit IgG gold particles, and the control region (C line) of the test strip contains goat anti-rabbit antibody. Regardless of whether the detection area shows color, the color is shown by the immobilized labeling of the rabbit IgG gold particles. If no color appears in this area, the test is invalid.
将上述两种方法结合使用，可提高抗体检测的敏感性。在开发尿液样品的准确检测方面，这项研究和开发导致了样品垫的发明，将样品放置在垫上并适当调整到反应***后，样品可以产生非常好的检测效果。The combination of the above two methods can improve the sensitivity of antibody detection. In terms of developing accurate detection of urine samples, this research and development led to the invention of the sample pad, which, when placed on the pad and properly adjusted to the reaction system, can produce very good detection results.
本实施例利用免疫层析原理，使用捕获法检测人尿液样本中新型冠状病毒SARS-CoV-2(也称2019-nCoV)抗体的存在。测试时，当样本中包含的SARS-CoV-2抗体密度大于等于检测下限时，抗体和抗原标记物耦连结合，并在检测区(T区)与二抗(抗μ链抗体/抗人IgG抗体)再次耦连，行成一个红色的反应条带，此时会判定结果为阳性，如无条带产生，则判定为阴性。在正常情况下，质控区域(C区)将始终变为彩色，以表明测试有效。质控区(C区)没有红线时，则表明检测无效。This example uses the principle of immunochromatography to detect the presence of antibodies to the novel coronavirus SARS-CoV-2 (also known as 2019-nCoV) in human urine samples using a capture method. During the test, when the density of the SARS-CoV-2 antibody contained in the sample is greater than or equal to the detection limit, the antibody and the antigen label are coupled and bound, and the secondary antibody (anti-μ chain antibody/anti-human IgG) is combined in the detection zone (T zone). Antibody) is coupled again to form a red reaction band, at this time the result will be judged as positive, if no band is produced, it will be judged as negative. Under normal circumstances, the quality control area (area C) will always be colored to indicate that the test is valid. When there is no red line in the quality control area (C area), it indicates that the detection is invalid.
本发明测试纸的检测卡包括在溶液中处理并烘干的玻璃纤维、聚酯薄膜或无纺布。The test card of the test paper of the present invention includes glass fiber, polyester film or non-woven fabric treated in solution and dried.
本实施例的试剂盒包括检测卡、干燥剂和尿杯，检测卡，检测卡由硝化纤维素膜(NC膜)、样品垫、胶体金连接垫、吸收膜、PVC背板组成，硝化纤维素膜包括抗μ链体/抗人IgG抗体、抗兔多克隆抗体，连接垫包括2019-nCoV重组抗原、兔IgG抗体。样品垫、胶体金连接垫、硝化纤维素膜和吸收膜均吸附在一支撑垫上，支撑垫由玻璃纤维制成。The kit of this embodiment includes a test card, a desiccant and a urine cup. The test card is composed of a nitrocellulose membrane (NC membrane), a sample pad, a colloidal gold connection pad, an absorbing film, and a PVC backing plate. The membrane includes anti-μchain/anti-human IgG antibody, anti-rabbit polyclonal antibody, and the junction pad includes 2019-nCoV recombinant antigen, rabbit IgG antibody. The sample pad, colloidal gold connection pad, nitrocellulose membrane and absorption membrane are all adsorbed on a support pad made of glass fiber.
1、检测卡的处理过程如下：1. The processing process of the test card is as follows:
试剂部分：Reagent part:
1)缓冲体系：用一至几种缓冲液混合为0.01～5M的复合缓冲体系，包括但不限于，Tris-盐酸、硼酸-硼砂、磷酸盐缓冲液、hepd缓冲体系；1) Buffer system: Mix one to several buffers into a composite buffer system of 0.01-5M, including but not limited to Tris-hydrochloric acid, boric acid-borax, phosphate buffer, and hepd buffer system;
2)大分子物质：0.1～3％的一至几种大分子物质，包括牛血清蛋白、酪蛋白、聚乙二醇、明胶；2) Macromolecular substances: 0.1-3% of one to several macromolecular substances, including bovine serum albumin, casein, polyethylene glycol, and gelatin;
3)盐含量：0.1％-5％氯化钠/氯化镁；3) Salt content: 0.1%-5% sodium chloride/magnesium chloride;
4)胶体保护成分：0.1％-5％，PVP；4) Colloidal protective ingredients: 0.1%-5%, PVP;
5)防腐剂：0.01％-1％，叠氮化钠，peoclin；5) Preservatives: 0.01%-1%, sodium azide, peoclin;
6)超纯水制备；6) Preparation of ultrapure water;
将玻璃纤维浸泡在溶液中后，放入25-50度的烘箱烘干后使用。After soaking the glass fiber in the solution, put it into an oven at 25-50 degrees to dry it before use.
制造工艺：Manufacturing process:
检测卡的制备工艺要求如下表1所示：The preparation process requirements of the test card are shown in Table 1 below:
表1Table 1
Figure PCTCN2020142092-appb-000001
Figure PCTCN2020142092-appb-000001
Figure PCTCN2020142092-appb-000002
Figure PCTCN2020142092-appb-000002
2、反应体系2. Reaction system
1)样品采集和处理1) Sample collection and processing
早晨收集首次排尿后的尿液，无需加工，可直接测试。Urine is collected in the morning after the first urination and can be tested directly without processing.
2)样品要求2) Sample requirements
女性：采样前使用香皂或含0.1％酒精的液体冲洗消毒外阴，用手指打开***，连续排尿并收集中段尿液，收集10～20mL尿液储存在无菌容器内。Female: Before sampling, use soap or liquid containing 0.1% alcohol to wash and disinfect the vulva, open the labia with fingers, continuously urinate and collect urine from the middle section, collect 10-20mL of urine and store it in a sterile container.
男性：采样前使用香皂或0.05％～0.1％的聚维酮碘(碘)溶液清洗消毒尿道口，擦拭干净后，拨开***，连续排尿并收集中段尿液，收集10～20mL尿液储存在无菌容器内。Male: Use soap or 0.05%-0.1% povidone-iodine (iodine) solution to clean and disinfect the urethral opening before sampling. After wiping it clean, remove the foreskin, urinate continuously and collect mid-section urine. Collect 10-20 mL of urine and store it in the in a sterile container.
样品采集后2小时内应进行检测。如果不能立即检测，可在2℃-8℃下保存2天；2天后，须在-20℃下保存最多7天。测试前，样品必须恢复室温，避免反复冻结和解冻。不建议使用热灭活样品。Testing should be performed within 2 hours of sample collection. If it cannot be detected immediately, it can be stored at 2°C-8°C for 2 days; after 2 days, it must be stored at -20°C for up to 7 days. Before testing, samples must be brought to room temperature to avoid repeated freezing and thawing. Heat inactivated samples are not recommended.
3)样本要求量3) Sample requirements
将检测卡直接***尿液样品中，确保样本量充足。Insert the test card directly into the urine sample to ensure sufficient sample volume.
3、制造流程3. Manufacturing process
如图1所示，本实施例的检测卡按照图1的工艺流程图在十万级洁净区域内进行，其中带★标记为工作过程的关键控制点。As shown in FIG. 1 , the detection card of this embodiment is carried out in a clean area of class 100,000 according to the process flow chart of FIG. 1 , wherein the marks marked with ★ are the key control points of the working process.
4、使用方法4. How to use
测试前，用于试验的样品、试验试剂和有关试验材料必须平衡到室温，试验必须在室温条件下进行。具体的检测步骤如下：Before the test, the samples, test reagents and related test materials used for the test must be equilibrated to room temperature, and the test must be carried out at room temperature. The specific detection steps are as follows:
1)将锡箔袋沿边缘打开，取出试纸条；1) Open the foil bag along the edge and take out the test strip;
2)使用尿杯收集尿液，或将冰冻的尿液样本恢复到室温，然后将其放入尿杯中，并为其分配编号；2) Use a urine cup to collect urine, or bring the frozen urine sample to room temperature, then put it in the urine cup and assign a number to it;
3)握住试纸蓝色的一端，将带MAX线的一侧***尿中。当***测试棒时，尿液表面不能超过最大值线；3) Hold the blue end of the test strip and insert the side with the MAX line into the urine. When the test stick is inserted, the urine surface cannot exceed the maximum line;
4)待白色膜区出现红色液体后，取下，取出试纸条，平放于平台上，膜面朝上。不要直接放置在风扇或空调的前面通风；4) After the red liquid appears in the white membrane area, remove it, take out the test strip, and place it flat on the platform with the membrane side up. Do not place it directly in front of a fan or air conditioner for ventilation;
5)10分钟后观察结果。15分钟后发生的结果没有临床意义见图2。当红色液体出现时，可将试纸收回，然后平放于桌面上。5) Observe the results after 10 minutes. Results that occurred after 15 minutes were not clinically meaningful as shown in Figure 2. When the red liquid appears, the test strip can be retracted and placed flat on the table.
5、结果说明5. Result description
如图3所示，当质控区(C)没有红线时，测试无效。建议拿新试纸重新检测，检测时注意试纸***尿样时需要足够深入且不超过max线。当检测区(T)和质控区(C)具有两条红色线时，说明为阳性。当仅在质控区(C)有一条红色线时，说明为阴性。其他情况均为异常，为无效测试，建议重新检测。As shown in Figure 3, when there is no red line in the quality control area (C), the test is invalid. It is recommended to take a new test strip for re-testing. When testing, note that the test strip needs to be inserted deep enough into the urine sample and not exceed the max line. When the detection area (T) and the quality control area (C) have two red lines, it is positive. When there is only one red line in the quality control area (C), it is negative. Other cases are abnormal, which is an invalid test, and it is recommended to re-test.
6、试剂盒性能测试6. Kit performance test
1)正向参考产品率：行业正向参考产品率必须为5/5；1) Positive reference product rate: The industry positive reference product rate must be 5/5;
2)负参考品率：行业负参考品率必须为10/10；2) Negative reference product ratio: The industry negative reference product ratio must be 10/10;
3)检测下限：行业检测下限参考产品S1必须为阴性，S2和S3必须为阳性；3) Lower detection limit: the reference product S1 of the industry detection lower limit must be negative, and S2 and S3 must be positive;
4)可重复性：使用2个行业可重复性对照品进行检测，每次检测10次，均为阳性；4) Repeatability: use 2 industry repeatability reference substances for detection, each test is 10 times, all are positive;
5)钩状效应：在新型冠状病毒临床阳性样本滴度范围内，本品检测结果未出现钩状效应；5) Hook effect: within the titer range of the new coronavirus clinical positive sample, the test results of this product did not show hook effect;
6)本发明的试剂盒检测结果不受破坏新型冠状病毒特异性IgM抗体的影响。6) The detection result of the kit of the present invention is not affected by the destruction of the novel coronavirus-specific IgM antibody.
在早期的临床检验中，晨尿阴性检出率达91％以上。In the early clinical examination, the negative detection rate of morning urine was over 91%.
最后有必要在此说明的是：以上实施例只用于对本发明的技术方案作进一步详细地说明，不能理解为对本发明保护范围的限制，本领域的技术人员根据本发明的上述内容作出的一些非本质的改进和调整均属于本发明的保护范围。Finally, it is necessary to explain here: the above embodiments are only used to further describe the technical solutions of the present invention in detail, and should not be construed as limiting the protection scope of the present invention. Non-essential improvements and adjustments belong to the protection scope of the present invention.
Figure PCTCN2020142092-appb-000003
Figure PCTCN2020142092-appb-000003
Figure PCTCN2020142092-appb-000004
Figure PCTCN2020142092-appb-000004
Figure PCTCN2020142092-appb-000005
Figure PCTCN2020142092-appb-000005
Figure PCTCN2020142092-appb-000006
Figure PCTCN2020142092-appb-000006
Figure PCTCN2020142092-appb-000007
Figure PCTCN2020142092-appb-000007
Figure PCTCN2020142092-appb-000008
Figure PCTCN2020142092-appb-000008
Figure PCTCN2020142092-appb-000009
Figure PCTCN2020142092-appb-000009
Figure PCTCN2020142092-appb-000010
Figure PCTCN2020142092-appb-000010
Figure PCTCN2020142092-appb-000011
Figure PCTCN2020142092-appb-000011
Figure PCTCN2020142092-appb-000012
Figure PCTCN2020142092-appb-000012
Figure PCTCN2020142092-appb-000013
Figure PCTCN2020142092-appb-000013
Figure PCTCN2020142092-appb-000014
Figure PCTCN2020142092-appb-000014
Figure PCTCN2020142092-appb-000015
Figure PCTCN2020142092-appb-000015

Claims (10)

  1. 一种用于检测新型冠状病毒抗体的无创取样检测试剂盒，其特征在于：所述检测试剂盒通过检测尿液中是否含有新型冠状病毒IgA抗体以快速判断受试者是否感染了新型冠状病毒，所述试剂盒包括检测卡，检测卡包括硝化纤维素膜(NC膜)、样品垫、胶体金连接垫、吸收膜和PVC背板，检测卡内各结构按照待测样品的通过顺序依次为：样品垫、胶体金连接垫、硝化纤维素膜和吸收膜，其中硝化纤维素膜包括μ链体/抗人IgG抗体、抗兔多克隆抗体，连接垫包括2019-nCoV重组抗原、兔IgG抗体，所述重组抗原为胶体金颗粒连接的胶体金抗体。A non-invasive sampling detection kit for detecting novel coronavirus antibodies, characterized in that: the detection kit can quickly determine whether a subject is infected with the novel coronavirus by detecting whether urine contains novel coronavirus IgA antibodies, The kit includes a detection card, the detection card includes a nitrocellulose membrane (NC membrane), a sample pad, a colloidal gold connection pad, an absorption film and a PVC backboard, and the structures in the detection card are in the order of passing the samples to be tested: Sample pad, colloidal gold connection pad, nitrocellulose membrane and absorption membrane, wherein the nitrocellulose membrane includes μ-chain/anti-human IgG antibody, anti-rabbit polyclonal antibody, and the connection pad includes 2019-nCoV recombinant antigen, rabbit IgG antibody, The recombinant antigen is a colloidal gold antibody linked to colloidal gold particles.
  2. 根据权利要求1所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒，其特征在于，所述重组抗原与IgA抗体至少配对90％。The non-invasive sampling detection kit for detecting novel coronavirus antibodies according to claim 1, wherein the recombinant antigen and IgA antibody are at least 90% paired.
  3. 根据权利要求1所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒，其特征在于，所述重组抗原与IgA抗体至少配对95％。The non-invasive sampling detection kit for detecting novel coronavirus antibodies according to claim 1, wherein the recombinant antigen and IgA antibody are at least 95% paired.
  4. 根据权利要求1所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒，其特征在于，所述所述序列如序列表SEQ ID NO:1所示。The non-invasive sampling detection kit for detecting a novel coronavirus antibody according to claim 1, wherein the sequence is shown in SEQ ID NO: 1 in the sequence listing.
  5. 根据权利要求1所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒，其特征在于，所述重组抗原还包括如序列表SEQ ID NO:2～10所示的序列，或包括上述序列的至少500个连续的氨基酸序列。The non-invasive sampling detection kit for detecting novel coronavirus antibodies according to claim 1, wherein the recombinant antigen further comprises the sequences shown in SEQ ID NOs: 2-10 in the Sequence Listing, or comprises the above-mentioned sequences of at least 500 contiguous amino acid sequences.
  6. 根据权利要求1所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒，其特征在于，所述检测卡上设有加样孔，待测样品由加样孔深入样品垫。The non-invasive sampling detection kit for detecting novel coronavirus antibodies according to claim 1, wherein the detection card is provided with a sample addition hole, and the sample to be tested penetrates into the sample pad through the sample addition hole.
  7. 根据权利要求1所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒，其特征在于，所述检测卡中的样品垫、胶体金连接垫、硝化纤维素膜和吸收膜均吸附在一支撑垫上。The non-invasive sampling detection kit for detecting novel coronavirus antibodies according to claim 1, wherein the sample pad, colloidal gold connection pad, nitrocellulose membrane and absorption membrane in the detection card are all adsorbed on one on the support pad.
  8. 一种采用权利要求1～7任一项所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒的检测方法，其特征在于，所述检测方法包括如下步骤：A detection method using the non-invasive sampling detection kit for detecting novel coronavirus antibodies according to any one of claims 1 to 7, wherein the detection method comprises the following steps:
    a)将待测样本置于样品垫的加样孔内，使待测样本由样品垫向胶体金连接垫流动；a) Place the sample to be tested in the sample filling hole of the sample pad, so that the sample to be tested flows from the sample pad to the colloidal gold connection pad;
    b)待测样本通过毛细管作用依次流过检测区和质控区，当待测序列相似度大于90％以上时，待测抗体可被重组抗原捕获，形成红色标记区域；b) The sample to be tested flows through the detection area and the quality control area in turn by capillary action. When the similarity of the sequence to be tested is greater than 90%, the antibody to be tested can be captured by the recombinant antigen to form a red marked area;
    c)肉眼观测检测区和质控区是否出现红色标记区域，以确定待测样本来源是 否感染新型冠状病毒。c) Visually observe whether the red marked area appears in the detection area and the quality control area to determine whether the source of the sample to be tested is infected with the new coronavirus.
  9. 根据权利要求8所述的新型冠状病毒的无创取样检测方法，其特征在于，所述检测样本为不经过二次加工处理的人体尿液。The method for non-invasive sampling and detection of novel coronavirus according to claim 8, wherein the detection sample is human urine that has not undergone secondary processing.
  10. 一种根据权利要求1～7任一项所述的用于检测新型冠状病毒抗体的无创取样检测试剂盒的应用，其特征在于，所述检测试剂盒用于快速检测新型冠状病毒的无创样品。An application of the non-invasive sampling detection kit for detecting novel coronavirus antibodies according to any one of claims 1 to 7, characterized in that the detection kit is used for rapid detection of non-invasive samples of novel coronavirus.
PCT/CN2020/142092 2020-12-31 2020-12-31 Non-invasive sampling test kit for testing sars-cov-2 antibody, and test method therefor WO2022141394A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
PCT/CN2020/142092 WO2022141394A1 (en) 2020-12-31 2020-12-31 Non-invasive sampling test kit for testing sars-cov-2 antibody, and test method therefor
CN202080107265.5A CN116670509A (en) 2020-12-31 2020-12-31 Noninvasive sampling detection kit for detecting novel coronavirus antibody and detection method thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2020/142092 WO2022141394A1 (en) 2020-12-31 2020-12-31 Non-invasive sampling test kit for testing sars-cov-2 antibody, and test method therefor

Publications (1)

Publication Number Publication Date
WO2022141394A1 true WO2022141394A1 (en) 2022-07-07

Family

ID=82258880

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/142092 WO2022141394A1 (en) 2020-12-31 2020-12-31 Non-invasive sampling test kit for testing sars-cov-2 antibody, and test method therefor

Country Status (2)

Country Link
CN (1) CN116670509A (en)
WO (1) WO2022141394A1 (en)

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111366734A (en) * 2020-03-20 2020-07-03 广州市康润生物科技有限公司 Method for screening new coronavirus through double indexes and predicting severe pneumonia
CN111505285A (en) * 2020-04-27 2020-08-07 东莞博识生物科技有限公司 SARS-CoV-2 detecting chip and its application
CN111690060A (en) * 2020-07-06 2020-09-22 深圳市亚辉龙生物科技股份有限公司 IgA antibody capable of specifically recognizing RBD protein and kit
CN211905393U (en) * 2020-09-22 2020-11-10 正元盛邦(天津)生物科技有限公司 Novel coronavirus specific antibody and neutralizing antibody combined detection test paper and device
US10844442B1 (en) * 2020-05-18 2020-11-24 Bret T. Barnhizer Rapid viral assay
CN112034179A (en) * 2020-08-10 2020-12-04 华中科技大学 Fluorescent reagent for detecting new coronavirus antibody and preparation method thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111366734A (en) * 2020-03-20 2020-07-03 广州市康润生物科技有限公司 Method for screening new coronavirus through double indexes and predicting severe pneumonia
CN111505285A (en) * 2020-04-27 2020-08-07 东莞博识生物科技有限公司 SARS-CoV-2 detecting chip and its application
US10844442B1 (en) * 2020-05-18 2020-11-24 Bret T. Barnhizer Rapid viral assay
CN111690060A (en) * 2020-07-06 2020-09-22 深圳市亚辉龙生物科技股份有限公司 IgA antibody capable of specifically recognizing RBD protein and kit
CN112034179A (en) * 2020-08-10 2020-12-04 华中科技大学 Fluorescent reagent for detecting new coronavirus antibody and preparation method thereof
CN211905393U (en) * 2020-09-22 2020-11-10 正元盛邦(天津)生物科技有限公司 Novel coronavirus specific antibody and neutralizing antibody combined detection test paper and device

Also Published As

Publication number Publication date
CN116670509A (en) 2023-08-29

Similar Documents

Publication Publication Date Title
ES2298244T5 (en) Method for the detection of influenza A / B viruses in saliva
CN111912980A (en) Rapid combined detection device for novel coronavirus antigen and antibody in saliva and preparation method thereof
CN211148669U (en) S-antigen-based novel coronavirus 2019-nCoV antibody rapid detection kit
CN103792373A (en) Colloidal gold immunochromatography test paper for antibodies of pseudorabies viruses of gE, gB and gD and preparation method
US11789020B2 (en) Neutralizing antibody testing and treatment
Vrublevskaya et al. A sensitive and specific lateral flow assay for rapid detection of antibodies against glycoprotein B of Aujeszky's disease virus
US20220244258A1 (en) Assay For Neutralizing Antibody Testing And Treatment
CN115629212A (en) Monkey pox virus antigen detection reagent, kit and preparation method
US11668710B2 (en) Assays for the detection of a blocking analyte
WO2022141394A1 (en) Non-invasive sampling test kit for testing sars-cov-2 antibody, and test method therefor
CN101738474A (en) Combined test reagent card for cytomegalovirus and rubella virus
WO2023087550A1 (en) Kit for detecting sars-cov-2 antigen and detection method
LU101824B1 (en) Novel coronavirus testing
CN109633162A (en) S-100 β protein detection kit
Shao Accurate interpretation of SARS-CoV-2 antigen detection by immunochromatography
CN103149356B (en) A kind of Test paper card utilizing sandwich method to detect Brucella abortus antigen
CN105769212A (en) Condom
WO2003023403A1 (en) A KIT FOR THE SIMULTANEOUS DIAGNOSIS OF FOUR KINDS OF DISEASES (ToRCH) RESULTING IN DEFORMITY FETUS AND A METHOD FOR PREPARATION OF IT
US20240142446A1 (en) Test kits, devices and methods for detecting hiv infection
US20220205998A1 (en) Assay for neutralizing antibody testing and treatment
CN211426520U (en) Combined measles and rubella detection card
US20230152315A1 (en) Kit for testing covid-19 antigen and method for testing same
US20220252588A1 (en) Neutralizing antibody testing and treatment
WO2022236891A1 (en) Non-invasive detection kit for detecting novel coronavirus antigen, and detection method thereof
CN113252907A (en) New crown detection kit

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20967733

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 202080107265.5

Country of ref document: CN

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20967733

Country of ref document: EP

Kind code of ref document: A1