JP2021165643A - Highly sensitive anti-HBV antibody detection method - Google Patents
Highly sensitive anti-HBV antibody detection method Download PDFInfo
- Publication number
- JP2021165643A JP2021165643A JP2020068102A JP2020068102A JP2021165643A JP 2021165643 A JP2021165643 A JP 2021165643A JP 2020068102 A JP2020068102 A JP 2020068102A JP 2020068102 A JP2020068102 A JP 2020068102A JP 2021165643 A JP2021165643 A JP 2021165643A
- Authority
- JP
- Japan
- Prior art keywords
- antibody
- hbv
- antigen
- hbv antibody
- immobilized
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000001514 detection method Methods 0.000 title claims description 8
- 239000000427 antigen Substances 0.000 claims abstract description 40
- 102000036639 antigens Human genes 0.000 claims abstract description 40
- 108091007433 antigens Proteins 0.000 claims abstract description 40
- 238000000034 method Methods 0.000 claims abstract description 15
- 239000007790 solid phase Substances 0.000 claims abstract description 8
- 230000035945 sensitivity Effects 0.000 abstract description 10
- 241000700721 Hepatitis B virus Species 0.000 description 31
- 239000003153 chemical reaction reagent Substances 0.000 description 24
- 239000000523 sample Substances 0.000 description 18
- 238000005259 measurement Methods 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 9
- 208000002672 hepatitis B Diseases 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 6
- 239000008280 blood Substances 0.000 description 6
- 239000010419 fine particle Substances 0.000 description 6
- 238000005406 washing Methods 0.000 description 6
- 230000002860 competitive effect Effects 0.000 description 5
- 235000002639 sodium chloride Nutrition 0.000 description 5
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 150000003839 salts Chemical class 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- NUFBIAUZAMHTSP-UHFFFAOYSA-N 3-(n-morpholino)-2-hydroxypropanesulfonic acid Chemical compound OS(=O)(=O)CC(O)CN1CCOCC1 NUFBIAUZAMHTSP-UHFFFAOYSA-N 0.000 description 3
- 206010059193 Acute hepatitis B Diseases 0.000 description 3
- 102000008186 Collagen Human genes 0.000 description 3
- 108010035532 Collagen Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 208000037628 acute hepatitis B virus infection Diseases 0.000 description 3
- 229920001436 collagen Polymers 0.000 description 3
- -1 for example Proteins 0.000 description 3
- 230000008105 immune reaction Effects 0.000 description 3
- 230000036046 immunoreaction Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 230000007420 reactivation Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 206010008909 Chronic Hepatitis Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 239000003018 immunosuppressive agent Substances 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 239000011347 resin Substances 0.000 description 2
- 229920005989 resin Polymers 0.000 description 2
- JIAARYAFYJHUJI-UHFFFAOYSA-L zinc dichloride Chemical compound [Cl-].[Cl-].[Zn+2] JIAARYAFYJHUJI-UHFFFAOYSA-L 0.000 description 2
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 description 1
- DVLFYONBTKHTER-UHFFFAOYSA-N 3-(N-morpholino)propanesulfonic acid Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 208000000419 Chronic Hepatitis B Diseases 0.000 description 1
- IGXWBGJHJZYPQS-SSDOTTSWSA-N D-Luciferin Chemical compound OC(=O)[C@H]1CSC(C=2SC3=CC=C(O)C=C3N=2)=N1 IGXWBGJHJZYPQS-SSDOTTSWSA-N 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- CYCGRDQQIOGCKX-UHFFFAOYSA-N Dehydro-luciferin Natural products OC(=O)C1=CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 CYCGRDQQIOGCKX-UHFFFAOYSA-N 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- BJGNCJDXODQBOB-UHFFFAOYSA-N Fivefly Luciferin Natural products OC(=O)C1CSC(C=2SC3=CC(O)=CC=C3N=2)=N1 BJGNCJDXODQBOB-UHFFFAOYSA-N 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- DDWFXDSYGUXRAY-UHFFFAOYSA-N Luciferin Natural products CCc1c(C)c(CC2NC(=O)C(=C2C=C)C)[nH]c1Cc3[nH]c4C(=C5/NC(CC(=O)O)C(C)C5CC(=O)O)CC(=O)c4c3C DDWFXDSYGUXRAY-UHFFFAOYSA-N 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010011756 Milk Proteins Proteins 0.000 description 1
- 102000014171 Milk Proteins Human genes 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- BUGBHKTXTAQXES-UHFFFAOYSA-N Selenium Chemical compound [Se] BUGBHKTXTAQXES-UHFFFAOYSA-N 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000919 ceramic Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 239000000084 colloidal system Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- TYJOJLOWRIQYQM-UHFFFAOYSA-L disodium;phenyl phosphate Chemical compound [Na+].[Na+].[O-]P([O-])(=O)OC1=CC=CC=C1 TYJOJLOWRIQYQM-UHFFFAOYSA-L 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 230000003100 immobilizing effect Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 239000006166 lysate Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000011147 magnesium chloride Nutrition 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 235000021239 milk protein Nutrition 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000013610 patient sample Substances 0.000 description 1
- 210000002381 plasma Anatomy 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 239000011669 selenium Substances 0.000 description 1
- 229910052711 selenium Inorganic materials 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000012798 spherical particle Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 239000011592 zinc chloride Substances 0.000 description 1
- 235000005074 zinc chloride Nutrition 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
本発明は、抗ＨＢＶ抗体の検出方法に関するものである。 The present invention relates to a method for detecting an anti-HBV antibody.
Ｂ型肝炎ウイルス（ＨＢＶ）は全世界での患者は２億５，７００万人（非特許文献１）と報告され、特に発展途上国においては、検査や治療を受けられる割合も未だ低いため、ウイルス性肝炎による死者は全世界で１３４万人と、結核やＨＩＶによる死亡者に匹敵する数となっている。近年増加してきている欧州型による慢性肝炎の増加や、既往感染者に対する免疫抑制剤の投与時にＨＢＶの再活性化による劇症化など、依然として、重要な公衆衛生問題であり続けている。 Hepatitis B virus (HBV) is reported to have 257 million patients worldwide (Non-Patent Document 1), and especially in developing countries, the rate of being tested and treated is still low. The death toll from viral hepatitis is 1.34 million worldwide, comparable to the death toll from tuberculosis and HIV. It continues to be an important public health problem, such as the increase in chronic hepatitis due to the European type, which has been increasing in recent years, and the fulminant effect due to the reactivation of HBV when immunosuppressive agents are administered to previously infected persons.
ＨＢＶは直径４２ｎｍの二重構造をもつ球形粒子で、外皮（エンベロープ）と芯（コア）から成ることが知られている。ヒト等にＨＢＶが感染するとＨＢＶコアに対する抗体（以下「抗ＨＢｃ抗体」）が産生されるが、抗ＨＢｃ抗体はＨＢＶ感染初期から感染後長期間にわたり血中に存在することが知られている（非特許文献２）。
また抗ＨＢｃ抗体は、Ｂ型肝炎表面抗原（以下「ＨＢｓ抗原」）が出現した後、血清中に出現するが、急性Ｂ型肝炎においてはＨＢｓ抗原が消失した後からＨＢｓ抗原に対する抗体（以下「抗ＨＢｓ抗体」）が出現するころまで持続する。これらの事実から、抗ＨＢｃ抗体は、ＨＢｓ抗原や抗ＨＢｓ抗体が血清中に見出されない時期にＨＢＶ感染を知見するうえで重要なマーカーということができる（非特許文献３）。また更には、急性Ｂ型肝炎時には抗ＨＢｃ抗体の抗体価が高くなることから、Ｂ型慢性肝炎の急性発症と急性Ｂ型肝炎を識別するうえでも重要なマーカーである（非特許文献４）。しかし、既存の抗ＨＢｃ抗体検出試薬において、陽性であるにも関わらず検出されないケースがあり、高感度化が求められている。また、２００６年に香港で発生した免疫抑制剤の使用に伴うＨＢＶの再活性化事例などへの対策として免疫抑制、化学療法により発生するＢ型肝炎対策ガイドライン（２０１１年改定）が策定されているが、その中でも抗ＨＢｃ抗体陽性かつ抗ＨＢｓ抗体陰性の事例は、再活性化リスクが高いことが知られている。既往感染者の抗ＨＢｃ抗体価は高値を示すが、感染から時間が経過すると抗ＨＢｃ抗体価が徐々に低下してくるため、ＨＢＶ−ＤＮＡ検出下限以上の患者においてもある時点から陰性を示す可能性があり、抗ＨＢｃ抗体検出試薬の感度向上はそうした点からも重要である。また抗ＨＢｃ抗体ばかりでなく、抗ＨＢＶ抗体全般においても、測定感度の向上は重要な課題である。
HBV is a spherical particle having a double structure with a diameter of 42 nm, and is known to consist of an outer skin (envelope) and a core (core). When humans and the like are infected with HBV, an antibody against the HBV core (hereinafter referred to as "anti-HBc antibody") is produced, and it is known that the anti-HBc antibody exists in the blood from the initial stage of HBV infection to a long period after infection (hereinafter, "anti-HBc antibody"). Non-Patent Document 2).
Further, the anti-HBc antibody appears in the serum after the appearance of the hepatitis B surface antigen (hereinafter, "HBs antigen"), but in acute hepatitis B, the antibody against the HBs antigen (hereinafter, "HBs antigen") after the HBs antigen disappears. It lasts until the time when "anti-HBs antibody") appears. From these facts, the anti-HBc antibody can be said to be an important marker for detecting HBV infection at a time when HBs antigen or anti-HBs antibody is not found in serum (Non-Patent Document 3). Furthermore, since the antibody titer of the anti-HBc antibody increases during acute hepatitis B, it is an important marker for distinguishing between acute onset of chronic hepatitis B and acute hepatitis B (Non-Patent Document 4). However, there are cases where the existing anti-HBc antibody detection reagent is not detected even though it is positive, and higher sensitivity is required. In addition, guidelines for measures against hepatitis B caused by immunosuppression and chemotherapy (revised in 2011) have been formulated as measures against cases of HBV reactivation associated with the use of immunosuppressants that occurred in Hong Kong in 2006. However, among them, cases of anti-HBc antibody positive and anti-HBs antibody negative are known to have a high risk of reactivation. The anti-HBc antibody titer of a previously infected person shows a high value, but since the anti-HBc antibody titer gradually decreases as time passes after infection, it is possible that even a patient above the lower limit of HBV-DNA detection shows a negative value from a certain point of time. Therefore, it is important to improve the sensitivity of the anti-HBc antibody detection reagent. Further, improving the measurement sensitivity is an important issue not only for anti-HBc antibody but also for anti-HBV antibody in general.
本発明は、検体中の抗ＨＢＶ抗体を検出する際に、感度が向上する方法を提供することを目的とする。 An object of the present invention is to provide a method for improving sensitivity when detecting an anti-HBV antibody in a sample.
本発明者らは、前記課題を解決すべく鋭意検討を行なった結果、反応系に抗ＩｇＧ抗体を共存させることにより、感度が向上することを見出し、本発明を完成するに至った。即ち、本発明は以下のとおりである。 As a result of diligent studies to solve the above problems, the present inventors have found that the sensitivity is improved by coexisting an anti-IgG antibody in the reaction system, and have completed the present invention. That is, the present invention is as follows.
（１）
ｉ）検体に由来する抗ＨＢＶ抗体、
ｉｉ）ＨＢＶ抗原を固相に固定化させた固相化ＨＢＶ抗原、及び
ｉｉｉ）抗ＨＢＶ抗体に標識を結合させた標識化抗ＨＢＶ抗体
を反応させて、
［ｉ）抗ＨＢＶ抗体−ｉｉ）固相化ＨＢＶ抗原−ｉｉｉ）標識化抗ＨＢＶ抗体］を有する複合体を形成させ、当該複合体中の標識を検出することにより検体に由来する抗ＨＢＶ抗体を検出する方法において、
ｉｉ）固相化ＨＢＶ抗原とｉｉｉ）標識化抗ＨＢＶ抗体とを反応させるのと同時に又はそれ以前に、
・ｉ）抗ＨＢＶ抗体とｉｉ）固相化ＨＢＶ抗原とを反応させ、かつ
・ｉｖ）抗ＩｇＧ抗体をｉ）抗ＨＢＶ抗体と共存させて、ｉ）抗ＨＢＶ抗体と反応させる、
ことを特徴とする、抗ＨＢＶ抗体の検出方法。
(1)
i) Anti-HBV antibody derived from the sample,
The reaction was carried out by reacting ii) an immobilized HBV antigen in which the HBV antigen was immobilized on a solid phase, and iii) a labeled anti-HBV antibody in which a label was bound to the anti-HBV antibody.
An anti-HBV antibody derived from a sample is obtained by forming a complex having [i) anti-HBV antibody-iii) immobilized HBV antigen-iii) labeled anti-HBV antibody] and detecting a label in the complex. In the method of detection
ii) At the same time as or before reacting the immobilized HBV antigen with the ii) labeled anti-HBV antibody.
・ I) Anti-HBV antibody and ii) Immobilized HBV antigen are reacted, and ・ iv) Anti-IgG antibody is made to coexist with i) Anti-HBV antibody, and i) is reacted with anti-HBV antibody.
A method for detecting an anti-HBV antibody.
以下、本発明を詳細に説明する。 Hereinafter, the present invention will be described in detail.
本発明は、上述の反応を行い、検体に由来する抗ＨＢＶ抗体を検出する方法である。 The present invention is a method for detecting an anti-HBV antibody derived from a sample by carrying out the above-mentioned reaction.
本発明において、ＨＢＶ抗原としては特に限定されるものではない。例えば、Ｂ型肝炎コア抗原(以下「ＨＢｃ抗原」)、Ｂ型肝炎エンベロープ抗原（以下「ＨＢｅ抗原」）、ＨＢｓ抗原等があげられる。好ましくはＨＢｃ抗原である。 In the present invention, the HBV antigen is not particularly limited. For example, hepatitis B core antigen (hereinafter “HBc antigen”), hepatitis B envelope antigen (hereinafter “HBe antigen”), HBs antigen and the like can be mentioned. It is preferably an HBc antigen.
また抗ＨＢＶ抗体としては、例えば、前述のＨＢＶ抗原に対する抗体があげられ、具体的には抗ＨＢｃ抗体、抗ＨＢｅ抗体、抗ＨＢｓ抗体等があげられ、中でも抗ＨＢｃ抗体が好ましい。 Examples of the anti-HBV antibody include an antibody against the above-mentioned HBV antigen, specifically, an anti-HBc antibody, an anti-HBe antibody, an anti-HBs antibody, and the like, and an anti-HBc antibody is preferable.
検体としては、例えばヒトの体液、具体的には血液、尿などをあげることができ、好ましい例として血液、中でも全血、血清、血漿等をあげることができる。 Examples of the sample include human body fluids, specifically blood, urine, and the like, and preferred examples include blood, especially whole blood, serum, plasma, and the like.
標識としては、１２５Ｉ、３Ｈなどの放射性物質、西洋わさびペルオキシダーゼ、β−Ｄ−ガラクトシダーゼ、アルカリホスファターゼなどの酵素、フルオレセインなどの蛍光物質、金コロイド、セレンコロイド、ルシフェリンなどの発光又は発色物質などを用いることができる。これらの標識は抗ＨＢＶ抗体に直接結合させてもよく、又はビオチン−アビジン等を介して間接的に結合させてもよい。 The labeling, radioactive substances such as 125 I, 3 H, horseradish peroxidase, beta-D-galactosidase, an enzyme such as alkaline phosphatase, fluorescent substances such as fluorescein, colloidal gold, selenium colloid, luminescent or chromogenic substances such as luciferin, etc. Can be used. These labels may be bound directly to the anti-HBV antibody or indirectly via biotin-avidin or the like.
固相としては、ビーズ、微粒子、容器自体等を使用することができる。特に微粒子が好ましく、ガラス、金属、セラミツクス等の無機物であってもよく、また高分子ポリマー等の有機物であってもよい。またそれらの微粒子は磁性体を含むものであってもよい。微粒子の粒子径は０．１から５０μｍが好ましく、さらには１から１０μｍが好ましい。 As the solid phase, beads, fine particles, the container itself, or the like can be used. Fine particles are particularly preferable, and they may be inorganic substances such as glass, metal and ceramics, and may be organic substances such as a high molecular polymer. Further, those fine particles may contain a magnetic substance. The particle size of the fine particles is preferably 0.1 to 50 μm, more preferably 1 to 10 μm.
ｉｉｉ）標識化抗ＨＢＶ抗体に用いられる抗体は、ポリクローナル抗体であってもモノクローナル抗体であってもよく、抗体を産生する実際上任意の動物種、例えばウサギ、ヤギ、ヒツジ、ブタ、ウマ、マウスまたはラットなど由来の抗体が使用できる。抗体の形態としては完全抗体や、それを酵素処理や化学処理により切断したＦ（ａｂ’）２ やＦａｂ’等のような抗体断片であってもよい。 iii) The antibody used for the labeled anti-HBV antibody may be a polyclonal antibody or a monoclonal antibody, and practically any animal species that produces the antibody, such as rabbit, goat, sheep, pig, horse, and mouse. Alternatively, an antibody derived from a rat or the like can be used. The form of the antibody may be a complete antibody or an antibody fragment such as F (ab') 2 or Fab' that is cleaved by an enzymatic treatment or a chemical treatment.
本発明に用いられるｉｖ）抗ＩｇＧ抗体は特に限定されるものではないが、ヒト検体を用いる場合は抗ヒトＩｇＧ抗体が好ましい。 The iv) anti-IgG antibody used in the present invention is not particularly limited, but when a human sample is used, an anti-human IgG antibody is preferable.
本発明は、競合法による反応系を用いる。なお、ＨＢＶ抗原は多価抗原（同じエピトープを複数個有する）であるため、同一の抗体が複数個結合することができる。 The present invention uses a reaction system based on a competitive method. Since the HBV antigen is a multivalent antigen (having a plurality of the same epitopes), the same antibody can bind to a plurality of the same antibodies.
本発明はｉ）抗ＨＢＶ抗体、ｉｉ）固相化ＨＢＶ抗原、及びｉｉｉ）標識化抗ＨＢＶ抗体を反応させるが、ｉｉ）固相化ＨＢＶ抗原とｉｉｉ）標識化抗ＨＢＶ抗体とを反応させるのと同時又はそれ以前に、
・ｉ）抗ＨＢＶ抗体とｉｉ）固相化ＨＢＶ抗原とを反応させることが必須であり、かつ
・ｉｖ）抗ＩｇＧ抗体をｉ）抗ＨＢＶ抗体と共存させることにより、ｉ）抗ＨＢＶ抗体と反応させることが必須である。
The present invention reacts i) an anti-HBV antibody, ii) a immobilized HBV antigen, and iii) a labeled anti-HBV antibody, but ii) reacts an immobilized HBV antigen with an ii) labeled anti-HBV antibody. At the same time or before
・ I) It is essential to react the anti-HBV antibody with ii) the immobilized HBV antigen, and ・ iv) by coexisting the anti-IgG antibody with the i) anti-HBV antibody, i) react with the anti-HBV antibody. It is essential to let them do it.
これにより、［ｉｖ）抗ＩｇＧ抗体−ｉ）抗ＨＢＶ抗体−ｉｉ）固相化ＨＢＶ抗原−ｉｉｉ）標識化抗ＨＢＶ抗体］を有する複合体が形成され、その複合体中の標識を検出することにより、検体に由来する抗ＨＢＶ抗体を検出する。このとき、ｉｖ）抗ＩｇＧ抗体は、ｉ）抗ＨＢＶ抗体と結合して、ｉ）抗ＨＢＶ抗体をかさ高く修飾することとなり、それによってｉｉｉ）標識化抗ＨＢＶ抗体がｉｉ）固相化ＨＢＶ抗原へ結合することを効率よく阻害することができ、その結果として検体に由来する抗ＨＢＶ抗体の量を感度良く求めることができると考えられる。 As a result, a complex having [iv) anti-IgG antibody-i) anti-HBV antibody-ii) immobilized HBV antigen-iii) labeled anti-HBV antibody] is formed, and the label in the complex is detected. Detects anti-HBV antibody derived from the sample. At this time, the iv) anti-IgG antibody binds to i) the anti-HBV antibody and i) modifies the anti-HBV antibody in a bulky manner, whereby iii) the labeled anti-HBV antibody becomes ii) the immobilized HBV antigen. It is considered that the binding to to can be efficiently inhibited, and as a result, the amount of anti-HBV antibody derived from the sample can be obtained with high sensitivity.
なお、ＨＢＶ抗原は多価抗原であるため、上述の複合体中のｉｉ）固相化ＨＢＶ抗原には、複数個のｉ）抗ＨＢＶ抗体が結合し、また複数個のｉｉｉ）標識化抗ＨＢＶ抗体が結合することもあるが、それらも含めて前述のように、［ｉ）抗ＨＢＶ抗体−ｉｉ）固相化ＨＢＶ抗原−ｉｉｉ）標識化抗ＨＢＶ抗体］を有する複合体や、［ｉｖ）抗ＩｇＧ抗体−ｉ）抗ＨＢＶ抗体−ｉｉ）固相化ＨＢＶ抗原−ｉｉｉ）標識化抗ＨＢＶ抗体］を有する複合体と表記した。 Since the HBV antigen is a polyvalent antigen, a plurality of i) anti-HBV antibodies are bound to the ii) immobilized HBV antigen in the above-mentioned complex, and a plurality of ii) labeled anti-HBVs are bound. Antibodies may bind, but as described above, including these, complexes having [i) anti-HBV antibody-iii) immobilized HBV antigen-iii) labeled anti-HBV antibody] and [iv) It was described as a complex having anti-IgG antibody-i) anti-HBV antibody-ii) immobilized HBV antigen-iii) labeled anti-HBV antibody].
一方、本発明における反応順序は上述の通りであるが、好ましくは以下の態様があげられる。即ち、まず、ｉｉ）固相化ＨＢＶ抗原とｉ）検体に由来する抗ＨＢＶ抗体とを混合することにより、ｉｉ）固相化ＨＢＶ抗原とｉ）検体に由来するＨＢＶ抗体が反応する。この後、未反応物をＢ／Ｆ分離や洗浄により除去することが好ましい。次いでその反応生成物に、ｉｉｉ）標識化抗ＨＢＶ抗体とｉｖ）抗ＩｇＧ抗体とを同時に加え、反応させる。この後、未反応物をＢ／Ｆ分離や洗浄により除去することが好ましい。最終的に、生成した［ｉｖ）抗ＩｇＧ抗体−ｉ）抗ＨＢＶ抗体−ｉｉ）固相化ＨＢＶ抗原−ｉｉｉ）標識化抗ＨＢＶ抗体］を有する複合体中の標識を用いて測定し、検体に由来する抗ＨＢＶ抗体の量を求めることができる。 On the other hand, the reaction sequence in the present invention is as described above, and the following aspects are preferably mentioned. That is, first, by mixing ii) the immobilized HBV antigen and i) the anti-HBV antibody derived from the sample, the ii) immobilized HBV antigen and the HBV antibody derived from the i) sample react with each other. After that, it is preferable to remove the unreacted product by B / F separation or washing. Then, iii) labeled anti-HBV antibody and iv) anti-IgG antibody are simultaneously added to the reaction product and reacted. After that, it is preferable to remove the unreacted product by B / F separation or washing. Finally, measurement was performed using a label in a complex having the generated [iv) anti-IgG antibody-i) anti-HBV antibody-ii) immobilized HBV antigen-iii) labeled anti-HBV antibody], and the sample was used. The amount of anti-HBV antibody derived can be determined.
本発明では、反応時に蛋白質、糖、緩衝液や塩類を共存させてもよく、蛋白質としては、例えばウシ血清アルブミン、コラーゲンペプチド、乳タンパク質等を使用することができる。蛋白質は０．１〜２０％（重量／容量）の濃度範囲とすることが好ましく、特に１〜１０％（重量／容量）の濃度範囲とすることが好ましい。糖であれば例えばスクロース、マンニトール、トレハロースやイノシトール等を使用することができる。緩衝液としては、例えばＴｒｉｓ、ＭＯＰＳＯ、ＭＯＰＳやＭＥＳ等を使用することができ、塩類としては、例えば塩化ナトリウム、塩化カリウム、塩化マグネシウム、塩化亜鉛、エチレンジアミン四酢酸二ナトリウム等を使用することができる。なお、反応時にはこれら以外にも、必要に応じて他の試薬成分等を共存させることもできる。 In the present invention, proteins, sugars, buffers and salts may coexist during the reaction, and as the proteins, for example, bovine serum albumin, collagen peptide, milk protein and the like can be used. The protein is preferably in the concentration range of 0.1 to 20% (weight / volume), and particularly preferably in the concentration range of 1 to 10% (weight / volume). As for sugar, for example, sucrose, mannitol, trehalose, inositol and the like can be used. As the buffer solution, for example, Tris, MOPSO, MOPS, MES and the like can be used, and as the salts, for example, sodium chloride, potassium chloride, magnesium chloride, zinc chloride, disodium ethylenediamine tetraacetate and the like can be used. .. In addition to these, other reagent components and the like can coexist as necessary during the reaction.
本発明によれば、抗ＩｇＧ抗体を用いて、ｉ）抗ＨＢＶ抗体をかさ高く修飾することがてき、それによってｉｉｉ）標識化抗ＨＢＶ抗体がｉｉ）固相化ＨＢＶ抗原へ結合することを効率よく阻害することができ、その結果、検体中の抗ＨＢＶ抗体の量を感度良く求めることができる。また、本発明は競合法でありながら、サンドイッチ法と同程度の測定感度を得ることができる。 According to the present invention, anti-IgG antibody can be used to i) bulk modify the anti-HBV antibody, thereby making it efficient for iii) labeled anti-HBV antibody to bind to ii) immobilized HBV antigen. It can be well inhibited, and as a result, the amount of anti-HBV antibody in the sample can be determined with high sensitivity. Further, although the present invention is a competitive method, it is possible to obtain the same measurement sensitivity as the sandwich method.
以下、実施例により本発明をさらに詳細に説明するが、本発明は本実施例により限定されるものではない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the present Examples.
［実施例１］
免疫測定装置として全自動エンザイムイムノアッセイ装置（ＡＩＡ−ＣＬ２４００、東ソー（株）製）と当該装置用抗ＨＢｃ抗体免疫反応試薬（試薬Ａ）を用い、２ステップ競合法により測定を行った。なお、抗ＨＢｃ抗体免疫反応試薬（試薬Ａ）は以下のようにして調製した。
[Example 1]
As an immunoassay device, a fully automatic enzyme immunoassay device (AIA-CL2400, manufactured by Tosoh Corporation) and an anti-HBc antibody immunoreactivity reagent (reagent A) for the device were used, and measurement was performed by a two-step competitive method. The anti-HBc antibody immune reaction reagent (reagent A) was prepared as follows.
（１）固相懸濁液の調製
ＨＢｃ抗原を磁性微粒子に固定化させ作製したＨＢｃ抗原固相化磁性微粒子を、コラーゲンペプチド、動物血清、糖、塩類を含むＭＯＰＳＯ緩衝液で希釈・混合し、固相懸濁液を作製し、樹脂製２穴容器の一方の穴に分注した。
(1) Preparation of solid phase suspension HBc antigen-immobilized magnetic fine particles prepared by immobilizing HBc antigen on magnetic fine particles are diluted and mixed with a MOPSO buffer solution containing collagen peptide, animal serum, sugar and salts, and mixed. A solid phase suspension was prepared and dispensed into one hole of a resin 2-hole container.
（２）標識抗体及び抗ＩｇＧ抗体を含む溶液の調製
アルカリ性フォスファターゼを抗ＨＢｃ抗体に結合させ作製したアルカリ性フォスファターゼ標識化抗ＨＢｃ抗体及び抗ヒトＩｇＧ抗体を、コラーゲンペプチド、動物血清、糖、塩類を含むＭＯＰＳＯ緩衝液で希釈・混合し、標識抗体及び抗ヒトＩｇＧ抗体を含む溶液を作製し、前述の樹脂製２穴容器の他方の穴に分注した。
(2) Preparation of solution containing labeled antibody and anti-IgG antibody An alkaline phosphatase-labeled anti-HBc antibody and anti-human IgG antibody prepared by binding alkaline phosphatase to an anti-HBc antibody, containing collagen peptide, animal serum, sugar and salts. A solution containing a labeled antibody and an anti-human IgG antibody was prepared by diluting and mixing with a MOPSO buffer, and dispensed into the other hole of the above-mentioned resin 2-hole container.
（３）抗ＨＢｃ抗体免疫反応試薬（試薬Ａ）の調製
上述の（１）（２）で作製した２穴容器を凍結乾燥し、抗ＨＢｃ抗体免疫反応試薬（試薬Ａ）を調製した。
(3) Preparation of Anti-HBc Antibody Immune Response Reagent (Reagent A) The 2-well container prepared in (1) and (2) above was lyophilized to prepare an anti-HBc antibody immunoreaction reagent (Reagent A).
（４）測定
上述の（３）の試薬Ａを用いて、２ステップ競合法により測定を行った。具体的には、試薬Ａの２穴のうち、固相懸濁液の凍結乾燥物が入っている穴に溶解液を入れて溶解し、そこへ検体（患者血清）を分注し、３７℃で６分間反応させ（第一反応）、未反応物をＢ／Ｆ分離及び洗浄液で３回洗浄することにより除去した。次に、検出用標識抗体溶液の凍結乾燥物が入っている穴に溶解液を入れて溶解し、それをもう一方の穴に分注し、３７℃で３分間反応させた（第二反応）。その後Ｂ／Ｆ分離及び洗浄液で３回洗浄して未反応物質を除去した後、発光基質（化合物名ＤＩＦＵＲＡＴ：３−（５−ｔｅｒｔ−ブチルー４，４−ジメチルー２，６，７−トリオキサビシクロ［３．２．０］ヘプト−１−イル）フェニルリン酸エステル ジナトリウム塩）を分注して４分間反応させ、固相に結合したアルカリホスファターゼ活性を発光量から測定し、陽性／陰性を判定した。
(4) Measurement Using the reagent A of (3) above, measurement was performed by a two-step competitive method. Specifically, of the two holes of Reagent A, the solution is put into the hole containing the freeze-dried product of the solid phase suspension to dissolve it, and the sample (patient serum) is dispensed into the hole to dispense the sample (patient serum) at 37 ° C. The reaction was carried out for 6 minutes (first reaction), and the unreacted material was removed by B / F separation and washing with a washing solution three times. Next, the lysate was placed in the hole containing the lyophilized product of the labeled antibody solution for detection to dissolve it, and the solution was dispensed into the other hole and reacted at 37 ° C. for 3 minutes (second reaction). .. Then, after removing the unreacted substance by B / F separation and washing with a washing solution three times, the luminescent substrate (compound name DIFURAT: 3- (5-tert-butyl-4,4-dimethyl-2,6,7-trioxabicyclo) [3.2.0] Hept-1-yl) phenyl phosphate disodium salt) was dispensed and reacted for 4 minutes, and the alkaline phosphatase activity bound to the solid phase was measured from the amount of luminescence, and positive / negative was obtained. Judged.
本測定は、競合法であるため、陰性コントロール即ち検体中に抗ＨＢｃ抗体が存在しない時の測定値を基準にする。また陽性コントロールは、抗ＨＢｃ抗体を検体希釈液にて希釈系列を作製して本測定法にて測定した際に、測定値に変化が見られなくなった濃度をさし、そのときの測定値を１００ＩＮＨ％と定めた。一方、検体中に抗ＨＢｃ抗体が存在する場合には、それによって、標識化抗ＨＢｃ抗体が固相化ＨＢｃ抗原に結合することが抑制される割合（ＩＮＨ（％））から抗ＨＢｃ抗体量を表す。それらは、以下の式に従って計算した。
ＩＮＨ（％）＝［（陰性コントロールの測定値−検体の測定値）／（陰性コントロールの測定値−陽性コントロールの測定値）］×１００
この計算で５０％に等しいか又は５０％より大きい値は陽性（＋）と判断し、それ以外は陰性（−）と判断した。
Since this measurement is a competitive method, it is based on the negative control, that is, the measured value when the anti-HBc antibody is not present in the sample. The positive control refers to the concentration at which no change is observed in the measured value when a dilution series of anti-HBc antibody is prepared with a sample diluent and measured by this measurement method, and the measured value at that time is used. It was set to 100 INH%. On the other hand, when the anti-HBc antibody is present in the sample, the amount of the anti-HBc antibody is determined from the ratio (INH (%)) at which the labeled anti-HBc antibody is suppressed from binding to the immobilized HBc antigen. show. They were calculated according to the following formula.
INH (%) = [(measured value of negative control-measured value of sample) / (measured value of negative control-measured value of positive control)] x 100
In this calculation, a value equal to or greater than 50% was judged to be positive (+), and other values were judged to be negative (-).
［比較例１］
実施例１と同様にして、但し抗ＨＢｃ抗体免疫反応試薬（試薬Ａ）の代わりに、抗ＨＢｃ抗体免疫反応試薬（試薬Ｂ）を用いて測定を行った。なお、抗ＨＢｃ抗体免疫反応試薬（試薬Ｂ）は、抗ＩｇＧ抗体を用いることなく作製した以外は、抗ＨＢｃ抗体免疫反応試薬（試薬Ａ）と同様にして作製した。
[Comparative Example 1]
The measurement was carried out in the same manner as in Example 1, except that the anti-HBc antibody immunoreaction reagent (reagent B) was used instead of the anti-HBc antibody immunoreaction reagent (reagent A). The anti-HBc antibody immune reaction reagent (reagent B) was prepared in the same manner as the anti-HBc antibody immune reaction reagent (reagent A) except that it was prepared without using an anti-IgG antibody.
［測定結果］
試薬Ｂの検出感度付近まで陰性血清で希釈した希釈陽性検体８例と陰性検体７例の測定を行い、その判定結果を比較した。結果を表１に示す。表中、括弧のついた値は、マイナスの値を表す。
[Measurement result]
Eight diluted positive samples and seven negative samples diluted with negative serum to the vicinity of the detection sensitivity of reagent B were measured, and the determination results were compared. The results are shown in Table 1. In the table, the values in parentheses represent negative values.
また、患者検体１３７例（陽性検体４７例、陰性検体９０例）］の測定を行いその測定結果を比較した。結果を図１に示す。 In addition, 137 patient samples (47 positive samples, 90 negative samples)] were measured and the measurement results were compared. The results are shown in FIG.
表１及び図１から明らかなように、抗ＩｇＧ抗体を使用した反応系である試薬Ａは、試薬Ｂに比べて、陽性検体をより感度よく測定することができた。 As is clear from Table 1 and FIG. 1, Reagent A, which is a reaction system using an anti-IgG antibody, was able to measure positive samples more sensitively than Reagent B.
Claims (1)
ｉｉ）ＨＢＶ抗原を固相に固定化させた固相化ＨＢＶ抗原、及び
ｉｉｉ）抗ＨＢＶ抗体に標識を結合させた標識化抗ＨＢＶ抗体
を反応させて、
［ｉ）抗ＨＢＶ抗体−ｉｉ）固相化ＨＢＶ抗原−ｉｉｉ）標識化抗ＨＢＶ抗体］を有する複合体を形成させ、当該複合体中の標識を検出することにより検体に由来する抗ＨＢＶ抗体を検出する方法において、
ｉｉ）固相化ＨＢＶ抗原とｉｉｉ）標識化抗ＨＢＶ抗体とを反応させるのと同時に又はそれ以前に、
・ｉ）抗ＨＢＶ抗体とｉｉ）固相化ＨＢＶ抗原とを反応させ、かつ
・ｉｖ）抗ＩｇＧ抗体をｉ）抗ＨＢＶ抗体と共存させて、ｉ）抗ＨＢＶ抗体と反応させる、
ことを特徴とする、抗ＨＢＶ抗体の検出方法。 i) Anti-HBV antibody derived from the sample,
The reaction was carried out by reacting ii) an immobilized HBV antigen in which the HBV antigen was immobilized on a solid phase, and iii) a labeled anti-HBV antibody in which a label was bound to the anti-HBV antibody.
An anti-HBV antibody derived from a sample is obtained by forming a complex having [i) anti-HBV antibody-iii) immobilized HBV antigen-iii) labeled anti-HBV antibody] and detecting a label in the complex. In the method of detection
ii) At the same time as or before reacting the immobilized HBV antigen with the ii) labeled anti-HBV antibody.
・ I) Anti-HBV antibody and ii) Immobilized HBV antigen are reacted, and ・ iv) Anti-IgG antibody is made to coexist with i) Anti-HBV antibody, and i) is reacted with anti-HBV antibody.
A method for detecting an anti-HBV antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020068102A JP2021165643A (en) | 2020-04-06 | 2020-04-06 | Highly sensitive anti-HBV antibody detection method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2020068102A JP2021165643A (en) | 2020-04-06 | 2020-04-06 | Highly sensitive anti-HBV antibody detection method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2021165643A true JP2021165643A (en) | 2021-10-14 |
Family
ID=78021943
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2020068102A Pending JP2021165643A (en) | 2020-04-06 | 2020-04-06 | Highly sensitive anti-HBV antibody detection method |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2021165643A (en) |
-
2020
- 2020-04-06 JP JP2020068102A patent/JP2021165643A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3510232B2 (en) | Virus detection or measurement method | |
| CN105548565A (en) | Kit for detecting trypanosoma cruzi antibody as well as preparation and application thereof | |
| CN102072957B (en) | Hepatitis C virus antibody diagnostic kit and preparation method thereof | |
| JPS6367661B2 (en) | ||
| CN101975859A (en) | Magnetic microparticle separation chemiluminescent immunoassay detection method for hepatitis B virus surface antigen | |
| JP2636331B2 (en) | One-step assay for antigen-specific antibodies and suitable reagents | |
| JPH02276969A (en) | Reagent for simultaneously detecting different markers of hepatitis | |
| WO2004088311A1 (en) | Diluent for norovirus or sapovirus specimen and method for detecting virus | |
| JP2945025B2 (en) | Anti-HBc antibody immunoassay | |
| JP4895727B2 (en) | Anti-HIV antibody detection reagent, reagent kit, reagent production method, and anti-HIV antibody detection method | |
| JP6048923B2 (en) | Detection method and detection kit for chronic hepatitis B | |
| JP2019053060A (en) | Immunoassay method for hepatitis B virus core antibody | |
| CN109613273A (en) | A kind of five indexes of hepatitis b time-resolved fluoroimmunoassay measurement in chromatography kit | |
| JP4292670B2 (en) | Immunoassay for anti-HBc antibody | |
| JP2021165643A (en) | Highly sensitive anti-HBV antibody detection method | |
| JPS60177265A (en) | Detection of immunoglobulin of antibody by class | |
| Yucel et al. | Development of sandwich ELISA systems for the diagnosis of hepatitis B virus surface antigen and its antibody in human sera | |
| JP4029298B2 (en) | Method for removing sticky microvesicles | |
| JPH05232111A (en) | Artificial standard and contrast serum and manufacture and use thereof | |
| JP3176570B2 (en) | HCV detection or measurement method | |
| JP2001133460A (en) | High-sensitivity immunity measurement reagent of hepatitis b virus surface antigen | |
| JP6513189B2 (en) | Kit for assaying human Fc-containing protein using indirect ELISA and method for assaying Fc-containing protein using the same | |
| JP2003083976A (en) | HBs ANTIGEN MEASURING REAGENT | |
| JP2009288149A (en) | Method of measuring hepatitis b virus core antigen or antibody against the same | |
| Korec et al. | Detection of antibodies against hepatitis B core antigen using the avidin-biotin system |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20230320 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20231201 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20231226 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20240213 |