WO2022215641A1 - Method for detecting virus-neutralizing antibodies - Google Patents

Method for detecting virus-neutralizing antibodies Download PDF

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WO2022215641A1
WO2022215641A1 PCT/JP2022/016414 JP2022016414W WO2022215641A1 WO 2022215641 A1 WO2022215641 A1 WO 2022215641A1 JP 2022016414 W JP2022016414 W JP 2022016414W WO 2022215641 A1 WO2022215641 A1 WO 2022215641A1
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virus
blood
filter paper
pseudotype
neutralizing antibodies
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French (fr)
Japanese (ja)
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芳智 森永
善裕 山本
仁史 川筋
英樹 谷
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国立大学法人富山大学
富山県
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/66—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving luciferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters

Definitions

  • the present invention relates to a method for testing virus neutralizing antibodies, and more particularly to a method for testing virus neutralizing antibodies, including SARS-CoV-2, in blood.
  • Antibodies are used as indicators to determine whether a person has acquired immunity against the causative microorganisms of infectious diseases. In order to confirm whether or not a person has acquired immunity against the causative microorganism, which is an antigen, it is not sufficient to simply check for the presence of antibodies. The presence or level (concentration, amount, signal intensity) of neutralizing antibodies should be examined.
  • BSL2 Biosafety Level 2
  • the present inventors prepared a pseudotype virus for SARS-CoV-2, which is known as the causative virus of the new coronavirus infection (COVID-19), and used not only normal serum but also whole blood containing blood cell components. as a patient sample, it has been confirmed that neutralizing antibodies can be evaluated (Chemiluminescence reduction neutralization test (CRNT method), Non-Patent Document 1).
  • This neutralizing antibody evaluation method itself is not limited to SARS-CoV-2, but is a technology based on co-cultivation of viruses and cells that can be generally performed even when new threat viruses emerge in the future. is.
  • a conventional method such as that described in Non-Patent Document 1 uses blood drawn from a subject's vein as a measurement sample.
  • Venous blood collection requires a person with blood collection skills, a blood collection site, and a blood collection container. Venous blood collection also places a heavy burden on the patient.
  • An object of the present invention is to provide a method for evaluating virus neutralization activity from a minute amount of dried blood.
  • the present inventor discovered that neutralizing antibodies can be evaluated by measuring a very small amount of blood by using filter paper blood collection, and completed the invention.
  • the present invention includes the embodiments described below.
  • Section 1 A method for evaluating a neutralizing antibody against a virus using dried blood adhered to a filter paper, wherein a blood sample obtained by extracting the dried blood adhered to the filter paper is coated with the coat protein of the virus of interest. and a pseudotype virus having a reporter gene integrated into its own gene to obtain a mixture; a step of infecting the mixture with a cultured cell line susceptible to the pseudotype virus; and detecting the presence or absence of a neutralizing antibody against the pseudotype virus in a blood sample based on the expression level of the reporter gene after the infection. or measuring the amount.
  • Section 2 A pseudotyped virus in a cultured cell line susceptible to the pseudotyped virus, wherein the neutralizing antibody in the blood sample is contacted with the spike protein of the pseudotyped virus in the mixture or a portion thereof that functions as an antigen for the neutralizing antibody. 2.
  • Section 3. The method of paragraph 2, wherein the spike protein is a spike protein derived from SARS-CoV-2.
  • Section 4 A kit for testing neutralizing antibodies of a virus using dried blood attached to a filter paper, comprising a pseudotype virus coated with a coat protein of a target virus and a cultured cell line sensitive to the pseudotype virus.
  • Item 5 The kit according to Item 4, wherein a reporter gene is incorporated into the pseudotype virus gene.
  • a method for evaluating neutralizing antibodies against a virus using dried blood attached to a filter paper comprising: a blood sample obtained by extracting the dried blood attached to the filter paper; A step of mixing a pseudotyped virus coated with a coat protein of a virus of interest and having a reporter gene integrated into its own gene to obtain a mixture, and transforming the pseudotyped virus in the mixture into a pseudotyped virus.
  • a method comprising the steps of infecting a susceptible cultured cell line, and detecting the presence or absence or quantification of neutralizing antibodies against the pseudotype virus in the blood sample based on the expression level of the reporter gene after infection. be.
  • the virus of interest refers to the virus that is the target of the method for evaluating neutralizing antibodies against viruses of this embodiment.
  • methods for measuring virus-neutralizing antibodies include methods for detecting the presence or absence of virus-neutralizing antibodies or determining the amount of virus-neutralizing antibodies.
  • Viruses that can be evaluated with this technique include, but are not limited to, SARS-CoV-2, SARS-CoV, MERS-CoV, and the like.
  • the blood collected from the subject is soaked into the filter paper and dried.
  • a filter paper to which such dried blood is adhered (hereinafter referred to as “dried blood filter paper") is used as a test specimen.
  • the amount of blood collected from the patient which is necessary for the virus neutralizing antibody test method according to this embodiment, is sufficient even at about 1 to 5 ⁇ l, and is dramatically larger than the amount required in the usual method of intravenous blood collection. Few.
  • the subject may be a healthy person, a person suspected of being infected with the virus, or a person already infected with the virus.
  • the subject may have symptoms or symptoms associated with viral infection, or may be suffering from such symptoms or diseases.
  • the filter paper may be any filter paper that can be used to collect blood from the subject.
  • the size of the filter paper can be any size.
  • the size of the filter paper is generally preferred for convenient transport and/or storage of the dried blood filter paper.
  • Dried blood adhering to the filter paper can be extracted from the filter paper by a known method.
  • the extraction temperature is preferably a temperature that suppresses denaturation of components in the blood, for example, 4 to 30°C, preferably about 4 to 25°C, but is not limited thereto.
  • the extraction time is, for example, about 30 minutes to 1 day, but is not limited to this.
  • extraction of dried blood from filter paper can be performed using an extractant for blood extraction.
  • Extraction solutions include, but are not limited to, buffers such as physiological saline, Tris buffer, phosphate buffer, and cell culture media.
  • the culture medium for pseudotype virus-susceptible culture cell lines when used as an extract, it matches the medium in which pseudotype virus-susceptibility culture cell lines were cultured, so the nutrients in the medium due to dilution of the medium Fluctuations are less likely to occur, and it is suitable for stabilizing medium conditions.
  • the extract may also contain non-human serum, inorganic salts, surfactants, etc. for the purpose of stabilizing components in blood.
  • a pseudotype virus artificially produced from plasmid DNA is used in order to reduce the risk of virus infection.
  • Pseudotyped viruses refer to viruses that are enveloped with the desired viral coat proteins (eg, envelope proteins, spike proteins, or portions thereof that serve as antigens for neutralizing antibodies of the virus).
  • VSV Vesicular stomatitis virus
  • retroviruses retroviruses
  • lentiviruses and the like are known as the original underlying viruses, and these viruses can also be used herein.
  • a VSV vector capable of producing a pseudotype virus has a reporter gene integrated into its own gene, and when a target cell is infected, the infectivity can be evaluated by the expression of the reporter gene. Reporter genes include luciferase, fluorescent proteins, colored proteins and the like.
  • luciferases examples include luciferases derived from various luminous organisms such as sea worms, black lobsters, luminous insects (fireflies, click beetles, etc.), luminous earthworms, lachias, sea pansy, and Aequorin jellyfish (aequorin).
  • Fluorescent proteins include genetically modified fluorescent proteins, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), DsRED, and red fluorescent protein. (RFP) and the like are exemplified. Colored proteins include phycocyanin and phycoerythrin. Among these, the luciferase gene is preferable because of its high signal detection sensitivity and high quantification.
  • Pseudotype viruses even virus species that must be handled at BSL3 or BSL4 (BSL: biosafety level), are produced using only the envelope protein, so they can be prepared at BSL2, which is a general infection experiment environment. It is advantageous in that it can be handled. For example, if you want to examine the presence or absence of neutralizing antibodies against the SARS-CoV-2 virus in the dried blood of a subject, use the spike protein of the SARS-CoV-2 virus or a modified spike protein lacking its C-terminal amino acid. The expressed VSV pseudotype virus can be used to specifically detect or determine the presence or absence of neutralizing antibodies against the SARS-CoV-2 virus in the dried blood of a subject.
  • a blood sample obtained by extracting dried blood attached to a filter paper is mixed with a pseudotype virus coated with the coat protein of the virus of interest, if the blood sample contains neutralizing antibodies against the virus, The neutralizing antibody in the blood sample is contacted with the pseudotyped virus coat protein in the resulting mixture or a portion thereof that functions as an antigen for the neutralizing antibody to pseudotype a cultured cell line susceptible to the pseudotyped virus. It becomes a state that inhibits infection by type virus.
  • the time for reacting the blood sample with the pseudotype virus coated with the envelope protein of the virus of interest is usually preferably 1 hour or longer.
  • the reaction temperature is usually preferably around 37°C.
  • the pseudotyped virus treated under the above reaction conditions is used to infect a pseudotyped virus-susceptible cultured cell line.
  • Cultured cell lines sensitive to pseudotype viruses can be selected as appropriate.
  • cultured cell lines highly susceptible to SARS-CoV-2 pseudotype viruses include VeroE6/TMPRSS2 cells.
  • the pseudotype virus in the mixture When used to infect a pseudotype virus-susceptible cultured cell line, it is allowed to adsorb for at least 1 hour at 37°C, then the culture medium is added and incubated at 37°C for 24 hours. It is preferable to culture for a long time.
  • the expression level of the reporter gene in the pseudotype virus can be calculated by measuring the luminescence level of the reporter gene using a known analytical instrument.
  • the reaction with the neutralizing antibody results in a state that inhibits infection with the pseudotype virus.
  • the infectious dose of pseudotyped viruses that can infect cultured cell lines susceptible to HIV is reduced.
  • the infection rate of a cultured cell line infected with a pseudotyped virus can be assessed by measuring the expression level of the reporter gene compared to a control (e.g., when the blood sample does not contain neutralizing antibody to the virus).
  • a commercially available kit can be used to measure the expression level of the reporter gene.
  • reporter gene when the reporter gene is luciferase, relative luminescence of luciferase is measured using commercially available kits such as PicaGene Luminescence Kit (TOYO B-Net Co., LTD, Tokyo, Japan) and GloMax Navigator System G2000 (Promega Corporation, Madison, WI). amount (RLU) can be measured.
  • the expression level of the reporter gene can be used as an indicator of the amount of pseudotype virus infection.
  • a virus-neutralizing antibody is tested using dried blood from a certain subject attached to filter paper.
  • the expression level of the reporter gene measured by the method of this embodiment is lower than the expression level of the reporter gene when a blood sample containing no virus-neutralizing antibody is used, the blood of such a subject has medium It can be determined that sum antibodies are present.
  • the expression level of the reporter gene measured by the method of this embodiment is the same as or higher than the expression level of the reporter gene when a blood sample containing no virus-neutralizing antibody is used, the subject It can be determined that the blood is free of neutralizing antibodies.
  • the virus-neutralizing antibody evaluation method of the present embodiment can evaluate the virus-neutralizing antibody with a high degree of precision comparable to that of intravenous blood collection as shown in Non-Patent Document 1, even with a very small amount of blood, such as several ⁇ L. be able to.
  • the extracted blood has sufficient activity to assess neutralizing antibodies to the virus.
  • the normal neutralizing antibody evaluation method using a blood sample obtained by intravenous blood collection normal method
  • the conventional method used a 96-well plate, the number of samples that could be processed simultaneously was limited to 44 samples, excluding the wells required for controls, when performing duplicate measurements.
  • the method for evaluating neutralizing antibodies against viruses according to the present embodiment can be performed with a very small amount of blood, so that more specimens can be treated simultaneously.
  • the neutralizing activity of viruses including new viruses such as SARS-CoV-2 or known viruses can be evaluated from a small amount of dried blood. Therefore, the neutralizing antibody evaluation method of this embodiment can be used, for example, to evaluate the preventive effect of vaccines against new viral infections including SARS-CoV-2 or to evaluate the status of collective immunity acquisition. be able to.
  • the neutralizing antibody evaluation method of this embodiment is an in vitro evaluation method that does not include human diagnostic methods.
  • kits for testing neutralizing antibody of a virus using dried blood adhered to filter paper wherein the pseudotype coats the coat protein of the virus of interest.
  • a kit is provided that includes the virus and a cultured cell line to infect the pseudotyped virus.
  • the kit of this aspect is preferably used to carry out the method of evaluating neutralizing antibodies against viruses using the dried blood adhered to the filter paper of the present invention.
  • a reporter gene is integrated into the gene of the pseudotype virus.
  • the dried blood collected from the subject and adhered to filter paper is examined, and the measured reporter gene expression level is higher than that of the control (for example, the blood sample contains virus neutralizing antibody). It can be determined that neutralizing antibodies are present in the blood of such a subject if the expression level of the reporter gene is lower than the expression level of the reporter gene in the absence of the antibody. If the expression level of the reporter gene measured using the kit of this embodiment is the same as or higher than the expression level of the reporter gene in the control, it is determined that there is no neutralizing antibody in the blood of the subject. can do.
  • the control for example, the blood sample contains virus neutralizing antibody
  • the kit of this embodiment further comprises a first container for housing a pseudotype virus coated with a viral coat protein of interest and a second container for housing a cultured cell line susceptible to the pseudotype virus. and may be provided.
  • the kit of this embodiment may further comprise a cell culture medium for culturing a cultured cell line susceptible to pseudotype virus, such culture medium being contained in the second container and good too.
  • the cell culture medium is as described with respect to the method for evaluating neutralizing antibodies against viruses.
  • the kit of this aspect may further comprise a filter paper for adhering blood collected from a subject, and an extraction liquid for extracting the dried blood adhering to the filter paper.
  • the extract is as described in relation to the method for evaluating the neutralizing antibody of the virus.
  • the kit of this embodiment may further comprise an instruction manual describing the procedure for carrying out the method of evaluating the virus-neutralizing activity of the present invention.
  • Example 1 Establishment of a measurement method for virus neutralizing antibodies using filter dried blood
  • blood collection filter paper blood collection filter paper manufactured by ADVANTEC
  • the blood recovery efficiency when immersed in physiological saline was measured by OD 560 , which is the absorbance of hemoglobin, and it was found to be inversely proportional to the amount of solvent. It was confirmed. Assuming that the amount of blood adsorbed on the filter paper was the lowest value of 0.33 ⁇ L/mm 2 , the amount of blood adsorbed on one filter paper and the dilution ratio recovered from the area of the filter paper and the amount of added solvent were calculated (Table 1).
  • a measles antibody kit (DENKA SEIKEN Diluted measles antibody levels were measured using virus antibody EIA "biopsy" measles IgG (manufactured by Co., Ltd.), and the correlation between the measles antibody levels in the diluted serum and the dilution ratio obtained by collecting blood on filter paper was evaluated.
  • a 96-well plate is used, and the cell lines cultured in it are infected with live virus.
  • the volume required per well is 1/2 of the volume required in the conventional method, with the medium containing cultured cells. It was confirmed that it was possible to measure 1/4 in blood samples and 1/9 in fluids containing pseudotype viruses.
  • Example 2 Measurement of Neutralizing Antibody Levels Against SARS-CoV-2 in Subjects Using Filter-Dried Blood Neutralizing antibody levels against the novel coronavirus SARS-CoV-2 were compared in blood and venous blood serum (26 cases, 28 specimens). The dried blood on the filter paper was extracted with a 4 mm punch, eluted with 100 ⁇ L of DMEM medium (manufactured by Nacalai Tesque), and the neutralizing antibody measured by the micro method was compared with the neutralizing antibody measured by the conventional method on the serum collected from the vein. .
  • DMEM medium manufactured by Nacalai Tesque
  • VSV vesicular stomatitis virus
  • S protein spike protein
  • SARS-CoV Pseudotype viruses enveloped with amino acid-deficient S protein showed high infectivity (J Gen Virol. 2005;86:2269-74; J Virol. 2009;83:712-21.).
  • a pseudotype virus encapsulating the S protein in which 19 amino acids at the C-terminal of the SARS-CoV-2 S protein were deleted was produced and used in the conventional method and the microvolume method of this example. See Tani et al. Virol J (2021) 18:16 for methods of generating pseudotyped viruses.
  • 293T cells are transfected with plasmid DNA expressing the SARS-CoV-2 S protein truncated for the C-terminal 19 amino acids.
  • the transfected 293T cells transformed into *G-VSV ⁇ G/Luc transiently enveloped with the VSV G protein, in which a reporter gene was integrated in place of its own envelope gene, which is the parental virus. Infect a certain amount. After allowing the virus to adsorb to the cells for 2 hours, uninfected virus is washed out with medium. After culturing for 24 hours, the culture supernatant is collected to obtain a pseudotype virus encapsulating the S protein of SARS-CoV-2. It has been shown that the S protein with 19 amino acid deletions is more enveloped by pseudotyped viruses and has higher infectivity than the intact S protein (Tani et al. Virol J (2021) 18 :16).
  • the serum is diluted with DMEM medium (manufactured by Nacalai Tesque) and mixed with a virus solution containing a pseudotype virus that coats the S protein of SARS-CoV-2 at a ratio of 9:1.
  • DMEM medium manufactured by Nacalai Tesque
  • virus solution containing a pseudotype virus that coats the S protein of SARS-CoV-2 at a ratio of 9:1.
  • filter paper-dried blood was diluted with DMEM medium (manufactured by Nacalai Tesque) to a serum equivalent of 200 times. After mixing, the mixture was allowed to stand at room temperature for 1 hour, and 22.5 ⁇ L of the mixture was added per well to a 384-well plate seeded with VeroE6/TMPRSS2 cells to prepare 2 wells. After 24 hours, 2.5 ⁇ L of luciferase substrate, a luciferase incorporated into the virus, was added, and luminescence was measured using a GloMax plate reader (manufactured by Promega). Calculated as a percentage of the comparison.
  • DMEM medium manufactured by Nacalai Tesque
  • the method of detecting a virus neutralizing antibody using filter paper-dried blood of the present invention reduces the risk of infection of subjects and medical workers, facilitates storage and transportation of collected blood samples, and allows the use of filter paper-dried blood for a long period of time. Because of its stability and ability to process multiple specimens, it can be widely used both in Japan and overseas to investigate the acquisition of neutralizing antibodies in subjects.

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Abstract

Provided is a method for evaluating virus-neutralizing antibodies using dried blood adhering to filter paper, the method including: a step for mixing a blood sample obtained by extracting the dried blood adhering to the filter paper and a pseudotype virus, which coats the coat protein of a target virus and in the genes of which a reporter gene is incorporated, to obtain a mixture; a step for infecting a susceptible cultured cell line with the psuedotype virus in the mixture; and a step for detecting the presence or measuring the amount of pseudotype-virus-neutralizing antibodies in the blood sample on the basis of the expression level of the reporter gene after infection.

Description

ウイルスの中和抗体の検出方法Method for detecting virus neutralizing antibody
　本発明は、ウイルスの中和抗体を検査するための方法に関し、より詳細には、血液中のSARS-CoV-2を含むウイルスの中和抗体を検査するための方法に関するものである。 The present invention relates to a method for testing virus neutralizing antibodies, and more particularly to a method for testing virus neutralizing antibodies, including SARS-CoV-2, in blood.
　感染症の原因微生物に対して免疫を獲得しているかを知るための指標として、抗体が利用される。抗原である原因微生物に対して免疫を獲得しているかどうかを確認するには、単なる抗体の有無を調べるだけでは不十分であり、病原微生物の病原性を中和する機能性を有する抗体である中和抗体の有無又はレベル（濃度、量、シグナル強度）を調べる必要がある。 　Antibodies are used as indicators to determine whether a person has acquired immunity against the causative microorganisms of infectious diseases. In order to confirm whether or not a person has acquired immunity against the causative microorganism, which is an antigen, it is not sufficient to simply check for the presence of antibodies. The presence or level (concentration, amount, signal intensity) of neutralizing antibodies should be examined.
　中和抗体の評価は、生きたウイルスが感受性のある細胞株に感染する現象が血液成分を混ぜることにより阻害されるかを捉えるものである。パンデミックの場合、病原性で未知の部分が多いウイルスは、一般的な感染実験環境であるバイオセーフティーレベル2（BSL2）での取り扱いが許可されないが、シュードタイプウイルスという、外殻は当該ウイルスで内部はBSL2で取り扱い可能な別のウイルスからなるウイルスを作製することで、BSL2環境での取り扱いが可能となる。 Evaluation of neutralizing antibodies captures whether the phenomenon of live virus infection of susceptible cell lines is inhibited by mixing blood components. In the case of a pandemic, viruses with unknown pathogenicity and many unknown parts are not allowed to be handled at Biosafety Level 2 (BSL2), which is a general infection experiment environment, but pseudotype viruses, the outer shell is the virus inside can be handled in a BSL2 environment by creating a virus composed of another virus that can be handled in BSL2.
　本発明者らは、新型コロナウイルス感染症（COVID-19）の原因ウイルスとして知られるSARS-CoV-2について、シュードタイプウイルスを作製し、通常の血清だけでなく、血球成分を含んだ全血を患者試料として用いて、中和抗体を評価可能であることを確認している（Chemiluminescence reduction neutralization test（CRNT法）、非特許文献1）。この中和抗体の評価方法自体は、SARS-CoV-2に限らず、今後新たに脅威となるウイルスが出現した場合でも一般的に行うことができる、ウイルスと細胞の共培養を基本とする技術である。 The present inventors prepared a pseudotype virus for SARS-CoV-2, which is known as the causative virus of the new coronavirus infection (COVID-19), and used not only normal serum but also whole blood containing blood cell components. as a patient sample, it has been confirmed that neutralizing antibodies can be evaluated (Chemiluminescence reduction neutralization test (CRNT method), Non-Patent Document 1). This neutralizing antibody evaluation method itself is not limited to SARS-CoV-2, but is a technology based on co-cultivation of viruses and cells that can be generally performed even when new threat viruses emerge in the future. is.
　非特許文献１に記載されているような従来の方法は、被検者の静脈より採血した血液を測定試料として用いるものである。静脈採血は、採血技術保有者、採血場所、採血容器が必要となる。静脈採血は患者の負担も大きい。また、採血を行うためには、採血者と被採血者との物理的接触機会と、一定の採血時間の確保が必要なため、パンデミックでは医療従事者及び／又は患者の感染リスクを伴う作業となる。これらは、多くの人で中和抗体を評価するうえでの障壁となると共に、被検者を限定してしまう。 A conventional method such as that described in Non-Patent Document 1 uses blood drawn from a subject's vein as a measurement sample. Venous blood collection requires a person with blood collection skills, a blood collection site, and a blood collection container. Venous blood collection also places a heavy burden on the patient. In addition, in order to collect blood, it is necessary to ensure physical contact between the person collecting blood and the person receiving blood, and to secure a certain amount of time for collecting blood. Become. These are obstacles in evaluating neutralizing antibodies in many people, and limit the number of subjects to be examined.
　本発明の課題は、微量の乾燥血液からウイルスの中和活性を評価する方法を提供することにある。 An object of the present invention is to provide a method for evaluating virus neutralization activity from a minute amount of dried blood.
　被検者の指先等から濾紙採血を行うことも可能であるが、得られる乾燥血液がSARS-CoV-2を含むウイルスの中和抗体に利用できるかは確認されていない。また、濾紙に付着する乾燥血液の血液量は微量であるため、従来の血清を用いた中和抗体の評価方法では測定するための十分な量が得られず、評価不可能であった。さらに、中和抗体の評価方法では活性を維持して血液中の抗体を回収することが重要であり、血液の乾燥などにより抗体が活性を失う中和抗体の評価は困難と考えられていた。 It is possible to collect blood from the subject's fingertips with filter paper, but it has not been confirmed whether the obtained dried blood can be used for neutralizing antibodies against viruses including SARS-CoV-2. In addition, since the amount of dried blood adhering to the filter paper is very small, the conventional method for evaluating neutralizing antibodies using serum cannot obtain a sufficient amount for measurement, making evaluation impossible. Furthermore, in the method of evaluating neutralizing antibodies, it is important to recover the antibodies in the blood while maintaining activity, and it was considered difficult to evaluate neutralizing antibodies that lose their activity due to drying of the blood.
　本発明者は、濾紙採血を利用することで、微量の血液を測定する方法で中和抗体を評価できることを見出し、発明を完成するに至った。 The present inventor discovered that neutralizing antibodies can be evaluated by measuring a very small amount of blood by using filter paper blood collection, and completed the invention.
　本発明は、以下に記載の実施形態を包含する。 The present invention includes the embodiments described below.
　項１．濾紙に付着した乾燥血液を用いてウイルスの中和抗体を評価する方法であって、　濾紙に付着した乾燥血液を抽出して得られた血液試料と、目的となるウイルスの外被タンパク質を外套し、かつ自身の遺伝子内にリポーター遺伝子が組み込まれたシュードタイプウイルスとを混合し、混合物を得る工程、
　前記混合物を、前記シュードタイプウイルスに感受性の培養細胞株に感染させる工程、及び
　前記感染後の前記リポーター遺伝子の発現レベルに基づいて、血液試料中の前記シュードタイプウイルスの中和抗体の有無を検出又は量を測定する工程、を含む方法。 Section 1. A method for evaluating a neutralizing antibody against a virus using dried blood adhered to a filter paper, wherein a blood sample obtained by extracting the dried blood adhered to the filter paper is coated with the coat protein of the virus of interest. and a pseudotype virus having a reporter gene integrated into its own gene to obtain a mixture;
a step of infecting the mixture with a cultured cell line susceptible to the pseudotype virus; and detecting the presence or absence of a neutralizing antibody against the pseudotype virus in a blood sample based on the expression level of the reporter gene after the infection. or measuring the amount.
　項２．血液試料中の中和抗体と、前記混合物中のシュードタイプウイルスのスパイクタンパク質又は前記中和抗体に対する抗原として機能するその一部とが接触し、シュードタイプウイルスに感受性の培養細胞株のシュードタイプウイルスによる感染を阻害する項１に記載の方法。 Section 2. A pseudotyped virus in a cultured cell line susceptible to the pseudotyped virus, wherein the neutralizing antibody in the blood sample is contacted with the spike protein of the pseudotyped virus in the mixture or a portion thereof that functions as an antigen for the neutralizing antibody. 2. A method according to paragraph 1 for inhibiting infection by
　項３．前記スパイクタンパク質はSARS-CoV-2由来のスパイクタンパク質である項２に記載の方法。 Section 3. 3. The method of paragraph 2, wherein the spike protein is a spike protein derived from SARS-CoV-2.
　項４．濾紙に付着した乾燥血液を用いてウイルスの中和抗体を検査するためのキットであって、目的となるウイルスの外被タンパク質を外套したシュードタイプウイルスと、前記シュードタイプウイルスに感受性の培養細胞株とを備えたキット。 Section 4. A kit for testing neutralizing antibodies of a virus using dried blood attached to a filter paper, comprising a pseudotype virus coated with a coat protein of a target virus and a cultured cell line sensitive to the pseudotype virus. A kit with
　項５．前記シュードタイプウイルスの遺伝子内にはリポーター遺伝子が組み込まれている項４に記載のキット。 Item 5. Item 5. The kit according to Item 4, wherein a reporter gene is incorporated into the pseudotype virus gene.
　本発明の中和抗体の評価方法によれば、微量の乾燥血液でもSARS-CoV-2のような新しいタイプのウイルスを含むウイルスの中和活性を評価することができる。 According to the method for evaluating neutralizing antibodies of the present invention, even a small amount of dried blood can be used to evaluate the neutralizing activity of viruses, including new types of viruses such as SARS-CoV-2.
希釈血清の麻疹抗体値と濾紙採血産出希釈率の比率のグラフ。Graph of ratio of measles antibody value of diluted serum to filter paper blood output dilution rate. 濾紙採血からの微量法と血清からの通常法によるSARS-CoV-2ウイルス中和抗体の測定値を示すグラフ。Graph showing measurement of SARS-CoV-2 virus neutralizing antibodies by the micro-method from filter blood and the conventional method from serum. 濾紙採血からの微量法と血清からの通常法によるSARS-CoV-2ウイルス中和抗体の測定値の検体の希釈との相関を示すグラフ。Graph showing the correlation of the SARS-CoV-2 virus-neutralizing antibodies measured by the micro-method from filter blood and the conventional method from serum to the dilution of the specimen.
　本発明のいくつかの態様によれば、濾紙に付着した乾燥血液を用いてウイルスの中和抗体を評価する方法であって、濾紙に付着した乾燥血液を抽出して得られた血液試料と、目的となるウイルスの外被タンパク質を外套し、かつ自身の遺伝子内にリポーター遺伝子が組み込まれたシュードタイプウイルスとを混合し、混合物を得る工程、前記混合物中の前記シュードタイプウイルスをシュードタイプウイルスに感受性の培養細胞株に感染させる工程、及び感染後のリポーター遺伝子の発現レベルに基づいて、血液試料中のシュードタイプウイルスに対する中和抗体の有無を検出又は量を測定する工程を含む方法が提供される。 According to some aspects of the present invention, there is provided a method for evaluating neutralizing antibodies against a virus using dried blood attached to a filter paper, comprising: a blood sample obtained by extracting the dried blood attached to the filter paper; A step of mixing a pseudotyped virus coated with a coat protein of a virus of interest and having a reporter gene integrated into its own gene to obtain a mixture, and transforming the pseudotyped virus in the mixture into a pseudotyped virus. A method is provided comprising the steps of infecting a susceptible cultured cell line, and detecting the presence or absence or quantification of neutralizing antibodies against the pseudotype virus in the blood sample based on the expression level of the reporter gene after infection. be.
　目的となるウイルスとは、本態様のウイルスの中和抗体の評価方法の対象となるウイルスを指す。 The virus of interest refers to the virus that is the target of the method for evaluating neutralizing antibodies against viruses of this embodiment.
　本明細書において、ウイルスの中和抗体を測定する方法には、ウイルスの中和抗体の有無を検出又はウイルスの中和抗体の量を決定する方法が含まれる。本手法で評価できるウイルスは、SARS-CoV-2、SARS-CoV、MERS-CoVなどを含むがこれらに限定されない。 As used herein, methods for measuring virus-neutralizing antibodies include methods for detecting the presence or absence of virus-neutralizing antibodies or determining the amount of virus-neutralizing antibodies. Viruses that can be evaluated with this technique include, but are not limited to, SARS-CoV-2, SARS-CoV, MERS-CoV, and the like.
　被検者から採取した血液を濾紙に染み込ませ、乾燥させる。かかる乾燥血液が付着した濾紙（以下、「乾燥血液濾紙」と称する） を試験検体として用いる。本態様によるウイルスの中和抗体の検査方法に必要な、患者から採取する血液の量は、約１～５μl程度でも十分であり、静脈採血による通常法で必要な量と比較して劇的に少ない。 The blood collected from the subject is soaked into the filter paper and dried. A filter paper to which such dried blood is adhered (hereinafter referred to as "dried blood filter paper") is used as a test specimen. The amount of blood collected from the patient, which is necessary for the virus neutralizing antibody test method according to this embodiment, is sufficient even at about 1 to 5 μl, and is dramatically larger than the amount required in the usual method of intravenous blood collection. Few.
　被検者は健常者であってもよいし、ウイルスの感染が疑われる者であってもよいし、ウイルスに既に感染している者であってもよい。或いはウイルス感染に伴う症状又は疾患の兆候が見られるか、かかる症状又は疾患を患っている者であってもよい。 The subject may be a healthy person, a person suspected of being infected with the virus, or a person already infected with the virus. Alternatively, the subject may have symptoms or symptoms associated with viral infection, or may be suffering from such symptoms or diseases.
　濾紙は被検者から血液を採取するために使用可能な任意の濾紙であってよい。濾紙のサイズは任意のサイズとすることができる。濾紙のサイズは、通常、乾燥血液濾紙を運搬及び／又は保存するのに便利なサイズであることが好ましい。 The filter paper may be any filter paper that can be used to collect blood from the subject. The size of the filter paper can be any size. The size of the filter paper is generally preferred for convenient transport and/or storage of the dried blood filter paper.
　濾紙に付着した乾燥血液の濾紙からの抽出は公知の方法により行うことができる。抽出温度は血液中の成分の変性が抑制される温度が好ましく、例えば４～３０℃、好ましくは４～２５℃程度が挙げられるがこれに限定されない。抽出時間は例えば３０分～１日程度が挙げられるが、これに限定されない。例えば、乾燥血液の濾紙からの抽出は血液抽出用の抽出液を用いて行うことができる。抽出液としては、生理食塩水、トリス緩衝液、リン酸緩衝液などの緩衝液；細胞培養用培地が挙げられるがこれらに限定されない。特に、シュードタイプウイルスに感受性の培養細胞株の培養用の培地を抽出液として用いると、シュードタイプウイルスに感受性の培養細胞株を培養した培地と一致するため、培地の希釈による培地中の栄養素の変動が生じにくく、培地条件の安定化の点で好適である。また、抽出液は、血液中の成分の安定化の目的で、非ヒト血清、無機塩類、界面活性剤などを含有してもよい。 Dried blood adhering to the filter paper can be extracted from the filter paper by a known method. The extraction temperature is preferably a temperature that suppresses denaturation of components in the blood, for example, 4 to 30°C, preferably about 4 to 25°C, but is not limited thereto. The extraction time is, for example, about 30 minutes to 1 day, but is not limited to this. For example, extraction of dried blood from filter paper can be performed using an extractant for blood extraction. Extraction solutions include, but are not limited to, buffers such as physiological saline, Tris buffer, phosphate buffer, and cell culture media. In particular, when the culture medium for pseudotype virus-susceptible culture cell lines is used as an extract, it matches the medium in which pseudotype virus-susceptibility culture cell lines were cultured, so the nutrients in the medium due to dilution of the medium Fluctuations are less likely to occur, and it is suitable for stabilizing medium conditions. The extract may also contain non-human serum, inorganic salts, surfactants, etc. for the purpose of stabilizing components in blood.
　本態様によるウイルスの中和抗体の評価方法では、ウイルスへの感染リスクを低減するため、人工的にプラスミドDNAから作製したシュードタイプウイルスを利用する。シュードタイプウイルスとは、目的とするウイルスの外被タンパク質（例えばエンベロープタンパク質、スパイクタンパク質、又はウイルスの中和抗体に対する抗原として機能するそれらの一部）を外套したウイルスを指す。元の基盤となるウイルスとして、水疱性口内炎ウイルス（VSV）やレトロウイルス、レンチウイルスなどが知られており、本明細書でもこれらのウイルスを用いることができる。シュードタイプウイルスを作製できるVSVベクターには、自身の遺伝子内にリポーター遺伝子が組み込まれており、標的細胞に感染するとリポーター遺伝子の発現により感染性を評価することができる。リポーター遺伝子としては、ルシフェラーゼ、蛍光タンパク質、着色タンパク質等が挙げられる。 In the evaluation method for virus neutralizing antibodies according to this embodiment, a pseudotype virus artificially produced from plasmid DNA is used in order to reduce the risk of virus infection. Pseudotyped viruses refer to viruses that are enveloped with the desired viral coat proteins (eg, envelope proteins, spike proteins, or portions thereof that serve as antigens for neutralizing antibodies of the virus). Vesicular stomatitis virus (VSV), retroviruses, lentiviruses, and the like are known as the original underlying viruses, and these viruses can also be used herein. A VSV vector capable of producing a pseudotype virus has a reporter gene integrated into its own gene, and when a target cell is infected, the infectivity can be evaluated by the expression of the reporter gene. Reporter genes include luciferase, fluorescent proteins, colored proteins and the like.
　ルシフェラーゼとしては、ウミボタル、ヒオドシエビ、発光昆虫（ホタル、ヒカリコメツキ等）、発光ミミズ、ラチア、ウミシイタケ、オワンクラゲ（エクオリン）等の各種発光生物由来のルシフェラーゼが例示される。蛍光タンパク質としては、遺伝子改変型の蛍光タンパク質が挙げられ、例えば、グリーン蛍光タンパク質（GFP）、黄色蛍光タンパク質（YFP），青色蛍光タンパク質（BFP）、シアン蛍光タンパク質（CFP）、DsRED、赤色蛍光タンパク質（RFP）等が例示される。着色タンパク質としては、フィコシアニン、フィコエリトリンが挙げられる。この中でも、シグナル検出の感度が高い点や定量性が高い点で、ルシフェラーゼ遺伝子が好ましい。
　シュードタイプウイルスは、BSL3やBSL4（BSL:バイオセーフティーレベル）で取り扱わなければならないウイルス種であっても外被タンパク質のみを用いて作製するものであるため、一般的な感染実験環境であるBSL2で取り扱い可能となる点で有利である。例えば、被検者の乾燥血液中のSARS-CoV-2ウイルスに対する中和抗体の有無を調べたい場合に、SARS-CoV-2ウイルスのスパイクタンパク質又はそのC末端のアミノ酸が欠損した改変スパイクタンパク質を発現するVSVシュードタイプウイルスを用いると、被検者の乾燥血液中のSARS-CoV-2ウイルスに対する中和抗体の有無を特異的に検出又は決定できる。 Examples of luciferases include luciferases derived from various luminous organisms such as sea worms, black lobsters, luminous insects (fireflies, click beetles, etc.), luminous earthworms, lachias, sea pansy, and Aequorin jellyfish (aequorin). Fluorescent proteins include genetically modified fluorescent proteins, such as green fluorescent protein (GFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), cyan fluorescent protein (CFP), DsRED, and red fluorescent protein. (RFP) and the like are exemplified. Colored proteins include phycocyanin and phycoerythrin. Among these, the luciferase gene is preferable because of its high signal detection sensitivity and high quantification.
Pseudotype viruses, even virus species that must be handled at BSL3 or BSL4 (BSL: biosafety level), are produced using only the envelope protein, so they can be prepared at BSL2, which is a general infection experiment environment. It is advantageous in that it can be handled. For example, if you want to examine the presence or absence of neutralizing antibodies against the SARS-CoV-2 virus in the dried blood of a subject, use the spike protein of the SARS-CoV-2 virus or a modified spike protein lacking its C-terminal amino acid. The expressed VSV pseudotype virus can be used to specifically detect or determine the presence or absence of neutralizing antibodies against the SARS-CoV-2 virus in the dried blood of a subject.
　濾紙に付着した乾燥血液を抽出して得られた血液試料と、目的となるウイルスの外被タンパク質を外套したシュードタイプウイルスとを混合すると、血液試料がウイルスの中和抗体を含む場合には、血液試料中の中和抗体と、得られた混合物中のシュードタイプウイルスの外被タンパク質又は中和抗体に対する抗原として機能するその一部とが接触し、シュードタイプウイルスに感受性の培養細胞株のシュードタイプウイルスによる感染を阻害する状態となる。 When a blood sample obtained by extracting dried blood attached to a filter paper is mixed with a pseudotype virus coated with the coat protein of the virus of interest, if the blood sample contains neutralizing antibodies against the virus, The neutralizing antibody in the blood sample is contacted with the pseudotyped virus coat protein in the resulting mixture or a portion thereof that functions as an antigen for the neutralizing antibody to pseudotype a cultured cell line susceptible to the pseudotyped virus. It becomes a state that inhibits infection by type virus.
　血液試料と、目的となるウイルスの外被タンパク質を外套したシュードタイプウイルスとを反応させる時間は通常1時間以上が好ましい。また、反応させる温度は通常、37℃前後が好ましい。 The time for reacting the blood sample with the pseudotype virus coated with the envelope protein of the virus of interest is usually preferably 1 hour or longer. Moreover, the reaction temperature is usually preferably around 37°C.
　次に、上記反応条件で処理したシュードタイプウイルスを、シュードタイプウイルスに感受性の培養細胞株に感染させる。シュードタイプウイルスに感受性の培養細胞株は、適宜選択することができる。例えばSARS-CoV-2のシュードタイプウイルスに感受性の高い培養細胞株としては、VeroE6/TMPRSS2細胞などが挙げられる。 Next, the pseudotyped virus treated under the above reaction conditions is used to infect a pseudotyped virus-susceptible cultured cell line. Cultured cell lines sensitive to pseudotype viruses can be selected as appropriate. For example, cultured cell lines highly susceptible to SARS-CoV-2 pseudotype viruses include VeroE6/TMPRSS2 cells.
　混合物中のシュードタイプウイルスを、シュードタイプウイルスに感受性の培養細胞株に感染させる場合、37℃の温度条件下で1時間以上吸着させ、その後培養培地を添加して37℃の温度条件下で24時間培養することが好ましい。 When the pseudotype virus in the mixture is used to infect a pseudotype virus-susceptible cultured cell line, it is allowed to adsorb for at least 1 hour at 37°C, then the culture medium is added and incubated at 37°C for 24 hours. It is preferable to culture for a long time.
　次に、感染後のリポーター遺伝子の発現レベルに基づいて、血液試料中のシュードタイプウイルスに対する中和抗体の有無を検出又は量を測定する。シュードタイプウイルスの中のリポーター遺伝子の発現レベルは、公知の分析機器を用いてリポーター遺伝子による発光量の測定により算出することができる。 Next, based on the expression level of the reporter gene after infection, the presence or absence of neutralizing antibodies against the pseudotype virus in the blood sample is detected or measured. The expression level of the reporter gene in the pseudotype virus can be calculated by measuring the luminescence level of the reporter gene using a known analytical instrument.
　血液試料がウイルスの中和抗体を含む場合、中和抗体との反応によりシュードタイプウイルスの感染を阻害する状態となり、血液試料がウイルスの中和抗体を含まない場合と比較して、シュードタイプウイルスに感受性のある培養細胞株に感染できるシュードタイプウイルスの感染量は減少する。シュードタイプウイルスに感染した培養細胞株の感染率は、対照（例えば血液試料がウイルスの中和抗体を含まない場合）と比較したリポーター遺伝子の発現レベルを測定することにより評価することができる。リポーター遺伝子の発現レベルの測定は、市販のキットを用いて行うことができる。例えばリポーター遺伝子がルシフェラーゼの場合、PicaGene Luminescence Kit (TOYO B-Net Co., LTD, Tokyo, Japan) 及びGloMax Navigator System G2000 (Promega Corporation, Madison,WI)などの市販のキットを用いてルシフェラーゼの相対発光量(RLU)を測定することができる。リポーター遺伝子の発現レベルは、シュードタイプウイルスの感染量の指標とすることができる。 When the blood sample contains a virus-neutralizing antibody, the reaction with the neutralizing antibody results in a state that inhibits infection with the pseudotype virus. The infectious dose of pseudotyped viruses that can infect cultured cell lines susceptible to HIV is reduced. The infection rate of a cultured cell line infected with a pseudotyped virus can be assessed by measuring the expression level of the reporter gene compared to a control (e.g., when the blood sample does not contain neutralizing antibody to the virus). A commercially available kit can be used to measure the expression level of the reporter gene. For example, when the reporter gene is luciferase, relative luminescence of luciferase is measured using commercially available kits such as PicaGene Luminescence Kit (TOYO B-Net Co., LTD, Tokyo, Japan) and GloMax Navigator System G2000 (Promega Corporation, Madison, WI). amount (RLU) can be measured. The expression level of the reporter gene can be used as an indicator of the amount of pseudotype virus infection.
　ある被検者由来の血液を濾紙に付着させた乾燥血液を用いてウイルスの中和抗体を検査すると仮定する。本態様の方法により測定されたリポーター遺伝子の発現レベルが、ウイルスの中和抗体を含まない血液試料を用いた場合のリポーター遺伝子の発現レベルよりも低い場合に、かかる被検者の血液には中和抗体が存在すると判定することができる。本態様の方法により測定されたリポーター遺伝子の発現レベルが、ウイルスの中和抗体を含まない血液試料を用いた場合のリポーター遺伝子の発現レベルと同じかそれよりも高い場合に、かかる被検者の血液には中和抗体が存在しないと判定することができる。 Suppose that a virus-neutralizing antibody is tested using dried blood from a certain subject attached to filter paper. When the expression level of the reporter gene measured by the method of this embodiment is lower than the expression level of the reporter gene when a blood sample containing no virus-neutralizing antibody is used, the blood of such a subject has medium It can be determined that sum antibodies are present. When the expression level of the reporter gene measured by the method of this embodiment is the same as or higher than the expression level of the reporter gene when a blood sample containing no virus-neutralizing antibody is used, the subject It can be determined that the blood is free of neutralizing antibodies.
　本態様のウイルスの中和抗体の評価方法は、驚くべきことに、数μLという微量の血液でも、非特許文献１に示すような静脈採血に匹敵する高い精度でウイルスの中和抗体を評価することができる。さらに、予想外なことに、採血した血液を濾紙乾燥血液の様式としても、抽出した血液がウイルスの中和抗体を評価するのに十分な活性を有する。また、静脈採血による血液試料を用いた通常の中和抗体評価法（通常法）では、全血60μLという比較的多量の採血が必要であったが、本態様のウイルスの中和抗体の評価方法によれば、静脈採血に伴う採血容器や医療従事者の労力は不要となり、被検者の負担も大幅に軽減される。 Surprisingly, the virus-neutralizing antibody evaluation method of the present embodiment can evaluate the virus-neutralizing antibody with a high degree of precision comparable to that of intravenous blood collection as shown in Non-Patent Document 1, even with a very small amount of blood, such as several μL. be able to. In addition, unexpectedly, even when the collected blood is in the form of filter-dried blood, the extracted blood has sufficient activity to assess neutralizing antibodies to the virus. In addition, in the normal neutralizing antibody evaluation method using a blood sample obtained by intravenous blood collection (normal method), it was necessary to collect a relatively large amount of blood of 60 μL of whole blood, but the virus neutralizing antibody evaluation method of this embodiment. According to the method, the blood collection container and the labor of the medical staff associated with intravenous blood collection are not required, and the burden on the examinee is greatly reduced.
　さらに、通常法では96ウェルプレートを利用していたため、2重測定する場合、コントロールに必要となるウェルを除いて収容可能な検体は最大44検体となり、同時処理可能な検体数に限りがあったが、本態様のウイルスの中和抗体の評価方法は、微量の血液で実施できるため、より多検体を同時処理することができる。 Furthermore, since the conventional method used a 96-well plate, the number of samples that could be processed simultaneously was limited to 44 samples, excluding the wells required for controls, when performing duplicate measurements. However, the method for evaluating neutralizing antibodies against viruses according to the present embodiment can be performed with a very small amount of blood, so that more specimens can be treated simultaneously.
　本態様の中和抗体の評価方法によれば、SARS-CoV-2のような新しいウイルス又は既知のウイルスを含むウイルスの中和活性を、微量の乾燥血液から評価することができる。このため、本態様の中和抗体の評価方法を、例えば、SARS-CoV-2を含む新しいウイルス感染症に対するワクチンの予防効果を評価したり、集団免疫獲得状況を評価したりする用途に利用することができる。本態様の中和抗体の評価方法は、ヒトの診断方法を含まないインビトロの評価方法である。 According to the neutralizing antibody evaluation method of this embodiment, the neutralizing activity of viruses including new viruses such as SARS-CoV-2 or known viruses can be evaluated from a small amount of dried blood. Therefore, the neutralizing antibody evaluation method of this embodiment can be used, for example, to evaluate the preventive effect of vaccines against new viral infections including SARS-CoV-2 or to evaluate the status of collective immunity acquisition. be able to. The neutralizing antibody evaluation method of this embodiment is an in vitro evaluation method that does not include human diagnostic methods.
　本発明の別のいくつかの態様によれば、濾紙に付着した乾燥血液を用いてウイルスの中和抗体を検査するためのキットであって、目的となるウイルスの外被タンパク質を外套したシュードタイプウイルスと、前記シュードタイプウイルスを感染させる培養細胞株とを備えたキットが提供される。本態様のキットは、本発明の濾紙に付着した乾燥血液を用いてウイルスの中和抗体を評価する方法を実施するために好適に用いられる。好ましくは、シュードタイプウイルスの遺伝子内にはリポーター遺伝子が組み込まれている。 According to some other aspects of the present invention, there is provided a kit for testing neutralizing antibody of a virus using dried blood adhered to filter paper, wherein the pseudotype coats the coat protein of the virus of interest. A kit is provided that includes the virus and a cultured cell line to infect the pseudotyped virus. The kit of this aspect is preferably used to carry out the method of evaluating neutralizing antibodies against viruses using the dried blood adhered to the filter paper of the present invention. Preferably, a reporter gene is integrated into the gene of the pseudotype virus.
　濾紙に付着した乾燥血液、目的となるウイルスの外被タンパク質を外套したシュードタイプウイルス、ならびにシュードタイプウイルスに感受性の培養細胞株については、上記のウイルスの中和抗体の評価方法の態様に関して説明した通りである。 Regarding the dried blood attached to the filter paper, the pseudotype virus coated with the target virus coat protein, and the pseudotype virus-susceptible cultured cell line, the method for evaluating the neutralizing antibody of the virus described above has been described. Street.
　かかる態様のキットを用いて、被検者から採取した血液を濾紙に付着させた乾燥血液を検査し、測定されたリポーター遺伝子の発現レベルが、対照（例えば血液試料がウイルスの中和抗体を含まない場合）のリポーター遺伝子の発現レベルよりも低い場合に、かかる被検者の血液には中和抗体が存在すると判定することができる。本態様のキットを用いて測定されたリポーター遺伝子の発現レベルが、対照のリポーター遺伝子の発現レベルと同じかそれよりも高い場合に、かかる被検者の血液には中和抗体が存在しないと判定することができる。 Using the kit of this embodiment, the dried blood collected from the subject and adhered to filter paper is examined, and the measured reporter gene expression level is higher than that of the control (for example, the blood sample contains virus neutralizing antibody). It can be determined that neutralizing antibodies are present in the blood of such a subject if the expression level of the reporter gene is lower than the expression level of the reporter gene in the absence of the antibody. If the expression level of the reporter gene measured using the kit of this embodiment is the same as or higher than the expression level of the reporter gene in the control, it is determined that there is no neutralizing antibody in the blood of the subject. can do.
　本態様のキットはさらに、目的となるウイルスの外被タンパク質を外套したシュードタイプウイルスを収容するための第１の容器と、シュードタイプウイルスに感受性の培養細胞株を収容するための第２の容器とを備えていてもよい。 The kit of this embodiment further comprises a first container for housing a pseudotype virus coated with a viral coat protein of interest and a second container for housing a cultured cell line susceptible to the pseudotype virus. and may be provided.
　本態様のキットはさらに、シュードタイプウイルスに感受性の培養細胞株を培養するための細胞培養用培地が含まれていてもよく、そのような培養用培地は上記第２の容器に入れられていてもよい。細胞培養用培地については上記のウイルスの中和抗体の評価方法の態様に関して説明した通りである。 The kit of this embodiment may further comprise a cell culture medium for culturing a cultured cell line susceptible to pseudotype virus, such culture medium being contained in the second container and good too. The cell culture medium is as described with respect to the method for evaluating neutralizing antibodies against viruses.
　本態様のキットはさらに、被検者から採取した血液を付着させるための濾紙と、該濾紙に付着した乾燥血液を抽出するための抽出液を備えていてもよい。抽出液については上記のウイルスの中和抗体の評価方法の態様に関して説明した通りである。 The kit of this aspect may further comprise a filter paper for adhering blood collected from a subject, and an extraction liquid for extracting the dried blood adhering to the filter paper. The extract is as described in relation to the method for evaluating the neutralizing antibody of the virus.
　本態様のキットはさらに、本発明のウイルスの中和活性を評価する方法を実施するための手順などを記載した取扱説明書を備えていてもよい。 The kit of this embodiment may further comprise an instruction manual describing the procedure for carrying out the method of evaluating the virus-neutralizing activity of the present invention.
　以下に実施例を挙げて本発明をより具体的に説明するが、本発明はこれらに限定されない。 The present invention will be described in more detail with reference to examples below, but the present invention is not limited to these.
実施例１　濾紙乾燥血液を用いたウイルスの中和抗体の測定方法の確立
　まず、中和抗体の評価に利用可能な血液量を確保できるかを調べるために、指先採血用器具（テルモ株式会社製　採血用穿刺針メディセーフ ファインタッチディスポ）を用いて採血量を調べた。針長0.8mmと1.5mmでは、採血量はそれぞれ15.3±1.4μL、18.8±1.2μL（各n=4、平均±標準誤差）であった。スタンプ法により採血用濾紙（ADVANTEC社製　採血用濾紙）に吸着させた場合、単位面積当たりの吸着血液量は0.33～0.40μL/mm2であった（n=3）。続いて、濾紙乾燥血液と溶出に用いる溶媒量との関係を知るために、生理食塩水に浸した場合の血液回収効率をヘモグロビンの吸光度であるOD560で測定したところ、溶媒量に反比例することを確認した。濾紙吸着血液量を最低値の0.33μL/mm2として、１枚の濾紙の吸着血液量と、濾紙面積と添加溶媒量から回収される希釈倍率とを算出した（表1）。 Example 1 Establishment of a measurement method for virus neutralizing antibodies using filter dried blood The amount of blood collected was examined using a puncture needle for blood collection (Medisafe Fine Touch Dispo). With needle lengths of 0.8 mm and 1.5 mm, the blood collection volumes were 15.3±1.4 μL and 18.8±1.2 μL, respectively (n=4 each, mean±standard error). When the sample was adsorbed onto blood collection filter paper (blood collection filter paper manufactured by ADVANTEC) by the stamp method, the amount of blood absorbed per unit area was 0.33 to 0.40 μL/mm 2 (n=3). Subsequently, in order to know the relationship between the dried blood on the filter paper and the amount of solvent used for elution, the blood recovery efficiency when immersed in physiological saline was measured by OD 560 , which is the absorbance of hemoglobin, and it was found to be inversely proportional to the amount of solvent. It was confirmed. Assuming that the amount of blood adsorbed on the filter paper was the lowest value of 0.33 μL/mm 2 , the amount of blood adsorbed on one filter paper and the dilution ratio recovered from the area of the filter paper and the amount of added solvent were calculated (Table 1).
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表1の算出した濾紙採血による希釈率の妥当性を確かめるために、濾紙採血用の血液を採血した患者からの実際の静脈採血から作成した希釈血清検体を比較対象として、麻疹抗体キット（デンカ生研社製、ウイルス抗体EIA「生検」麻疹IgG）により希釈麻疹抗体値を測定し、希釈血清における麻疹抗体値と濾紙採血による希釈率との相関関係を評価した。その結果、ADVANTEC社製採血用濾紙（濾紙A）ならびにWhatman社製採血用（濾紙B）濾紙ともに、6倍希釈濾紙採血（算出値）は、麻疹抗体値測定における8倍希釈血清に、50倍希釈濾紙採血（算出値）は、麻疹抗体値測定における32倍希釈血清に近似した値を示し、算出式の妥当性を確認した（図1）。 In order to confirm the appropriateness of the dilution ratio calculated in Table 1 for the blood drawn from the filter paper, a measles antibody kit (DENKA SEIKEN Diluted measles antibody levels were measured using virus antibody EIA "biopsy" measles IgG (manufactured by Co., Ltd.), and the correlation between the measles antibody levels in the diluted serum and the dilution ratio obtained by collecting blood on filter paper was evaluated. As a result, for both ADVANTEC blood collection filter paper (filter paper A) and Whatman blood collection filter paper (filter paper B), 6-fold diluted filter paper blood collection (calculated value) Diluted filter paper blood collection (calculated value) showed a value similar to 32-fold diluted serum in measles antibody level measurement, confirming the validity of the calculation formula (Fig. 1).
　なお、通常法による中和抗体評価では、96ウェルプレートを利用し、その中で培養した細胞株に生きたウイルスを感染させる。改良法である微量法では、384ウェルプレートを利用することで、1ウェルあたりに必要とする量を、通常法で必要とする量と比較して、培養細胞を含む培養液で1/2、血液試料で1/4、シュードタイプウイルスを含む液で1/9として測定可能であることを確かめた。 In addition, in neutralizing antibody evaluation by the conventional method, a 96-well plate is used, and the cell lines cultured in it are infected with live virus. In the improved microvolume method, by using a 384-well plate, the volume required per well is 1/2 of the volume required in the conventional method, with the medium containing cultured cells. It was confirmed that it was possible to measure 1/4 in blood samples and 1/9 in fluids containing pseudotype viruses.
　以上より、4 mmパンチ大の濾紙採血を100μLで溶出した場合を例にとると、血清200倍希釈に相当する濃度での中和抗体測定が、標準法では3回分、微量法では14回分確保できることが確認された。 Based on the above, taking the case of eluting 100 μL of blood collected from a 4-mm punch-sized filter paper, neutralizing antibody measurement at a concentration equivalent to a 200-fold dilution of serum is ensured for 3 times with the standard method and 14 times with the micro-volume method. confirmed to be possible.
実施例２　濾紙乾燥血液を用いた被検者におけるSARS-CoV-2に対する中和抗体値の測定
　COVID-19に罹患していると診断された患者の協力を得て、同日に採取した濾紙乾燥血液と静脈採血血清での新型コロナウイルスSARS-CoV-2に対する中和抗体値を比較した（26症例、28検体）。濾紙乾燥血液を4 mmパンチで抜き取り、100μLのDMEM培地（ナカライテスク社製）で溶出して微量法で測定した中和抗体と、静脈採血血清を通常法で測定した中和抗体とを比較した。 Example 2 Measurement of Neutralizing Antibody Levels Against SARS-CoV-2 in Subjects Using Filter-Dried Blood Neutralizing antibody levels against the novel coronavirus SARS-CoV-2 were compared in blood and venous blood serum (26 cases, 28 specimens). The dried blood on the filter paper was extracted with a 4 mm punch, eluted with 100 μL of DMEM medium (manufactured by Nacalai Tesque), and the neutralizing antibody measured by the micro method was compared with the neutralizing antibody measured by the conventional method on the serum collected from the vein. .
　以前に報告したように、水疱性口内炎ウイルス（vesicular stomatitis virus, VSV）を基盤としたSARS-CoV-2シュードタイプウイルスを作製するにあたり、SARS-CoVのスパイクタンパク質（Sタンパク質）のC末端の19アミノ酸を欠損させたSタンパク質を外套したシュードタイプウイルスで感染性が高かったことから（J Gen Virol.2005;86:2269-74; J Virol. 2009;83:712-21.）、本実施例においてもSARS-CoV-2のSタンパク質のC末端の19アミノ酸を欠損させたSタンパク質を外套したシュードタイプウイルスを作製し、本実施例の通常法及び微量方法に用いた。シュードタイプウイルスの作製方法についてはTani et al. Virol J (2021) 18:16を参照されたい。簡単に説明すると、293T細胞にC末端の19アミノ酸を欠損させたSARS-CoV-2のSタンパク質を発現するプラスミドDNAをトランスフェクションする。24時間後に、該トランスフェクトした293T細胞に、親ウイルスである、自身のエンベロープ遺伝子の代わりにリポーター遺伝子が組み込まれており、一過性にVSVのGタンパク質を外套した*G-VSVΔG/Lucを一定量感染させる。2時間ウイルスを細胞に吸着させた後、感染しなかったウイルスを培地で洗い出す。24時間培養後、培養上清を回収し、SARS-CoV-2のSタンパク質を外套したシュードタイプウイルスを得る。19アミノ酸を欠損させたSタンパク質は欠損させていないSタンパク質よりもより多くシュードタイプウイルスに外套されており、感染性も高いことが明らかとなっている（Tani et al. Virol J (2021) 18:16）。 As we previously reported, to generate the vesicular stomatitis virus (VSV)-based SARS-CoV-2 pseudotyped virus, the C-terminal 19 of the spike protein (S protein) of SARS-CoV Pseudotype viruses enveloped with amino acid-deficient S protein showed high infectivity (J Gen Virol. 2005;86:2269-74; J Virol. 2009;83:712-21.). Also, a pseudotype virus encapsulating the S protein in which 19 amino acids at the C-terminal of the SARS-CoV-2 S protein were deleted was produced and used in the conventional method and the microvolume method of this example. See Tani et al. Virol J (2021) 18:16 for methods of generating pseudotyped viruses. Briefly, 293T cells are transfected with plasmid DNA expressing the SARS-CoV-2 S protein truncated for the C-terminal 19 amino acids. Twenty-four hours later, the transfected 293T cells transformed into *G-VSVΔG/Luc transiently enveloped with the VSV G protein, in which a reporter gene was integrated in place of its own envelope gene, which is the parental virus. Infect a certain amount. After allowing the virus to adsorb to the cells for 2 hours, uninfected virus is washed out with medium. After culturing for 24 hours, the culture supernatant is collected to obtain a pseudotype virus encapsulating the S protein of SARS-CoV-2. It has been shown that the S protein with 19 amino acid deletions is more enveloped by pseudotyped viruses and has higher infectivity than the intact S protein (Tani et al. Virol J (2021) 18 :16).
　静脈採血血清を用いた通常法では、血清をDMEM培地（ナカライテスク社製）で希釈し、SARS-CoV-2のSタンパク質を外套したシュードタイプウイルスを含むウイルス液と9:1に混合後、VeroE6/TMPRSS2細胞（NIBIOHN JCRB細胞バンクから入手）を収容した96ウェルプレートに1ウェルあたり100μL添加し、2ウェル準備する。24時間後にウイルスに組み込まれた発光酵素であるルシフェラーゼの基質を10μL添加し、発光をGloMaxプレートリーダー（プロメガ社製）で測定し、2重測定の平均値を、血清非含有の測定値と比較した場合のパーセンテージとして算出する。詳しくは、Tani et al. Virol J (2021) 18:16を参照されたい。 In the usual method using intravenous blood serum, the serum is diluted with DMEM medium (manufactured by Nacalai Tesque) and mixed with a virus solution containing a pseudotype virus that coats the S protein of SARS-CoV-2 at a ratio of 9:1. Add 100 μL per well to a 96-well plate containing VeroE6/TMPRSS2 cells (obtained from the NIBIOHN JCRB Cell Bank) to prepare 2 wells. After 24 hours, 10 μL of luciferase substrate, a luciferase incorporated into the virus, was added, luminescence was measured with a GloMax plate reader (manufactured by Promega), and the average value of duplicate measurements was compared with the value without serum. calculated as a percentage of the For more information, see Tani et al. Virol J (2021) 18:16.
　微量法では、濾紙乾燥血液をDMEM培地（ナカライテスク社製）で血清換算200倍相当に希釈し、この希釈液57μLとSARS-CoV-2のSタンパク質を外套したシュードタイプウイルスを含むウイルス液3μLとを混合後1時間室温静置し、VeroE6/TMPRSS2 細胞を播種した384ウェルプレートに該混合物を1ウェルあたり22.5μL添加し、これを2ウェル準備した。24時間後にウイルスに組み込まれた発光酵素であるルシフェラーゼの基質を2.5μL添加し、発光をGloMaxプレートリーダー（プロメガ社製）で測定し、2重測定の平均値を、血清非含有の測定値と比較した場合のパーセンテージとして算出した。 In the micro-quantity method, filter paper-dried blood was diluted with DMEM medium (manufactured by Nacalai Tesque) to a serum equivalent of 200 times. After mixing, the mixture was allowed to stand at room temperature for 1 hour, and 22.5 μL of the mixture was added per well to a 384-well plate seeded with VeroE6/TMPRSS2 cells to prepare 2 wells. After 24 hours, 2.5 μL of luciferase substrate, a luciferase incorporated into the virus, was added, and luminescence was measured using a GloMax plate reader (manufactured by Promega). Calculated as a percentage of the comparison.
　濾紙採血からの中和抗体値（縦軸）と、静脈採血検体からの中和抗体値（横軸）は、正の相関を示した（図2）。十分に中和抗体を保有していることを確認した3例については、更に検体を200倍、800倍、3200倍、12800倍に希釈して相関性を確認した（図3）。この検証に用いた濾紙乾燥血液は採血後3～31日間、常温で保存されたものであり、約1か月は安定であることを確認した。 A positive correlation was shown between the neutralizing antibody value from filter paper blood (vertical axis) and the neutralizing antibody value from venous blood sample (horizontal axis) (Fig. 2). For the 3 cases confirmed to possess sufficient neutralizing antibodies, the samples were further diluted 200-fold, 800-fold, 3200-fold, and 12800-fold to confirm the correlation (Fig. 3). The filter paper-dried blood used for this verification was stored at room temperature for 3 to 31 days after blood collection, and was confirmed to be stable for about one month.
　本発明の濾紙乾燥血液を用いたウイルスの中和抗体の検出方法は、被検者及び医療従事者の感染リスクの軽減、採血した血液試料の保管・輸送の簡便性、濾紙乾燥血液の長期間の安定性、及び多検体処理能から、国内外で広く被検者における中和抗体獲得状況の調査に利用することができる。 The method of detecting a virus neutralizing antibody using filter paper-dried blood of the present invention reduces the risk of infection of subjects and medical workers, facilitates storage and transportation of collected blood samples, and allows the use of filter paper-dried blood for a long period of time. Because of its stability and ability to process multiple specimens, it can be widely used both in Japan and overseas to investigate the acquisition of neutralizing antibodies in subjects.

Claims (5)

  1. 　濾紙に付着した乾燥血液を用いてウイルスの中和抗体を評価する方法であって、
    　濾紙に付着した乾燥血液を抽出して得られた血液試料と、目的となるウイルスの外被タンパク質を外套し、かつ自身の遺伝子内にリポーター遺伝子が組み込まれたシュードタイプウイルスとを混合し、混合物を得る工程、
    　前記混合物を、前記シュードタイプウイルスに感受性の培養細胞株に感染させる工程、及び
    　前記感染後の前記リポーター遺伝子の発現レベルに基づいて、血液試料中の前記シュードタイプウイルスの中和抗体の有無を検出又は量を測定する工程、を含む方法。     A method for evaluating neutralizing antibodies of viruses using dried blood attached to filter paper,
    A blood sample obtained by extracting dried blood adhering to a filter paper is mixed with a pseudotype virus that coats the coat protein of the virus of interest and that has a reporter gene integrated in its own gene, resulting in a mixture. a step of obtaining
    a step of infecting the mixture with a cultured cell line susceptible to the pseudotype virus; and detecting the presence or absence of a neutralizing antibody against the pseudotype virus in a blood sample based on the expression level of the reporter gene after the infection. or measuring the amount.
  2. 　血液試料中の中和抗体と、前記混合物中のシュードタイプウイルスのスパイクタンパク質又は前記中和抗体に対する抗原として機能するその一部とが接触し、シュードタイプウイルスに感受性の培養細胞株のシュードタイプウイルスによる感染を阻害する請求項１に記載の方法。 A pseudotyped virus in a cultured cell line susceptible to the pseudotyped virus, wherein the neutralizing antibody in the blood sample is contacted with the spike protein of the pseudotyped virus in the mixture or a portion thereof that functions as an antigen for the neutralizing antibody. 2. The method of claim 1, which inhibits infection by
  3. 　前記スパイクタンパク質はSARS-CoV-2由来のスパイクタンパク質である請求項２に記載の方法。 The method according to claim 2, wherein the spike protein is a spike protein derived from SARS-CoV-2.
  4. 　濾紙に付着した乾燥血液を用いてウイルスの中和抗体を検査するためのキットであって、目的となるウイルスの外被タンパク質を外套したシュードタイプウイルスと、前記シュードタイプウイルスに感受性の培養細胞株とを備えたキット。 A kit for testing neutralizing antibodies of a virus using dried blood attached to a filter paper, comprising a pseudotype virus coated with a coat protein of a target virus and a cultured cell line sensitive to the pseudotype virus. A kit with
  5. 　前記シュードタイプウイルスの遺伝子内にはリポーター遺伝子が組み込まれている請求項４に記載のキット。 The kit according to claim 4, wherein a reporter gene is incorporated into the pseudotype virus gene.
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SCHMIDT FABIAN, WEISBLUM YISKA, MUECKSCH FRAUKE, HOFFMANN HANS-HEINRICH, MICHAILIDIS ELEFTHERIOS, LORENZI JULIO C. C., MENDOZA PIL: "Measuring SARS-CoV-2 neutralizing antibody activity using pseudotyped and chimeric viruses", BIORXIV, 9 June 2020 (2020-06-09), pages 1 - 44, XP055841655, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2020.06.08.140871v1.full.pdf> [retrieved on 20210916], DOI: 10.1101/2020.06.08.140871 *
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