JP2007145775A - Method for detecting norovirus gi in high sensitivity - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a monoclonal or polyclonal antibody to synthetic peptide, and to provide a method for detecting norovirus GI infection using the antibody. <P>SOLUTION: The monoclonal or polyclonal antibody to peptide is provided, comprising a sequence:Gly Glu Phe Thr Ile Ser Pro Asn Asn Thr Pro Gly Asp (Sequence No.1). A reagent for detecting norovirus GI containing the antibody is provided. A method for detecting the norovirus GI, a detection kit for the method, and a hybridoma that produces the monoclonal antibody are also provided, respectively. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a monoclonal or polyclonal antibody against a synthetic peptide having a specific sequence, a detection reagent, detection method and detection kit for Norovirus GI infection using this antibody.

  Norovirus is conventionally referred to as SRSV (Small Round Structured Virus) and also referred to as NLV (Norwalk-like virus), and depending on its genotype, GI (genogroup I) and GII Classified as (genogroup II). Norovirus is a small virus with a diameter of about 30 nanometers (one nano is one billionth) and grows only in the human intestine. A virus that causes food poisoning with symptoms such as diarrhea, vomiting, abdominal pain, nausea, and fever. It adheres to the internal organs of oysters and bivalves that live near the river mouth where sewage flows. It can be prevented by heating at 70 ° C. for 1 minute, but food poisoning occurs due to inadequate heating or raw consumption. Recently, human-to-human infection due to the excrement of virus carriers has increased.

  In the event of food poisoning, in order to keep the damage to a minimum, the cause is examined for specimens such as the feces of patients and suspected foods. Each bacterium has its own characteristics, so it is possible to find the target bacterium using a medium suitable for the bacterium, but in general, viruses do not grow on a synthetic medium like bacteria, so live cells will be used. . However, norovirus GI cannot be grown in cultured cells. Therefore, as a method of examination, a method of directly finding virus particles by an electron microscope, or amplifying a DNA or RNA fragment of a Norovirus GI gene collected from a specimen, and then amplifying the amplified product using electrophoresis or a spectrofluorometer A detection method is used.

Recently, a detection method of norovirus GI by enzyme immunoassay (ELISA) using a monoclonal antibody has been reported. The monoclonal antibody used is expressed in a baculovirus expression system using ORF2 encoding NV capsid protein. Monoclonal antibodies prepared using virus-like particles (VLPs) as antigens, and detection kits using these monoclonal antibodies are Genogroup I (GI) and Genogroup II (Genogroup II). , GII) are detected (Non-Patent Document 1).
“Japanese Clinical”, Vol. 60, No. 6, pp. 1188 to 1193 (2002), Japan Clinical Co., Ltd.

  The electron microscope method and the method using gene fragments are skilled and time consuming to make a detectable sample such as sample processing, and the detection process also takes time, and a large amount of samples cannot be processed. There are disadvantages such as. However, early investigation of the cause is necessary for the expansion of damage and the resolution of social anxiety, and food poisoning caused by oysters contaminated with norovirus GI develops with a trace amount of virus. The norovirus GI detection method has been awaited. In response, an ELISA method using a monoclonal antibody is being developed. The present inventors have intensively studied for a method capable of processing in a shorter time, simple operation and capable of simultaneously processing multiple specimens, and completed the present invention.

The first of the present invention is the following amino acid sequence comprising the 66th to 78th amino acid sequences common in the capsid region of genotype 1-14 of Norovirus GI as a suitable region used for the antigen:
Gly Glu Phe Thr Ile Ser Pro Asn Asn Thr Pro Gly Asp (SEQ ID NO: 1)
Is a monoclonal or polyclonal antibody prepared by adding a Cys residue to the N-terminus of the amino acid sequence, further adding KLH as a carrier to the Cys residue, and using this as an immunogen. The antibody can recognize or bind to Norovirus GI, Norovirus GI antigen or antigenic fragment. As the norovirus GI, 14 genotypes such as Norwalk strain, Chiba strain, Saitama strain and the like are known, but the antibody of the present invention does not affect the norovirus GI regardless of these genotypes. Can recognize and combine.

  The second of the present invention is a norovirus GI detection reagent containing the above monoclonal or polyclonal antibody.

    The detection reagent of the present invention includes a diagnostic agent for diagnosing a patient suspected of being infected with Norovirus GI and a test agent for examining foods suspected of being contaminated with Norovirus GI.

  A third aspect of the present invention is a method for detecting norovirus GI, wherein a specimen is brought into contact with the above monoclonal or polyclonal antibody or the above detection reagent and detected by ELISA.

  Specimens include specimens collected from patients suspected of being infected with Norovirus GI, such as stool, or specimens that are part of foodstuffs suspected of being contaminated with Norovirus GI.

  The ELISA used in the present invention includes a fluorescent ELISA.

  The 4th of this invention is a kit used for implementation of said norovirus GI detection method. In particular, the enzyme containing 1% skim milk as a blocking agent, and the enzyme linked to the above antibody and the substrate of the enzyme are (1) peroxidase and o-phenylenediamine or (2) β-galactosidase-avidin and 4-methylumbelliferyl- A kit containing β-D-galactoside.

  A fifth aspect of the present invention is a hybridoma that produces the above monoclonal antibody, and this hybridoma is assigned to the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology on March 16, 2005, and has the accession number FERM P-20461. And was deposited.

  The monoclonal or polyclonal antibody of the present invention is very sensitive. Therefore, according to the ELISA method using this, it is possible to detect Norovirus GI early in food, for example, oysters, etc., quickly, simply and with high sensitivity, and damage can be minimized. Further, since the antibody of the present invention can recognize and bind to Norovirus GI regardless of genotype, it can be tested without omission. In the detection system of the present invention, since norovirus GI is often contained in fish and shellfish, it is expected to be utilized by an organization that performs microbial inspection of fresh food. Moreover, it can be used not only for food inspection but also for stool inspection.

  To construct a highly sensitive and accurate immunoassay system, it is necessary to use high-quality antibodies. Therefore, an amino acid region considered to be optimal as an antibody epitope was selected from the biological information of Norovirus GI published on various databases, and a hybridoma producing a monoclonal antibody against this region was prepared.

Design of Synthetic Peptide Norovirus capsid is a collection of one protein called N protein, and N protein is divided into three regions, P2, P1, and S. The P1 and P2 regions are the regions exposed on the surface of the virus particles and are considered to be the regions that are most likely to react with the antibody. On the other hand, the S region is located inside the virus particle surface, but the amino acid sequence is relatively highly conserved among strains. In order to create an antibody capable of reacting with as many strains as possible, a synthetic peptide was prepared based on the amino acid sequence of the most conserved region among strains existing in the S region.

As a result, the 66th to 78th amino acid sequences were conserved in many strains of Norovirus G1, and the reactivity of the monoclonal antibody was the highest in the confirmation by production of the monoclonal antibody. Therefore, a synthetic peptide was prepared for the site, a Cys residue was added to the N-terminus of the prepared synthetic peptide, and KLH was added as a carrier to the Cys residue, which was used as an immunogen. The amino acid sequence of the synthetic peptide is as follows.
Gly Glu Phe Thr Ile Ser Pro Asn Asn Thr Pro Gly Asp SEQ ID NO: 1
The amino acid sequence to which Cys residue and carrier KLH are added is as follows.
KLH-Cys Gly Glu Phe Thr Ile Ser Pro Asn Asn Thr Pro Gly Asp SEQ ID NO: 2

Immunization schedule The synthetic peptide prepared above is diluted with physiological saline and mixed with an adjuvant. This antigen solution is administered intraperitoneally to mice. Further immunization is carried out, the antibody titer in the mouse serum is measured by ELISA, and this operation is repeated until the blood antibody titer becomes 64000 times. Synthetic peptides are also administered intraperitoneally 3 days before cell fusion.

Cell fusion After final immunization, the spleen is removed and dispersed into individual cells in DMEM medium. After centrifugation, DMEM medium is added to suspend splenocytes, and lysis buffer is added to lyse the red blood cells. After centrifugal washing again, the suspension is suspended in DMEM medium and the number of cells is counted. Then, the spleen cells and mouse myeloma cell P3U1 strain are mixed in a centrifuge tube. After centrifugation, the supernatant is removed and washed with DMEM medium. After centrifugation again to remove the supernatant completely, a DMEM medium containing polyethylene glycol (PEG) 6000 and DMSO is added. After centrifugal washing and removing the supernatant, the number of mouse myeloma cells is adjusted, and DMEM medium containing fetal calf serum (FCS) is added. After culturing at 37 ° C. for 1 day, the cells are collected and washed the next day, the number of mouse myeloma cells is adjusted, and 15% FCS / DMEM medium containing hypoxanthine, aminopterin, and thymidine (HAT) is added. Dispense into 96-well plates and incubate at 37 ° C. for 10-14 days to grow only hybridomas. After screening for a hybridoma producing the desired antibody by ELISA, positive clones are transferred to a 48-well plate, and 15% FCS / HT / DMEM medium containing no aminopterin is added to continue the culture.

  After the cells are sufficiently grown, screening by ELISA is performed again, and positive clones are cloned using the limiting dilution method. This screening and cloning is performed 2-3 times to make a single clone. As a result, a hybridoma that produces an antibody against an amino acid sequence common to Norovirus GI is obtained.

Method for producing monoclonal antibody The obtained hybridoma is cultured for about 1 week. After culturing, the medium is centrifuged to separate the supernatant, and then ammonium sulfate is added to a 50% saturation concentration to carry out salting out. After standing overnight, the mixture is centrifuged, the supernatant is discarded, and the precipitate is dissolved in PBS and dialyzed against PBS. This is purified by affinity chromatography to obtain a purified antibody against the amino acid sequence common to the Norovirus GI strain.

  The above monoclonal antibody is used for ELISA. Normal ELISA (enzyme immunoassay: enzyme immunoassay, EIA or ELISA) and fluorescent ELISA (FELISA) may be used. A fluorescence ELISA is preferable. The combination of ELISA label, substrate, and enzyme is appropriately determined.

  For example, as a detection system of a sandwich fluorescence ELISA system, a monoclonal antibody is first immobilized on a plate. After blocking, the sample is reacted. Thereafter, a mouse monoclonal antibody or rabbit polyclonal antibody labeled with biotin is reacted, avidin-labeled galactosidase or peroxidase is reacted, and finally a substrate solution (for example, 4-methylumbelliferyl-β-D-galactoside or o-phenylenediamine is reacted). ) Is added. The fluorescence intensity of each well is measured with a fluorescence ELISA microplate reader (see FIG. 1). As the blocking agent, for example, bovine serum albumin or skim milk is used. Alternatively, an antibody labeled with peroxidase without using an avidin-biotin bond can be used.

  Prepare the detection system of the above-mentioned fluorescence ELISA system as a part of an appropriate kit.

EXAMPLES Hereinafter, although this invention is demonstrated concretely based on an Example, it is only for description and it does not restrict | limit this invention.
Example 1
Preparation of anti-NV capsid protein monoclonal and polyclonal antibodies A-1. Preparation of mouse monoclonal antibody Synthetic peptide KLH-Cys Gly Glu Phe Thr Ile Ser Pro Asn Asn Thr Pro Gly Asp (SEQ ID NO: 2) is used as an immunogen, the antigen amount is 20 μg per mouse, and the adjuvant is RIBI Adjuvant System R-700 Immunization was performed at intervals of about 3 weeks by intraperitoneal inoculation using Spleen cells and myeloma cells of mice with increased antibody titers were fused with polyethylene glycol by a conventional method. After screening positive antibody-producing hybridomas by ELISA, cloning was performed 2-3 times by the limiting dilution method. The cloned antibody-producing hybridoma was cultured in a large amount in a serum-free medium, and the monoclonal antibody was purified by affinity chromatography. Some were labeled with biotin.

In mice immunized with a synthetic peptide designed from the strain-specific region of the amino acid sequence that can be an epitope of the Norovirus GI strain, cell fusion was performed using a mouse with a sufficiently high antibody titer after the third immunization. (Table 1).

  As a result, a plurality of hybridomas were obtained. When the antibody produced from each hybridoma was reacted with a synthetic peptide, the best clone 1 showed the best reaction. After mass-culturing this clone, the antibody was purified and the construction of a sandwich fluorescence ELISA system was examined.

A-2. Preparation of Rabbit Polyclonal Antibody Immunization was performed at intervals of about 4 weeks by subcutaneous inoculation using a synthetic peptide as an immunogen, an antigen amount of 50 μg per rabbit, and an adjuvant using RIBI Adjuvant System R-730. Whole blood was collected from rabbits with increased antibody titers. After blood collection, serum is separated and IgG is purified by the caprylic acid method, a part of which is described in Example 2 B-2. Biotin labeling was performed as described in.

Rabbits immunized with a synthetic peptide designed from the consensus sequence of Norovirus GI strain were boosted after the fourth immunization and whole blood collected (Table 2).

Example 2
Peroxidase labeling of secondary antibody B-1. Peroxidase labeling of mouse monoclonal antibody 0.2 ml (1.14 mg / ml) of mouse monoclonal antibody prepared in A-1 of Example 1 was labeled with peroxidase using Peroxidase labeling kit-SH.

  About 0.2 ml (1.3 mg / ml) of peroxidase-labeled mouse monoclonal antibody was prepared.

B-2. Biotin labeling of rabbit polyclonal antibody 1 ml (1.39 mg / ml) of rabbit polyclonal antibody prepared in A-2 of Example 1 was dialyzed overnight against sodium bicarbonate buffer (pH 8.5), and protein quantification was performed by the Bradford method. It was. Thereafter, EZ-Link-Sulfo-NHS-LC-Biotin dissolved in ultrapure water was mixed with 128 μl (64 μg) (50 μg biotin: 1 mg antibody) in 1 ml (1.28 mg / ml) of antibody. After standing on ice for 2 hours, it was dialyzed overnight at 4 ° C. against PBS (pH 7.4).

  About 1 ml (0.98 mg / ml) of a rabbit polyclonal antibody labeled with biotin was prepared from about 60 ml of serum.

Example 3
Confirmation of reaction of anti-NV GI mouse monoclonal antibody and anti-NV GI rabbit polyclonal antibody with peptide
A synthetic peptide prepared to 1 μg / mL with PBS was immobilized on an ELISA plate at 37 ° C. for 2 hours. Blocking was performed overnight at 4 ° C. using 1% skim milk as a blocking agent. A virus extract from Norovirus patient stool was used as a sample. Peroxidase-labeled anti-NV GI mouse monoclonal antibody and biotin-labeled anti-NV GI rabbit polyclonal antibody were diluted with PBS from 5 μg / mL to 2.5, 1.25, 0.63, 0.32, 0.16, 0.08, 0.04, 0.02, 0.01. For 2 hours. The plate reacted with the biotin-labeled anti-NV GI rabbit polyclonal antibody was reacted with avidin-labeled peroxidase at 37 ° C. for 1 hour. The substrate (o-phenylenediamine) was reacted at 37 ° C. for 30 minutes and measured with a microplate reader.

Results The peroxidase-labeled anti-NV GI mouse monoclonal antibody gave good results up to 0.01 μg / mL. Biotin-labeled anti-NV GI rabbit polyclonal antibody showed a reaction up to 0.32 μg / mL (FIG. 2).

Norovirus GI detection ELISA
Anti-NV GI mouse monoclonal antibody was prepared at 5 μg / ml in PBS on an ELISA plate and immobilized at 4 ° C. overnight. The reaction was performed at 37 ° C. for 2 hours using 1% skim milk as a blocking agent. As a sample, a viral extract from Norovirus patients flights use was prepared as viral load in PBS is ^{10,10 2, 10 3, 10 4} , 10 5 / well. The reaction was carried out at 37 ° C for 2 hours. Peroxidase-labeled anti-NV GI mouse monoclonal antibody or biotin-labeled anti-NV GI rabbit polyclonal antibody prepared at 500 ng / mL in PBS was reacted at 37 ° C. for 2 hours. The plate reacted with the biotin-labeled anti-NV GI rabbit polyclonal antibody was reacted with avidin-labeled peroxidase at 37 ° C. for 1 hour. Substrate (o-phenylenediamine) was reacted at 37 ° C. for 30 minutes, and then measured with a plate reader.

Results Detection antibody anti NV GI mouse monoclonal antibody, when using anti-NV GI rabbit polyclonal antibody, the absorbance value was detected high at both 10 ⁵ particles / well (Figure 3).

Confirmation of anti-NV GI mouse monoclonal antibody, anti-NV GI rabbit polyclonal antibody and genotype
Anti-NV GI mouse monoclonal antibody prepared at 5 μg / mL was immobilized on an ELISA plate at 4 ° C. overnight. 1% skim milk was reacted at 37 ° C. for 1 hour for blocking. Three types of GI / 1, GI / 9, and GI / 12 were prepared at 10 ⁵ particles / well using a patient stool extract virus solution with known genotype of Norovirus GI, and reacted at 37 ° C. for 2 hours. As a control, GII patient fecal extract virus solution was reacted in the same manner. Next, peroxidase-labeled anti-NV GI mouse monoclonal antibody or biotin-labeled anti-NV GI rabbit polyclonal antibody prepared at 500 ng / mL was reacted at 37 ° C. for 2 hours. The plate reacted with the biotin-labeled anti-NV GI rabbit polyclonal antibody was reacted with avidin-labeled peroxidase at 37 ° C. for 1 hour. The substrate (o-phenylenediamine) was reacted and then measured with a microplate reader.

Results When anti-NV GI mouse monoclonal antibody and anti-NV GI rabbit polyclonal antibody were reacted with the detection antibody, the absorbance of GII reacted as a control was detected low. Each norovirus GI genotype reacted as a sample also showed color development, confirming that it was clearly reacting (FIG. 4). Since this antibody is an antibody produced by using the amino acid sequence of the Norovirus GI capsid region as a synthetic peptide and the common part as a synthetic peptide, it was confirmed that it reacted with Norovirus GI regardless of genotype. (Figure 4).

Comparison of Norovirus GI Detection ELISA and Fluorescence ELISA Anti-NV GI mouse monoclonal antibody was prepared at 5 μg / ml in PBS on an ELISA plate and immobilized at 4 ° C. overnight. The blocking agent is 1% skim milk for 1 hour at 37 ° C. Samples of patient stool virus extract are prepared with PBS so that the viral load is 10, 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ / well And allowed to react at 37 ° C. for 2 hours. As a control, GII patient fecal extract virus solution was reacted in the same manner. As a biotin-labeled antibody, a mouse monoclonal antibody was prepared at 500 ng / ml and reacted at 37 ° C. for 2 hours. The examination was performed using β-galactosidase-avidin as the enzyme and 4-methylumbelliferyl-β-D-galactoside as the substrate.

Results In fluorescence ELISA, the fluorescence intensity of GII reacted as a control was measured to be slightly higher, but in the well reacted with the patient fecal extract virus solution, a difference from the control was observed from 10 ⁴ particles / well (FIG. 5).

Discussion As a detection system of the sandwich fluorescence ELISA system, first, a monoclonal antibody is immobilized on a plate. After blocking, the sample virus extract is reacted. Thereafter, mouse monoclonal antibody or rabbit polyclonal antibody labeled with biotin is reacted, and avidin-labeled galactosidase is reacted. Finally, the substrate solution 4-methylumbelliferyl β-D-galactoside is added. The fluorescence intensity of each well is measured with a fluorescence ELISA microplate reader. As can be seen by comparing a normal ELISA and a fluorescence ELISA, it was found that the fluorescence ELISA is more sensitive and can be detected even at a low concentration of virus.

  According to the ELISA method using the monoclonal or polyclonal antibody of the present invention, norovirus GI can be detected from foods quickly, conveniently and with high sensitivity, and the damage of food poisoning can be minimized. Since the detection system of the present invention often contains norovirus GI in fish and shellfish, it can be used in institutions that perform microbial testing of fresh foods, and can be used not only for food testing but also for stool testing. It is.

FIG. 1 shows an example of a detection system of a sandwich fluorescence ELISA system used in the method of the present invention. FIG. 2 shows a graph for confirming the reaction between the antibody and the synthetic peptide (Example 3). FIG. 3 shows a graph of confirmation of the reaction between the antibody (mono) and Norovirus GI from the Norovirus patient's stool (Example 3). FIG. 3 shows a graph for confirming the reaction between the antibody (poly) and Norovirus GI from the stool of a Norovirus patient (Example 3). FIG. 4 shows a graph of the reaction confirmation between the antibody (mouse monoclonal) and GI / 1, GI / 9 and GI / 12 (Example 3). FIG. 4 shows a graph for confirming the reaction of the antibody (rabbit polyclonal) with GI / 1, GI / 9 and GI / 12 (Example 3). FIG. 5 shows a graph of Norovirus GI detection fluorescence ELISA (F-ELISA) (Example 3).

Claims (9)

The following sequence:
A monoclonal or polyclonal antibody against a peptide containing Gly Glu Phe Thr Ile Ser Pro Asn Asn Thr Pro Gly Asp.   The antibody according to claim 1, which recognizes or binds Norovirus GI, Norovirus GI antigen or antigenic fragment.   The antibody according to claim 1 or 2, which is a monoclonal antibody.   A norovirus GI detection reagent comprising the antibody according to any one of claims 1 to 3.   A method for detecting Norovirus GI, comprising contacting a specimen suspected of containing Norovirus GI with the antibody according to any one of Claims 1 to 3 or the reagent according to Claim 4, and detecting the Norovirus GI.   A norovirus GI detection kit for performing the method of detecting norovirus GI according to claim 5.   A hybridoma producing the monoclonal antibody according to claim 3.   The hybridoma according to claim 8, which is the accession number FERM P-20461. A monoclonal antibody produced by the hybridoma of claim 9.

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