WO2017208425A1 - Liquide de prétraitement pour test de détection d'anticorps, kit de test de détection d'anticorps, et procédé de test de détection d'anticorps - Google Patents

Liquide de prétraitement pour test de détection d'anticorps, kit de test de détection d'anticorps, et procédé de test de détection d'anticorps Download PDF

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Publication number
WO2017208425A1
WO2017208425A1 PCT/JP2016/066456 JP2016066456W WO2017208425A1 WO 2017208425 A1 WO2017208425 A1 WO 2017208425A1 JP 2016066456 W JP2016066456 W JP 2016066456W WO 2017208425 A1 WO2017208425 A1 WO 2017208425A1
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WO
WIPO (PCT)
Prior art keywords
antibody
target
antigen
antibody test
pretreatment liquid
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Application number
PCT/JP2016/066456
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English (en)
Japanese (ja)
Inventor
清美 海野
周子 森
Original Assignee
日立化成株式会社
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Publication date
Application filed by 日立化成株式会社 filed Critical 日立化成株式会社
Priority to PCT/JP2016/066456 priority Critical patent/WO2017208425A1/fr
Publication of WO2017208425A1 publication Critical patent/WO2017208425A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals

Definitions

  • the present invention relates to a pretreatment liquid for antibody testing, an antibody testing kit, and an antibody testing method.
  • An antibody test for detecting antibodies against bacteria and viruses in a sample collected from a subject is known as a method for testing bacterial and virus infections.
  • Patent Document 1 a method described in Patent Document 1 has been proposed as an antibody test for detecting an anti-Chlamydia pneumoniae antibody.
  • a specimen sample is brought into contact with a Chlamydia pneumoniae antigen immobilized on a solid phase carrier, and the produced antigen-antibody complex is brought into contact with a labeled antibody. Pneumonier antibody is measured.
  • false positives may occur that determine that a subject who is not actually infected is infected. If the test result is positive, it is necessary to perform a more accurate test such as an antigen test. If the false positive rate is high, unnecessary time and cost are required.
  • the inventors of the present invention are effective in reducing the false positive rate in antibody testing by treating a sample collected from a subject with a pretreatment liquid having a specific composition in advance. As a result, the present invention was completed.
  • the present invention is a pretreatment solution for a sample collected from a subject for antibody testing, which recognizes an antibody that recognizes a heavy chain and a light chain of a non-target antibody and an Fc heavy chain of a non-target antibody. And a pretreatment solution containing the antibody.
  • the mass ratio of the antibody recognizing the heavy chain and light chain of the non-target antibody to the antibody recognizing the Fc heavy chain of the non-target antibody in the pretreatment solution may be 0.05 to 0.75.
  • the present invention also provides an antibody test kit comprising a carrier holding an antigen recognized by a target antibody, a labeled secondary antibody that recognizes the target antibody, and a pretreatment liquid.
  • the present invention provides a step of pretreating a sample collected from a subject with a pretreatment liquid, and reacting the pretreated sample with an antigen held on a carrier, whereby the antigen and target antibody are reacted on the carrier.
  • an antibody test method comprising a step of forming an antigen-antibody complex, a step of reacting the formed antigen-antibody complex with a labeled secondary antibody, and a step of detecting a label of the labeled secondary antibody.
  • the false positive rate in antibody testing is reduced.
  • One embodiment of the present invention is a pretreatment solution of a sample collected from a subject for antibody testing, wherein an antibody that recognizes a heavy chain and a light chain of a non-target antibody, and an Fc An antibody that recognizes a chain.
  • a specific antibody (target antibody) in a sample collected from a subject can be detected.
  • a specific antibody (target antibody) in a sample collected from a subject can be detected.
  • the subject is infected with Chlamydia pneumoniae (positive).
  • the present invention is applicable not only to anti-Chlamydia pneumoniae antibodies but also to antibody tests against anti-Chlamydia trachomatis antibodies and various other antibodies.
  • Target antibody means an antibody to be detected in an antibody test, and may be selected from the group consisting of anti-IgM antibody, anti-IgG antibody and anti-IgA antibody.
  • Non-target antibody means an antibody other than the antibody to be detected in the antibody test. The non-target antibody is usually a different class of antibody from the target antibody, and may be selected from the group consisting of anti-IgM antibody, anti-IgG antibody and anti-IgA antibody. For example, when the target antibody is an anti-IgM antibody, the non-target antibody may be an anti-IgG antibody or an anti-IgA antibody.
  • An antibody that recognizes the heavy and light chains of the non-target antibody and an antibody that recognizes the Fc heavy chain of the non-target antibody are not only full-length antibodies having two complete light chains and two complete heavy chains, Also includes antibody fragments of full-length antibodies.
  • the antibody fragment can be, for example, F (ab ′) 2 , F (ab ′), Fab, or Fv.
  • the antibody that recognizes the heavy chain and light chain of the non-target antibody and the antibody that recognizes the Fc heavy chain of the non-target antibody may be polyclonal antibodies.
  • the mass ratio of the antibody recognizing the heavy chain and light chain of the non-target antibody to the antibody recognizing the Fc heavy chain of the non-target antibody in the pretreatment solution may be 0.05 or more, 0.15 or more It may be 0.20 or more.
  • the ratio may be 0.75 or less, 0.35 or less, and 0.25 or less.
  • the pretreatment liquid may be, for example, serum containing an antibody that recognizes the heavy chain and light chain of the non-target antibody and an antibody that recognizes the Fc heavy chain of the non-target antibody.
  • the sample collected from the subject is not particularly limited as long as it is a body fluid, and may be, for example, blood, serum, throat swab, tears, saliva, urine, or sweat.
  • An antibody test kit includes a carrier holding an antigen recognized by a target antibody, a labeled secondary antibody that recognizes the target antibody, and the pretreatment liquid.
  • the antigen is not particularly limited as long as it has a site recognized by the target antibody.
  • the antigen may be, for example, a commonly used protein that can be an antigen.
  • the antigen may be an allergen or a protein derived from the outer membrane or outer membrane of bacteria and viruses.
  • a carrier having a general material and shape may be used as the carrier holding the antigen.
  • the carrier is not particularly limited, and may be, for example, a microtiter plate, a bead, a paper disk, or a thread.
  • the labeled secondary antibody that recognizes the target antibody is obtained by labeling a secondary antibody that recognizes the target antibody with a labeling substance.
  • the label is not particularly limited as long as it is generally used as a label, and may be, for example, an enzyme, a fluorescent substance, a luminescent substance, biotin, or a radioisotope.
  • alkaline phosphatase can be used as the label.
  • the labeled secondary antibody that recognizes the target antibody may be provided in the form of a solution dissolved in water or a buffer solution.
  • the antibody test kit may further include other components such as a negative control sample, a positive control sample, and a washing solution.
  • the cleaning liquid may be used, for example, for cleaning the carrier and may be used for diluting the pretreatment liquid.
  • the washing solution may be a buffer solution.
  • the kit may further include components such as a substrate, a diluent, and a reaction stop solution.
  • the diluent may be, for example, a buffer solution that dissolves the substrate.
  • the reaction stop solution is not particularly limited as long as it stops the reaction between the substrate and the enzyme and does not affect the antibody test result.
  • An antibody testing method includes a step of pretreating a sample collected from a subject with a pretreatment liquid (pretreatment step), and reacting the pretreated sample with an antigen held on a carrier.
  • pretreatment step a step of pretreating a sample collected from a subject with a pretreatment liquid
  • pretreatment step a step of reacting the pretreated sample with an antigen held on a carrier.
  • a step of forming an antigen-antibody complex of an antigen and a target antibody on a carrier (antigen-antibody complex formation step), and a step of reacting the formed antigen-antibody complex with a labeled secondary antibody (labeling step)
  • labeling step a step of detecting a labeled product of the labeled secondary antibody
  • the pretreatment step may be performed by mixing a sample collected from the subject and the pretreatment liquid.
  • the sample and the pretreatment liquid may be diluted with a washing liquid, water, or a buffer in advance.
  • these control samples may be pretreated by mixing with a pretreatment solution.
  • the antigen-antibody complex formation step may be performed by dispensing the pretreated sample on a microtiter plate in which the antigen is retained in the well and incubating the microtiter plate. After the incubation, the reagent in the well may be removed, and the well may be washed with a washing solution, water, or a buffer.
  • the labeling step may be performed by dispensing a solution containing the labeled secondary antibody onto a microtiter plate in which an antigen-antibody complex is formed in the well and incubating the microtiter plate. After the incubation, the reagent in the well may be removed, and the well may be washed with a washing solution, water, or a buffer. By this step, an antigen-target antibody-labeled secondary antibody complex is formed in the well.
  • the detection step may be performed by detecting a labeled product of the labeled secondary antibody by a known method suitable for each labeled product. Since the amount of the label in the microtiter plate depends on the amount of the target antibody, the target antibody can be quantitatively detected by quantitatively analyzing the amount of the label. For example, when the label is alkaline phosphatase, first, p-nitrophenyl phosphate, which is a substrate for alkaline phosphatase, is added into the well in which the antigen-target antibody-labeled secondary antibody complex has been formed. The reaction between alkaline phosphatase and p-nitrophenyl phosphate may be stopped by adding a reaction stop solution. The target antibody can be quantitatively detected by measuring the absorbance of the produced p-nitrophenol.
  • Example 1 Using the anti-Chlamydia pneumoniae IgM antibody detection kit “Hitazyme C. pneumoniae Ab-IgM” (manufactured by Hitachi Chemical Co., Ltd.), 8 healthy persons (healthy persons A to H) and 8 patients with respiratory diseases (patients) Serum samples collected from IP) were tested for anti-Chlamydia pneumoniae IgM antibodies.
  • 8 healthy persons healthy persons A to H
  • 8 patients with respiratory diseases patients
  • Serum samples collected from IP were tested for anti-Chlamydia pneumoniae IgM antibodies.
  • anti-human IgG (Fc) goat serum catalog number: 100G, manufactured by International Immunology
  • anti-human IgG (H + L) goat A mixed solution with serum catalog number: IGG-000-10015, manufactured by CAPRICORN
  • the mass ratio of anti-human IgG (H + L) antibody to anti-human IgG (Fc) antibody in the pretreatment solution is as shown in Table 1.
  • the other numerical values in Table 1 are indexes calculated according to the kit protocol.
  • the relationship between the index value and the determination result of the antibody test is as shown in Table 2.
  • Example 2 The healthy subjects Q to Z were tested for anti-Chlamydia pneumoniae IgM antibody in the same manner as in Example 1.
  • Table 3 shows the composition of the pretreatment liquid and the calculated index.
  • Example 3 For the positive control sera a and b, an antibody test for anti-Chlamydia pneumoniae IgM antibody was performed twice in the same manner as in Example 1.
  • the positive control sera a and b are sera whose IgM antibody titers by the micro fluorescent antibody method (Micro-IF method) are 128 times and 16 times, respectively.
  • Table 4 shows the composition of the pretreatment liquid and the average value of the calculated indexes.

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  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention fournit un liquide de prétraitement pour un échantillon prélevé sur un patient qui est destiné à un test de détection d'anticorps, et qui contient un anticorps identifiant les chaînes lourdes et les chaînes légères d'un anticorps non cible, et un anticorps identifiant les chaînes lourdes Fc d'un anticorps non cible. Ce liquide de prétraitement permet de réduire le taux de faux positifs dans un test de détection d'anticorps.
PCT/JP2016/066456 2016-06-02 2016-06-02 Liquide de prétraitement pour test de détection d'anticorps, kit de test de détection d'anticorps, et procédé de test de détection d'anticorps WO2017208425A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/JP2016/066456 WO2017208425A1 (fr) 2016-06-02 2016-06-02 Liquide de prétraitement pour test de détection d'anticorps, kit de test de détection d'anticorps, et procédé de test de détection d'anticorps

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/JP2016/066456 WO2017208425A1 (fr) 2016-06-02 2016-06-02 Liquide de prétraitement pour test de détection d'anticorps, kit de test de détection d'anticorps, et procédé de test de détection d'anticorps

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WO2017208425A1 true WO2017208425A1 (fr) 2017-12-07

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024085141A1 (fr) * 2022-10-20 2024-04-25 Ｔｏｐｐａｎホールディングス株式会社 Dispositif fluidique et procédé de détection de molécule cible

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02152999A (ja) * 1988-07-25 1990-06-12 Nippon Kayaku Co Ltd 免疫学的測定法における非特異的反応の除去・抑制に用いるモノクローナル抗体由来物質、その製造方法及びその使用法
JPH09511569A (ja) * 1993-12-20 1997-11-18 エンテロン， エル．ピー． 微生物抗原に関連する疾患の検出および処置のための方法および組成物
JP2004301684A (ja) * 2003-03-31 2004-10-28 Denka Seiken Co Ltd ノロウイルス又はサポウイルス検体用希釈液及びウイルス検出試薬

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH02152999A (ja) * 1988-07-25 1990-06-12 Nippon Kayaku Co Ltd 免疫学的測定法における非特異的反応の除去・抑制に用いるモノクローナル抗体由来物質、その製造方法及びその使用法
JPH09511569A (ja) * 1993-12-20 1997-11-18 エンテロン， エル．ピー． 微生物抗原に関連する疾患の検出および処置のための方法および組成物
JP2004301684A (ja) * 2003-03-31 2004-10-28 Denka Seiken Co Ltd ノロウイルス又はサポウイルス検体用希釈液及びウイルス検出試薬

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
TATE, JILL ET AL.: "Interferences in Immunoassay", THE CLINICAL BIOCHEMIST REVIEWS, vol. 25, 2004, pages 105 - 120, XP055268650 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024085141A1 (fr) * 2022-10-20 2024-04-25 Ｔｏｐｐａｎホールディングス株式会社 Dispositif fluidique et procédé de détection de molécule cible

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