WO2014073464A1 - 三日熱マラリアと熱帯熱マラリアの双方を検出するペプチドおよび抗体検査材料 - Google Patents
三日熱マラリアと熱帯熱マラリアの双方を検出するペプチドおよび抗体検査材料 Download PDFInfo
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- WO2014073464A1 WO2014073464A1 PCT/JP2013/079666 JP2013079666W WO2014073464A1 WO 2014073464 A1 WO2014073464 A1 WO 2014073464A1 JP 2013079666 W JP2013079666 W JP 2013079666W WO 2014073464 A1 WO2014073464 A1 WO 2014073464A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56905—Protozoa
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
- C07K16/20—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans from protozoa
- C07K16/205—Plasmodium
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/44—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from protozoa
- G01N2333/445—Plasmodium
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/10—Detection of antigens from microorganism in sample from host
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a peptide for detecting a malaria parasite, in particular, a peptide antigen for measuring antibody titers of both Plasmodium falciparum and P. falciparum and an antibody test material containing the peptide. More specifically, the present invention relates to a peptide capable of binding to an antibody against both Plasmodium falciparum and Plasmodium falciparum in blood samples of humans and other animals, and an antibody test material containing the peptide.
- Non-Patent Document 1 WHO World Malaria Report 2011 (Non-Patent Document 1), malaria is estimated to have 216 million affected, 655,000 fatalities in 2010. 91% of deaths are reported from the African region, and an additional 86% are children under 5 years of age. With the progress of global measures, the number of affected persons and deaths in 2000 decreased by 17% and 26% respectively. Malaria has a wide range of endemic areas not only in developing countries but also in India, China, Brazil, Thailand, etc., which are rapidly emerging economically. Therefore, malaria is still one of the most important infectious diseases in the world, even when epidemic measures are progressing. In Japan, malaria is designated as a class 4 infectious disease, for which all reports are required.
- Plasmodium parasites that cause malaria in humans include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ⁇ ovale, Plasmodium ovale, And some of the five types of Salmalaria (Plasmodiumiknowlesi). Malaria parasites that enter the body due to the bite of mosquitoes that carry protozoa quickly enter the hepatocytes from the blood (primary intrahepatic stage), divide and proliferate in the hepatocytes, and are then released into the blood, resulting in red blood cells The protozoa that invades and repeats divisional proliferation (intrared blood cell cycle) is further propagated by other mosquitoes. Symptoms of fever from malaria are caused by an intraerythrocyte cycle. In particular, P. falciparum poses a risk of increasing severity and death if treatment is delayed compared to the other four.
- Kits that measure malaria infection history are needed in large quantities, for example, to control malaria infection in the Philippines (such as Palawan) and prove that the epidemic is disappearing.
- the IFAT method is still used because measurement kits are very expensive. This method also requires approximately one day of preparation of a test slide glass and observation with a fluorescence microscope. Since the fluorescence microscope is also very expensive (one million yen), only about 100 people were observed in one epidemic area survey.
- Non-patent Document 3 Similar research has been attempted for a long time by Mamoru Suzuki, Kumiko Sato (1980s-90s) and others in the Department of Parasitology and Gunma University School of Medicine. However, it has not been put to practical use because it is difficult to distinguish it from the response to normal serum with no history of malaria infection. That is, this method is only for research and is not suitable for practical use.
- Non-patent Document 1 a diagnostic material for malaria infection using a malaria parasite-derived antigen, a malaria vaccine (Patent Document 1), a method for producing a malaria antigen peptide (Patent Document 2), and a microparticle encapsulating a malaria antigen.
- Patent Document 3 A manufacturing method (Patent Document 3) is reported.
- an antigen against malaria it is known to use a peptide of a protein that has a well-conserved sequence in the species of Plasmodium and has few antigenic variations.
- Lactate Dehydrogenase (LDH, lactate dehydrogenase) peptides are known as antigens for generating antibodies (Non-patent Documents 4 and 5).
- An object of the present invention is to provide a novel antigen peptide that can be used as an antibody test material for Plasmodium.
- the present inventor has intensively studied to solve the above problems. As a result, it was found that the antibody titer of malaria parasite can be efficiently measured by using a peptide containing the amino acid sequence of SEQ ID NO: 3 as an antigen peptide, and the present invention has been completed.
- the present invention provides the following.
- a material for testing an antibody for malaria parasite comprising a peptide comprising the amino acid sequence of SEQ ID NO: 3 as an active ingredient.
- the antibody test material according to ⁇ 1> which is used for testing antibodies against Plasmodium falciparum and Plasmodium falciparum.
- ⁇ 3> The antibody test material according to ⁇ 1> or ⁇ 2>, wherein the peptide is immobilized on a carrier.
- the carrier is a polymer obtained by polymerizing the following compound (I) and compound (II).
- X represents halogen or -OY
- Y represents an alkyl group, aromatic group, pyridyl group, quinolyl group, succinimide group, maleimide group, benzoxazole group, benzothiazole group, or benzotriazole group, and these groups
- the hydrogen atom in may be substituted with a halogen.
- a test or diagnostic agent for malaria parasite infection comprising the antibody test material according to any one of ⁇ 1> to ⁇ 4>.
- ⁇ 6> A test kit or diagnostic kit for malaria parasite infection comprising the antibody test material according to any one of ⁇ 1> to ⁇ 4>.
- ⁇ 7> A method for examining or diagnosing a Plasmodium infection comprising the step of reacting the antibody test material according to any one of ⁇ 1> to ⁇ 4> with a sample of a subject having a Plasmodium infection.
- the novel antigenic peptide of the present invention is a type of antigenic peptide that can measure the current infection status and recent infection history of P. falciparum malaria and S. falciparum malaria that are particularly infected.
- the novel antigenic peptide of the present invention is less affected by the past (old) malaria infection history, and is a fever patient (a patient suspected of malaria) that is not malaria infection even in endemic areas, falciparum malaria and three-day fever malaria. It is possible to determine who is infected. Therefore, it can also be used for a simple inspection kit for the purpose of surveying endemic areas.
- the novel antigen peptide of the present invention can be immobilized on polymer nanoparticles, and the antibody titer of malaria parasite can be measured efficiently.
- FIG. 1 It is a schematic diagram of the structure of LDH derived from Plasmodium falciparum and LDH derived from Plasmodium falciparum.
- the sequence and position of the artificial antigen peptide used in the Examples are shown.
- Each peak in the figure is attributed to 7.9 min as the target product, and 8.9 min to the target product as a compound that has undergone dehydration reaction. Small peaks with elution times earlier than 7.9 min and 8.9 min overlap each other. These are attributed to a deletion sequence of 18 residues or less. The peak at 13-14 min is attributed to the product with the Arg residue protecting group remaining. It is a figure which shows the measurement result of the antibody titer of the plasma of the malaria patient and the fever patient which were extract
- Sensitivity when ROC analysis was performed by measuring 10 sera of P. falciparum and 10 sera of fever patients collected in the Philippines endemic area by the method of the present invention (pLDH microparticles) and the conventional method (AD22 microparticles) -It is a figure which shows the difference of a false positive. 10 sera from P. falciparum, 10 sera from P. falciparum and 10 sera from fever patients collected in the Philippines endemic area using the method of the present invention (pLDH microparticles) and the conventional method (AD22 microparticles) And it is a figure which shows the difference of a sensitivity and a false positive at the time of performing ROC analysis.
- the present invention relates to an antibody test material against Plasmodium that contains a peptide containing the amino acid sequence of SEQ ID NO: 3 as an active ingredient, that is, an antigen peptide.
- the present inventors focused on Lactate Dehydrogenase (LDH, lactate dehydrogenase), a protein that has a well-conserved sequence and has few antigenic mutations in four types of Plasmodium species that infect humans. went. In particular, we searched for a type of antigenic peptide that can measure the infection history of tropical fever and S. falciparum malaria, which are especially infected.
- LDH Lactate Dehydrogenase
- the peptide containing the amino acid sequence of SEQ ID NO: 3 includes the peptide of SEQ ID NO: 3 and peptide analogs generated by substitution, deletion and / or insertion of the amino acids constituting the peptide of SEQ ID NO: 3.
- the peptide analog refers to a peptide having the same activity as the peptide of the present invention with respect to an immunological response in which amino acids constituting the peptide of SEQ ID NO: 3 are substituted, deleted, and / or inserted.
- the number of amino acids to be substituted, deleted and / or inserted is not particularly limited, but is preferably 1 to 3, more preferably 1 to 2.
- the antigen peptide containing the amino acid sequence of SEQ ID NO: 3 is preferably a peptide having 19 to 21 residues.
- the peptide may be labeled with a fluorescent substance or the like, or may contain an unnatural amino acid.
- the sequence of the peptide of the present invention can be prepared by any synthetic method such as an organic chemical method or a biochemical method based on the sequence.
- a method for preparing the peptide antigen of the present invention by an organic chemical method for example, (1) the peptide antigen of the present invention is divided into several parts and synthesized separately, and the peptides are bound by combining them.
- synthesis may be performed using a synthesis procedure other than the methods disclosed in the examples of the present invention.
- any peptide synthesis method that can be used by those skilled in the art, such as synthesis of the peptide compound of the present invention by an automatic peptide synthesizer, may be used.
- the peptide of the present invention can also be obtained by a biochemical method (that is, recombinant DNA technology). For example, by expressing an LDH protein using an E. coli expression system using an LDH protein expression vector in which a DNA encoding the entire or partial sequence of the malaria parasite LDH gene is inserted downstream of the promoter of the E. coli expression vector. Achieved.
- This expression vector can be constructed according to a known method (for example, Sambrook and Russel, MOLECULAR CLONING: A LABORATORY MANUAL, 3rd edition (2001)).
- a peptide compound having a partial LDH sequence can be obtained by transforming Escherichia coli using this vector based on a known method, and producing, recovering and purifying the protein.
- the peptide introduced into the fine particles may be one in which a plurality of sequences are linked in a linear or branched manner.
- carrier molecules that can be used to link peptide sequences include natural proteins such as tetanus toxoid, ovalbumin, serum albumin, hemocyanin and the like.
- the carrier is hemocyanin
- the amino groups of the peptide and the carrier can be exchanged with glutaraldehyde by the method of Boquet et al. (P. It can also be combined.
- a synthetic polymer carrier called MAP (multiple antigenic peptide) or lysine dendrimer can also be used.
- MAP lysine dendrimer
- a target cross-linked product can be prepared by reacting lysine in a stepwise manner to a resin in which a ⁇ -alanine-cysteine (S-acetamidomethyl) dipeptide is immobilized.
- a conjugate of one lysine per dipeptide is obtained as a dimeric branched peptide
- a lysine 3-residue conjugate obtained by further reacting lysine is obtained by further reacting lysine as a tetrameric branched peptide.
- a conjugate of lysine 7 residues can be used as a cross-linked octamer.
- the octamer can also be obtained by oxidatively deprotecting the acetamidomethyl group of the cysteine residue with iodine to form a disulfide bond.
- a multimeric peptide can be synthesized by sequentially reacting the structural amino acids of the target peptide to these cross-linked products by a conventional method.
- the peptide antigen of the present invention bound to an anti-LDH antibody can be detected using a detection system known in the art, such as a fluorescence ELISA method or an assay using an agglutination reaction. From this, it was found that the peptide antigen of the present invention can be used as a novel peptide antigen that can be used as an immunodiagnostic material for infection with P. falciparum malaria and P. falciparum malaria. Therefore, the peptide sequence of the present invention is useful as a diagnostic material for diagnosing malaria infection, in particular, as an artificial antigen that reacts very easily with serum antibodies from malaria patients.
- the peptide antigen of the present invention can be provided as an immunodiagnostic material by binding to, immobilizing or adsorbing to a solid phase surface.
- a solid surface include, but are not limited to, a solid surface such as a film, latex particle, polymer fine particle, plastic plate, or microbead.
- the compounds of the present invention bound to polymer microparticles can be used for agglutination reactions as described in detail below.
- the present invention is an antibody test material in which the peptide of the present invention is immobilized on a carrier comprising a polymer obtained by polymerizing the following compound (I) and compound (II). About.
- X represents halogen (chlorine, fluorine, bromine, etc.) or -OY, where Y is an alkyl group, aromatic group, pyridyl group, quinolyl group, succinimide group, maleimide group, benzoxazole group, benzothiazole group, or A benzotriazole group is shown, and a hydrogen atom in these groups may be substituted with a halogen (chlorine, fluorine, bromine or the like).
- alkyl group examples include groups such as methyl group, ethyl group, n-propyl group, isopropyl group, n-butyl group, t-butyl group, isobutyl group and sec-butyl group; Examples include groups such as a phenyl group, 1-naphthyl group and 2-naphthyl group; examples of the pyridyl group include groups such as a 2-pyridyl group, a 3-pyridyl group and a 4-pyridyl group.
- Examples of the quinolyl group include groups such as 2-quinolyl group, 3-quinolyl group, 4-quinolyl group, 5-quinolyl group, 6-quinolyl group, 7-quinolyl group, and 8-quinolyl group.
- Examples of the benzoxazole group include 2-benzoxazole group; examples of the benzothiazole group include 2-benzothiazole. It is like; examples of the benzotriazole group, for example, 1-benzotriazole group. Of these, phenyl, 3-pyridyl, 8-quinolyl, succinimide (OSu), 2-benzothiazole, and 1-benzotriazole (OBt) are more active in active esters. This is preferable.
- the polymerization method can be carried out by ordinary radical polymerization, and a method using radiation ( ⁇ rays) or a polymerization initiator is exemplified.
- a known radical polymerization initiator can be used, for example, azobisisobutyronitrile (AIBN), 1,1′-azobis (cyclohexanecarbonitrile) (ABCN), etc. Can do.
- the solvent may be any solvent that can dissolve each compound and allow the polymerization reaction to proceed.
- MIBK methyl ethyl isobutyl ketone
- dimethylformamide dimethyl
- dimethyl include acetamide, N-methylpyrrolidone, and mixed solvents thereof.
- the particle size of the fine particles obtained by the polymerization reaction is preferably 0.1 to 10 ⁇ m.
- the peptide can be introduced onto the surface of the fine particles by a reaction between the active ester group -OY of the fine particles and the amino group of the peptide.
- the amino group of the peptide may be a terminal amino group or a side chain amino group.
- a linker or carrier may be added to the end of the peptide and bound to the amino group of the linker or carrier.
- the ratio of the peptide introduced into the microparticles is preferably 0.05 to 2% by weight.
- the present invention relates to a test agent or diagnostic agent for malaria parasite infection containing the antibody test material of the present invention. Moreover, this invention relates to the test kit or diagnostic kit of the malaria parasite infection which contains the antibody test material of this invention.
- the peptide sequence of the present invention is an artificial antigen that is highly reactive with serum antibodies, particularly in patients with malaria infected with P. falciparum and P. falciparum, and a test agent or diagnostic agent and test for testing or diagnosing malaria infection Useful as a kit or diagnostic kit.
- the test drug or diagnostic drug and test kit or diagnostic kit of the present invention are used in test drugs or diagnostic drugs and test kits or diagnostic kits known to those skilled in the art, such as buffer solutions. It can be configured by each element.
- an antibody against malaria parasite in a sample can be detected.
- the antibody test material of the present invention can detect, for example, a malaria parasite-derived anti-LDH antibody in a blood sample of a subject, and can be used as a material to test malaria diagnosis / infection history survey and confirmation of antibody titer maintenance after vaccine administration. can do.
- a sample blood, serum, plasma or the like of a subject can be used.
- the detection method is not particularly limited as long as it is a method for detecting a binding reaction, and examples thereof include an ELISA method, an assay using an agglutination reaction, a fluorescence detection method, a luminescence detection method, a visible ultraviolet absorption detection method, and an electrochemical detection method.
- an assay by an agglutination reaction using polymer fine particles on which the peptide antigen of the present invention is immobilized is preferable. Fine particles are aggregated by the reaction of the peptide antigen with the antibody, and the antibody can be detected visually.
- the antibody that can be detected is an antibody to the peptide sequence introduced into the microparticle (anti-peptide antibody, eg, anti-pLDH antibody), or a protein containing this peptide sequence (anti-protein antibody, eg, anti-Plasmodium falciparum LDH antibody that recognizes the pLDH sequence, Anti-Plasmodium vivax LDH antibody) and antibodies against homologous peptide sequences and proteins (antibodies made from closely related proteins and peptide sequences) with amino acid residues mutated.
- anti-peptide antibody eg, anti-pLDH antibody
- anti-protein antibody eg, anti-Plasmodium falciparum LDH antibody that recognizes the pLDH sequence, Anti-Plasmodium vivax LDH antibody
- antibodies against homologous peptide sequences and proteins antibodies made from closely related proteins and peptide sequences
- homologous peptide sequences in which amino acid residues are mutated include, for example, sequences derived from closely related proteins, sequences derived from mutated proteins, and high homology (amino acid homology> 60%). Examples include amino acid sequences derived from proteins.
- a test material was prepared in which the antigen peptide of P. falciparum (Comparative Example 1: (AD22) 4 -MAP) 3 mg) was chemically bonded to the surface of 300 mg of fine particles via an active ester group (succinimide group). At this time, the chemical bond was reacted at 37 ° C. for 4 hours, and the physical adsorption was performed at 37 ° C. for 20 hours.
- ⁇ Aggregation test Pulmonary falciparum patient plasma (Pf patient), normal volunteer plasma (normal), suspected malaria blood collection as a fever patient, collected at the National Institute of International Medical Research Center, but received rapid detection kit for antigen detection and smear observation with microscope The agglutination test was performed using plasma of patients diagnosed as negative from the above (fever patients). In a 96-well plate, add 8 ⁇ l each of 50 ⁇ L of subject serum diluted 16-2048 times with phosphate buffered saline (PBS), and add 50 ⁇ L of phosphate buffer control to one well, Finally, 25 ⁇ L (0.1 mg / mL) of the fine particles were added.
- PBS phosphate buffered saline
- the 96-well plate was shaken and stirred for 1 minute, and then allowed to stand at room temperature for 8 hours for reaction to detect agglutination.
- Example 1 The present inventors consider a peptide antigen sequence that is a protein derived from a malaria parasite and that can distinguish sera of patients in endemic areas, and the sequence is well conserved in (four types of protozoan species that infect humans). Antigen design was carried out focusing on lactate dehydrogenase (LDH, lactate dehydrogenase), a protein with few antigenic mutations. In particular, we searched for a type of antigenic peptide that can measure the infection history of tropical fever and S. falciparum malaria, which are especially infected.
- LDH lactate dehydrogenase
- P. falciparum peculiar sequence partial peptide 18 residues SEQ ID NO: 4
- P. falciparum malaria peculiar sequence peptide 18 residues pvLDH
- P. falciparum LDH P. falciparum LDH
- the condensing agent equimolar HCTU / HOBt / DIEA and a protected amino acid were used, and each reaction time was 30 min.
- 3% DBU / DMF condition b
- the resin was cut out from the resin. This solution was collected, the resin was washed with CH 2 Cl 2 , and the peptide was obtained by drying under reduced pressure.
- the obtained peptide was washed with cold diethyl ether and dried by drying under reduced pressure to obtain a crude product (the yield of the crude product is described in Table 2).
- the crude product was subjected to 15, 20, 25, 30% acetonitrile aqueous solution (0.1% trifluoroacetic) by solid phase extraction of ODS column (Waters SepPack ODS-5g). acid) was eluted with 50 mL each, and 10 mL was fractionated. Thereafter, each fraction was measured by HPLC and ESI-MS, and the target product was confirmed with a fraction of 20% -2, and the target product was obtained by lyophilization (the yield after purification is described in Table 2).
- 3 and 4 show the HPLC chromatogram and ESI-MS spectrum of the purified product, respectively.
- HPLC condition analyzer Shimadzu LC-2010C-HT Analytical column: YMC-PACK ODS (4 x 100 mm, particle size 3 ⁇ m) Solvent conditions: 10% -70% acetonitrile aqueous solution (0.1% trifluoroacetic acid) Analysis time: 30 minutes, flow rate: 1 mL / min ESI-MS condition analyzer: AP-SCIEX API-2000 Solvent: 10% acetonitrile aqueous solution (0.1% trifluoroacetic acid) Flow rate: 10 mL / min
- the antigen peptide and the polymer microparticles were reacted in an incubator at 37 ° C. for 24 hours in the same manner as in Comparative Example 1 except that they were not converted to MAP. Thereafter, the mixture was centrifuged to block the reaction. During the reaction, the reaction solution was sampled at reaction times of 4 hours and 24 hours, and the progress of the reaction was confirmed by detecting the generated HOSu by reverse phase HPLC.
- Plasma fever and P. falciparum mixed infection patient plasma Pf, Pv mixed infection patient
- P. falciparum malaria patient plasma Pf patient
- P. falciparum malaria patient plasma An agglutination test was performed using each sample of Pv patients) and plasma (fever patients) who had received blood from suspected malaria as a fever patient.
- To a 96-well plate 25 ⁇ L each of plasma samples diluted 16-32768 times with phosphate buffered saline (PBS-0.1% tween20) was added to 12 wells, and finally 25 ⁇ L of nanoparticles modified with pLDH antigen were added. (0.1 mg / mL) was added in portions.
- the 96-well plate was shaken and stirred for 1 minute, and then allowed to stand at room temperature for 8 hours for reaction to detect agglutination.
- the antibody titer indicating a significant difference was successfully measured for malaria patients (Pf, Pv mixed infection patients, Pf patients, Pv patients) and fever patients who were not malaria.
- the obtained antibody titer was compared with the ELISA method which is a conventional antibody titer test method.
- the ELISA method was carried out by using diluted plasma of primary antibodies (dilution ratios 1/64, 1/256, 25 ⁇ L each) on a NUNC 96-well microplate (immobilizer amino) to which antigen peptides (10 ⁇ g each) were bound.
- HRP-modified anti-human IgG antibody (dilution ratio 1/1000, 100 ⁇ L) and ABTS (concentration 0.7 mg / mL, 300 ⁇ L) were used as detection reagents.
- the absorbance at 405 nm in a microplate reader when the plasma dilution rate was 1/64, 1/256 was used. As shown in FIG. 8 (dilution rate 64 times) and FIG. 9 (dilution rate 256 times), it was found that the antibody titer measurement using nanoparticles was sufficiently correlated with the conventional ELISA measurement method.
- Table 15 show differences in sensitivity and false positives when ROC analysis is performed by measuring 10 sera of falciparum malaria patients and 10 sera of fever patients collected in Philippine endemic areas.
- Table 5 and FIG. 16 show the sensitivity and sensitivity when ROC analysis was performed on 10 sera of P. falciparum, 10 sera of P. falciparum, and 10 sera of fever. Shows false positive differences.
- Table 3 and FIG. 14 that the method of the present invention is superior in sensitivity and specificity as compared with the conventional method.
- Table 4 and FIG. 15 that the method of the present invention is superior in sensitivity and specificity as compared with the conventional method.
- Table 5 and FIG. 16 that the method of the present invention is superior in both sensitivity and specificity as compared with the conventional method.
- the method of the present invention using a single antigen is slightly inferior in specificity as compared with the IFAT method using the entire protozoan antigen.
- the method of the present invention is capable of analyzing multiple specimens simultaneously (IFAT measures each patient sample with a fluorescence microscope) and is stable even at room temperature because the test material is a synthetic material (protozoal antigen used in IFAT method) Erythrocyte samples require strict refrigerated storage).
- the method of the present invention is simple, it is particularly suitable for distinguishing serum samples without a history of malaria.
- the IFAT method is convenient for finding examples of high antibody titers.
- malaria antibody titers are often low in endemic areas where the number of patients is decreasing due to the progress of malaria countermeasures as in the Philippines, and as a result, the method of the present invention is used in clinical settings, endemic areas, It is judged that there is a great practical advantage such as epidemiological survey.
- the antigen microparticles prepared according to the present invention are expected to be able to carry out a great number of tests simultaneously as an alternative to the IFAT method, which conventionally took about one day to diagnose one specimen. Furthermore, since the present invention can obtain a result in 5 hours if there is a serum sample of 3 ⁇ L, it is expected to investigate the epidemiological survey of local populations in endemic areas and the temporal transition of epidemics between groups. .
- the IFAT method is a technique for diagnosing the current infection status and recent history based on the high antibody titer against Plasmodium.
- IFAT is no longer implemented in Japan, the development of an IFAT alternative method to reliably diagnose “whether it was malaria or not” about past fever during stay in malaria endemic areas and after stay is clinical It is strongly desired from the field.
- the present invention provides a technique that meets such a demand.
- the novel antigen peptide produced by the present invention is useful in fields such as medicine, diagnosis and research.
- it can be used for diagnosis of infection with P. falciparum and T. falciparum, determination of immune status, and determination of the presence or absence of epidemic (especially observing the end of the epidemic).
- the present invention can be applied to the following test kits. Malaria inspection kit that can be used in trendy areas and bedsides that do not require freezing and refrigeration equipment for transport and storage, and does not require special equipment or power supply during measurement.
- An infection test kit used to diagnose malaria patients in endemic areas and imported malaria patients in non-endemic areas. (This is useful for malaria countermeasures for endemic residents and for inspection of overseas travelers who have returned from endemic areas.)
Abstract
Description
わが国では、マラリアは全数届出が義務づけられている4類感染症に指定されている。1959年に土着のマラリアは制圧されたが、その後わが国の経済成長によって人の移動が盛んになり、日本人渡航者が流行地で感染する例や、流行地から日本への入国者が国内で、発症する“輸入マラリア”の症例が1980年代から急増した。そして2000年には最多の154例の報告があったが、現在は渡航者のマラリア予防知識の普及などにより、年間50-60症例に抑えられている。また隣国の韓国では、いったん制圧された“土着マラリア”が再興し、2000年には4000症例まで増加、現在でも年間1000-2000症例で推移している。韓国から日本への輸入マラリア症例も報告されている(非特許文献2)。よってマラリア対策は流行地のみならず、日本国の水際でも重要な課題となっている。
したがって、マラリア感染を検出したりマラリアワクチンの効果を確かめたりするための血清抗体価を簡便に測定できる試薬の開発が求められている。
しかしながら、マラリア感染症の現在の感染状況及び最近の感染履歴を測定できる、すなわち、流行地調査などを目的として、簡易検査キットに用いる新しい抗原が、さらに求められていた。
マラリアに対する抗原として、マラリア原虫種内で配列がよく保存されていて抗原変異の少ない蛋白質のペプチドを用いることが知られている。抗体を生成するための抗原として、Lactate Dehydrogenase(LDH、乳酸脱水素酵素)ペプチドが知られている(非特許文献4,5)。
<1>配列番号3のアミノ酸配列を含むペプチドを有効成分とする、マラリア原虫の抗体検査材料。
<2>熱帯熱マラリア原虫及び三日熱マラリア原虫の抗体検査用である、<1>に記載の抗体検査材料。
<3>ペプチドが、担体に固定化されている、<1>又は<2>に記載の抗体検査材料。
<4>担体が、下記化合物(I)と化合物(II)を重合反応により得られる重合体である、<3>に記載の抗体検査材料。
<5><1>~<4>のいずれかの抗体検査材料を含む、マラリア原虫感染症の検査薬又は診断薬。
<6><1>~<4>のいずれかの抗体検査材料を含む、マラリア原虫感染症の検査キット又は診断キット。
<7><1>~<4>のいずれかの抗体検査材料を、マラリア原虫感染症の被験者の試料と反応させる工程を含む、マラリア原虫感染症の検査方法又は診断方法。
本発明の新規抗原ペプチドは、特に感染者の多い熱帯熱マラリアと三日熱マラリアの現在の感染状況及び最近の感染履歴を測定できるタイプの抗原ペプチドである。本発明の新規抗原ペプチドは、過去の(古い)マラリアの感染履歴の影響が少なく、流行地においてもマラリア感染ではない熱発患者(マラリアの疑いがある患者)と、熱帯熱マラリアと三日熱マラリアの感染患者とを判定することが可能である。したがって、流行地調査などを目的とした、簡易検査キットに用いることも可能である。
本発明の新規抗原ペプチドは、高分子ナノ微粒子に固定化することが可能であり、マラリア原虫の抗体価を効率よく測定することができる。
(1)マラリア原虫に対する抗体検査材料
本発明は、配列番号3のアミノ酸配列を含むペプチドを有効成分とする、すなわち、抗原ペプチドとする、マラリア原虫に対する抗体検査材料に関する。
本発明者らは、ヒトに感染する4種類のマラリア原虫種内では配列がよく保存されていて抗原変異の少ない蛋白質であるLactate Dehydrogenase(LDH、乳酸脱水素酵素)に着目して、抗原設計を行った。特に感染者の多い熱帯熱と三日熱マラリアの感染履歴を測定できるタイプの抗原ペプチドを探索した。その結果、三日熱マラリア原虫由来LDHと熱帯熱マラリア原虫由来LDHの共通配列部分ペプチド19残基(pLDH)(配列番号3)が得られたものである。
配列番号3のアミノ酸配列を含むペプチドとしては、配列番号3のペプチド並びに配列番号3のペプチドを構成するアミノ酸が置換、欠失および/また挿入することにより生成されるペプチド類縁体が含まれる。ペプチド類縁体とは、配列番号3のペプチドを構成するアミノ酸が置換、欠失および/また挿入され、免疫学的応答に関して本発明のペプチドと同様の活性を有するペプチドのことをいう。置換、欠失および/また挿入されるアミノ酸の個数は特に制限されないが、1~3個が好ましく、1~2個がより好ましい。
配列番号3のアミノ酸配列を含む抗原ペプチドは、好ましくは19~21残基のペプチドが好ましい。
ペプチドは蛍光物質等で標識されたものでもよいし、非天然アミノ酸を含むものでもよい。
本発明は、本発明のペプチドが、下記化合物(I)と化合物(II)を重合反応により得られる重合体からなる担体に固定化されている、抗体検査材料に関する。
重合開始剤としては、公知のラジカル重合開始剤を使用することができ、例えば、アゾビスイソブチロニトリル(AIBN)、1,1'-アゾビス(シクロヘキサンカルボニトリル)(ABCN)などを使用することができる。
微粒子へのペプチドの導入割合は好ましくは重量比として0.05~2%である。
本発明は、本発明の抗体検査材料を含む、マラリア原虫感染症の検査薬又は診断薬に関する。また、本発明は、本発明の抗体検査材料を含む、マラリア原虫感染症の検査キット又は診断キットに関する。
本発明のペプチド配列は、特に熱帯熱マラリア及び三日熱マラリア感染マラリア患者の血清抗体と、非常に反応しやすい人工抗原であり、マラリア感染を検査又は診断するための検査薬又は診断薬及び検査キット又は診断キットとして有用である。
本発明の検査薬又は診断薬及び検査キット又は診断キットには、本発明の抗体検査材料以外に、緩衝液等、当業者に公知の検査薬又は診断薬及び検査キット又は診断キットに用いられている各要素によって構成することができる。
本発明の抗体検査材料を用いることにより、試料中のマラリア原虫に対する抗体を検出することができる。
本発明の抗体検査材料により、例えば、被験者の血液試料中のマラリア原虫由来抗LDH抗体を検出でき、マラリアの診断・感染履歴の調査やワクチン投与後の抗体価維持の確認を検査する材料として利用することができる。
試料は、被験者の血液、血清、血漿等を使用することができる。
ペプチド抗原が抗体と反応することにより微粒子が凝集し、目視により抗体の検出が可能である。
下記化合物(i)(市販品、中村化学製)0.4gと化合物(ii)(メタクリル酸クロリドとHOSu(N-ヒドロキシスクシンイミド)の反応生成物を使用)を0.1gを溶媒プロピオン酸エチル10mL中、室温25℃で3時間γ線照射下(30kGy)で反応させ、重合反応を行った。
微粒子表面に結合させるペプチドは以下のものを用いた。
AD22(MAP化した分子量として1.4 kD)Ala Ser Glu Phe Tyr Asn Ser Glu Asn Lys Thr Tyr Asp Leu Asp Phe Lys Thr Pro Asn Asn Asp(配列番号6)
この抗原ペプチド配列に基づき、(AD22)4-MAP (MAP = multiple antigenic peptide))(分子量1.4kD)(図13)を、手動合成装置を用いて合成し、SepPack(waters社製の使い捨てODSカラム)にて精製した。
続いて、活性エステル基(スクシンイミド基)を介して300mgの微粒子表面に熱帯熱マラリア原虫の抗原ペプチド(比較例1:(AD22)4-MAP)3mg)を化学結合させた検査材料を作製した。
このとき化学結合には37℃4時間、さらに物理吸着に37℃20時間の条件にて反応させた。
上記ペプチドの化学結合に伴って遊離するHOSu(N-ヒドロキシスクシンイミド)量をHPLCによってモニターすることで、反応の進行を確認した。
国立国際医療研究センター研究所において保管されている熱帯熱マラリア患者血漿(Pf患者)、正常なボランティア血漿(正常)、熱発患者としてマラリア疑い採血を受けたが抗原検出迅速診断キットと顕微鏡によるスメアー観察から陰性と診断された患者血漿(熱発患者)を用いて凝集試験を行った。
96穴プレートに、8個のウェルに、リン酸緩衝生理食塩水(PBS)で16~2048倍希釈した被験者血清を50μLずつ、および1個のウェルにリン酸緩衝液のコントロールを50μLを加え、最後に上記微粒子を25μL(0.1mg/mL)ずつ加えた。96穴プレートを1分間振動撹拌後に、室温で8時間静置して反応させることで、凝集反応を検出した。
その結果、Pf患者と正常、Pf患者と熱発患者について一定の違いを表す陽性凝集像と陰性凝集像を得ることに成功した。
本発明者らは、マラリア原虫由来の蛋白質であって、流行地患者血清の区別が可能なペプチド抗原配列を考え、(ヒトに感染する4種類の)原虫種内では配列がよく保存されていて抗原変異の少ない蛋白質であるLactate Dehydrogenase(LDH、乳酸脱水素酵素)に着目して抗原設計を行った。特に感染者の多い熱帯熱と三日熱マラリアの感染履歴を測定できるタイプの抗原ペプチドを探索した。
アミノ酸配列
(Gly-Phe-Thr-Lys-Ala-Pro-Gly-Lys-Ser-Asp-Lys-Glu-Trp-Asn-Arg-Asp-Asp-Leu-Leu)
(配列番号3)-Lys
組成式C102H161O32N29(mw. 2304.19)
pLDH抗原の合成はFmoc固相合成法によりShimadzu PSSM8自動ペプチド合成装置を用いて行った。固相合成で使用した樹脂はFmoc-Lys(Boc)-PEG-resin(0.18 mmol / g)を54 mg用いた。この樹脂を室温にて、3時間DMFで膨潤させた後(条件a)、脱Fmoc反応、保護アミノ酸の縮合反応、を繰り返して行った。合成に用いたFmoc保護アミノ酸の重量を表1に示した(各0.10 mmol、10 当量)。縮合剤は保護アミノ酸と等モルのHCTU/HOBt/DIEAを用い、各反応時間を30 minとした。脱Fmocには3 %DBU/DMF(条件b)を用いた。全アミノ酸縮合後、DMFとCH2Cl2によって樹脂を洗浄、trifluoroacetic acid : H2O : triisopropylsilane = 95 : 2.5 : 2.5の混合試薬2 mLを加えて、室温で20時間静置し(条件c)、樹脂からの切り出しを行った。この溶液を回収し、CH2Cl2で樹脂の洗浄を行い、減圧乾燥によってペプチドを得た。得られたペプチドを冷ジエチルエーテルで洗浄を行い、減圧乾燥によって乾燥させ、粗生成物を得た(粗生成物の収量は表2に記載)。この粗生成物をHPLC(図2)、ESI-MSにより目的物を確認後、ODSカラムの固相抽出(Waters SepPack ODS-5g)によって、15, 20, 25, 30 % アセトニトリル水溶液(0.1 % trifluoroacetic acid)を各50 mLで溶出させ、10 mLずつ分取した。その後、各フラクションをHPLCとESI-MSによる測定を行い、20 %-2のフラクションで目的物を確認し、凍結乾燥によって目的物を得た(精製後の収量は表2に記載)。図3、図4にそれぞれ精製物のHPLCクロマトグラムとESI-MSスペクトルを示した。
分析装置:Shimadzu LC-2010C-HT
分析カラム: YMC-PACK ODS(4 x 100 mm、粒子径3μm)
溶媒条件: 10%-70% アセトニトリル水溶液(0.1 % trifluoroacetic acid)
分析時間: 30分、流速: 1 mL/min
ESI-MS条件
分析装置:AP-SCIEX API-2000
溶媒: 10% アセトニトリル水溶液(0.1 % trifluoroacetic acid)
流速: 10 mL/min
表2に記載のように、3つの条件a~cについて変更することで、目的物の収量が12 %から50 %へ大幅に向上した。HPLCクロマトグラムの比較(図2と図6)から、切り出し反応の温度と時間(条件c)によってArg残基側鎖が残ることなく目的物の収量が大幅に向上したことがわかる。さらに(条件a)膨潤時間の延長によって固相反応樹脂へ十分にDMF溶媒を親和させることで反応試薬が十分に樹脂内へ行き渡り、(条件b)脱Fmoc試薬をpiperidineからDBUへ変更することで、脱Fmoc反応収率が向上し、合成中のペプチド鎖にFmoc基が残ることがないため、結果として欠損配列の生成が抑えられたことがわかった。
抗原ペプチドの高分子微粒子への化学修飾は、MAP化しなかったこと以外は上記比較例1と同様にして、抗原ペプチドと高分子微粒子をそれぞれインキュベーター内37℃で24時間反応させた。その後遠心分離し反応をブロッキングした。途中、反応時間4時間、24時間に反応溶液のサンプリングを行い、生成したHOSuを逆相HPLCにより検出することで反応の進行を確認した。
フィリピン大学マニラ校公衆衛生学部において保管されている、熱帯熱・三日熱マラリア混合感染患者血漿(Pf,Pv混合感染患者)、熱帯熱マラリア患者血漿(Pf 患者)、三日熱マラリア患者血漿(Pv 患者)、熱発患者としてマラリア疑い採血を受けた血漿(熱発患者)、の各試料を用いて凝集試験を行った。96穴プレートに、12個のウェルに、リン酸緩衝生理食塩水(PBS-0.1% tween20)で16~32768倍希釈した血漿試料を25μLずつを加え、最後にpLDH抗原を修飾したナノ微粒子を25μL(0.1mg/mL)ずつ加えた。96穴プレートを1分間振動撹拌後に、室温で8時間静置して反応させることで、凝集反応を検出した。その結果、図7に示すように、マラリア患者(Pf,Pv混合感染患者、Pf 患者、Pv 患者)とマラリアではない熱発患者について有意な違いを表す抗体価を測定することに成功した。
本発明は、このような要望に対応する技術を提供するものである。
より具体的には、以下の検査キットに適用が可能である。
輸送や保管に冷凍冷蔵設備を必要せず、測定時に特別な設備や電源を必要としない流行地やベッドサイドでも使用可能なマラリア検査キット。
流行地のマラリア患者や非流行地における輸入マラリア患者の診断に用いる感染検査キット。(流行地住民へのマラリア対策、流行地から帰国した海外渡航者の検査に有用である。)
Claims (7)
- 配列番号3のアミノ酸配列を含むペプチドを有効成分とする、マラリア原虫の抗体検査材料。
- 熱帯熱マラリア原虫及び三日熱マラリア原虫の抗体検査用である、請求項1に記載の抗体検査材料。
- ペプチドが、担体に固定化されている、請求項1又は2に記載の抗体検査材料。
- 請求項1~4のいずれか一項の抗体検査材料を含む、マラリア原虫感染症の検査薬又は診断薬。
- 請求項1~4のいずれか一項の抗体検査材料を含む、マラリア原虫感染症の検査キット又は診断キット。
- 請求項1~4のいずれか一項の抗体検査材料を、マラリア原虫感染症の被験者の試料と反応させる工程を含む、マラリア原虫感染症の検査方法又は診断方法。
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CN107001435A (zh) * | 2014-11-28 | 2017-08-01 | 国立研究开发法人日本医疗研究开发机构 | 使用了恶性疟原虫的烯醇化酶蛋白质的部分序列的人工抗原及其制造方法 |
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WO2018043584A1 (ja) * | 2016-08-31 | 2018-03-08 | 栄研化学株式会社 | 異なる方式で抗原を固定化した抗原担持不溶性担体粒子を用いる抗体測定法、抗体測定用試薬 |
JPWO2018043584A1 (ja) * | 2016-08-31 | 2019-06-24 | 栄研化学株式会社 | 異なる方式で抗原を固定化した抗原担持不溶性担体粒子を用いる抗体測定法、抗体測定用試薬 |
TWI757328B (zh) * | 2016-08-31 | 2022-03-11 | 日商榮研化學股份有限公司 | 使用以相異方式將抗原固定化之承載抗原不溶性承載粒子之抗體測定法、抗體測定用試劑 |
JP2018163064A (ja) * | 2017-03-27 | 2018-10-18 | 栄研化学株式会社 | 異なる担持方式で固定化した抗体担持不溶性担体粒子を用いる抗原測定法、抗原測定用試薬、及び、測定用キット |
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