WO2017208425A1 - Pretreatment liquid for antibody test, antibody test kit, and antibody test method - Google Patents
Pretreatment liquid for antibody test, antibody test kit, and antibody test method Download PDFInfo
- 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
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- antibody
- target
- antigen
- antibody test
- pretreatment liquid
- Prior art date
Links
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.
Abstract
The present invention provides a pretreatment liquid for a sample collected from a subject for an antibody test, the pretreatment liquid including antibodies for recognizing heavy chains and light chains of non-target antibodies, and antibodies for recognizing Fc heavy chains of non-target antibodies. Through use of this pretreatment liquid, the false-positive rate in an antibody test is reduced.
Description
本発明は、抗体検査のための前処理液及び抗体検査キット、並びに抗体検査方法に関する。
The present invention relates to a pretreatment liquid for antibody testing, an antibody testing kit, and an antibody testing method.
細菌及びウイルスの感染を検査する方法として、被験者から採取したサンプル中の細菌及びウイルスに対する抗体を検出する抗体検査が知られている。
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.
例えば、抗クラミジア・ニューモニエ抗体を検出する抗体検査として特許文献１に記載された方法が提案されている。この方法では、固相担体に固定化したクラミジア・ニューモニエ抗原に検体試料を接触させ、生成した抗原抗体複合体を標識抗体と接触させて、標識抗体上の標識物量から検体試料中の抗クラミジア・ニューモニエ抗体を測定する。
For example, a method described in Patent Document 1 has been proposed as an antibody test for detecting an anti-Chlamydia pneumoniae antibody. In this method, 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.
抗体検査では、実際には感染していない被験者を感染していると判定してしまう偽陽性が生じることがある。検査結果が陽性であった場合、抗原検査など、より正確性の高い検査を行う必要があり、偽陽性率が高いと、不必要な時間及びコストがかかってしまう。
In antibody testing, 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.
このような状況に鑑み、本発明者らは鋭意検討を行った結果、被験者から採取されたサンプルを予め特定の組成の前処理液で処理することが、抗体検査における偽陽性率の低減に有効であることを見出し、本発明を完成させた。
In view of such a situation, as a result of intensive studies, 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.
すなわち、本発明は、抗体検査のための、被験者から採取されたサンプルの前処理液であって、非標的抗体の重鎖及び軽鎖を認識する抗体と、非標的抗体のＦｃ重鎖を認識する抗体と、を含む、前処理液を提供する。前処理液における、非標的抗体の重鎖及び軽鎖を認識する抗体と非標的抗体のＦｃ重鎖を認識する抗体との質量の比は、０．０５～０．７５であってよい。
That is, 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.
また、本発明は、標的抗体によって認識される抗原が保持された担体と、標的抗体を認識する標識二次抗体と、前処理液と、を備える抗体検査キットを提供する。
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.
さらに、本発明は、被験者から採取されたサンプルを前処理液で前処理する工程と、前処理したサンプルと担体上に保持された抗原とを反応させて、担体上に抗原と標的抗体との抗原抗体複合体を形成させる工程と、形成された抗原抗体複合体を標識二次抗体と反応させる工程と、標識二次抗体の標識物を検出する工程と、を含む抗体検査方法を提供する。
Furthermore, 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. Provided is 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.
本発明によれば、抗体検査における偽陽性率が低減される。
According to the present invention, the false positive rate in antibody testing is reduced.
以下、本発明を実施するための形態について詳細に説明する。ただし、本発明は以下の実施形態に限定されるものではない。
Hereinafter, embodiments for carrying out the present invention will be described in detail. However, the present invention is not limited to the following embodiments.
本発明の一の実施形態は、抗体検査のための、被験者から採取されたサンプルの前処理液であって、非標的抗体の重鎖及び軽鎖を認識する抗体と、非標的抗体のＦｃ重鎖を認識する抗体と、を含む前処理液である。
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.
抗体検査によれば、被験者から採取されたサンプル中の特定の抗体（標的抗体）を検出することができる。例えば、クラミジア・ニューモニエの抗体検査では、被験者から採取されたサンプル中に、特定のクラスの抗クラミジア・ニューモニエ抗体が所定量検出された場合、被験者がクラミジア・ニューモニエに感染している（陽性）と判定することができる。本発明は、抗クラミジア・ニューモニエ抗体に限らず、抗クラミジア・トラコマティス抗体及びその他様々な抗体に対する抗体検査に適用することができる。
According to the antibody test, a specific antibody (target antibody) in a sample collected from a subject can be detected. For example, in an antibody test for Chlamydia pneumoniae, if a specific class of anti-Chlamydia pneumoniae antibody is detected in a sample collected from the subject, the subject is infected with Chlamydia pneumoniae (positive). Can be determined. 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.
「標的抗体」は、抗体検査における検出対象の抗体を意味し、抗ＩｇＭ抗体、抗ＩｇＧ抗体及び抗ＩｇＡ抗体からなる群より選ばれてよい。「非標的抗体」は、抗体検査における検出対象の抗体以外の抗体を意味する。非標的抗体は、通常、標的抗体とは異なるクラスの抗体であり、抗ＩｇＭ抗体、抗ＩｇＧ抗体及び抗ＩｇＡ抗体からなる群より選ばれてよい。例えば、標的抗体が抗ＩｇＭ抗体である場合、非標的抗体は、抗ＩｇＧ抗体又は抗ＩｇＡ抗体であってよい。
“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.
非標的抗体の重鎖及び軽鎖を認識する抗体、及び、非標的抗体のＦｃ重鎖を認識する抗体は二つの完全な軽鎖と二つの完全な重鎖とを有する全長抗体だけでなく、全長抗体の抗体断片も含む。抗体断片は、例えば、Ｆ（ａｂ’）２、Ｆ（ａｂ’）、Ｆａｂ、又はＦｖであってよい。また、非標的抗体の重鎖及び軽鎖を認識する抗体、及び、非標的抗体のＦｃ重鎖を認識する抗体は、ポリクローナル抗体であってよい。
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.
前処理液における、非標的抗体の重鎖及び軽鎖を認識する抗体と非標的抗体のＦｃ重鎖を認識する抗体との質量の比は、０．０５以上であってよく、０．１５以上であってよく、０．２０以上であってよい。また、上記比は、０．７５以下であってよく、０．３５以下であってよく、０．２５以下であってよい。上記比をこのような範囲に設定することで、抗体検査において、偽陰性率を上昇させずに、偽陽性率を低減しやすくなる。
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. By setting the ratio in such a range, it is easy to reduce the false positive rate without increasing the false negative rate in antibody testing.
前処理液は、例えば、非標的抗体の重鎖及び軽鎖を認識する抗体と、非標的抗体のＦｃ重鎖を認識する抗体と、を含む血清であってもよい。
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.
被験者から採取されたサンプルは、体液であれば特に限定されず、例えば、血液、血清、咽頭ぬぐい液、涙、唾液、尿、又は汗であってよい。
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.
本発明の他の実施形態に係る抗体検査キットは、標的抗体によって認識される抗原が保持された担体と、標的抗体を認識する標識二次抗体と、上記前処理液と、を備える。
An antibody test kit according to another embodiment of the present invention includes a carrier holding an antigen recognized by a target antibody, a labeled secondary antibody that recognizes the target antibody, and the pretreatment liquid.
抗原は、標的抗体によって認識される部位を有するものであれば特に限定されない。抗原は、例えば、一般に用いられる、抗原となり得るタンパク質であってよく、具体的には、アレルゲン、又は細菌及びウイルスの外膜若しくは外膜由来のタンパク質であってよい。
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. Specifically, the antigen may be an allergen or a protein derived from the outer membrane or outer membrane of bacteria and viruses.
抗原が保持された担体としては、一般的な材質及び形状の担体を使用してよい。担体は、特に限定されず、例えば、マイクロタイタプレート、ビーズ、ペーパーディスク、又は糸であってよい。
As the carrier holding the antigen, a carrier having a general material and shape may be used. The carrier is not particularly limited, and may be, for example, a microtiter plate, a bead, a paper disk, or a thread.
標的抗体を認識する標識二次抗体は、標的抗体を認識する二次抗体を標識物で標識したものである。標識物は、一般に標識物として使用されているものであれば特に限定されず、例えば、酵素、蛍光物質、発光物質、ビオチン、又は放射性同位元素であってよい。標識物としては、例えば、アルカリフォスファターゼを使用することができる。標的抗体を認識する標識二次抗体は、水又は緩衝液に溶解されて、溶液の状態で提供されてもよい。
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. For example, 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.
抗体検査キットは、陰性対照サンプル、陽性対照サンプル、及び洗浄液など、その他の構成をさらに備えていてもよい。洗浄液は、例えば、担体の洗浄に使用されてよく、前処理液の希釈に使用されてもよい。洗浄液は、緩衝液であってよい。標識物が酵素である場合、キットは、基質、希釈液、及び反応停止液などの構成をさらに備えていてもよい。希釈液は、例えば、基質を溶解する緩衝液であってよい。反応停止液は、基質と酵素との反応を停止し、抗体検査の結果に影響を与えないものであれば、特に限定されない。
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. When the label is an enzyme, 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.
本発明の一実施形態に係る抗体検査方法は、被験者から採取されたサンプルを前処理液で前処理する工程（前処理工程）と、前処理したサンプルと担体上に保持された抗原とを反応させて、担体上に抗原と標的抗体との抗原抗体複合体を形成させる工程（抗原抗体複合体形成工程）と、形成された抗原抗体複合体を標識二次抗体と反応させる工程（標識工程）と、標識二次抗体の標識物を検出する工程（検出工程）と、を含む。
An antibody testing method according to an embodiment of the present invention 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. 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) And a step of detecting a labeled product of the labeled secondary antibody (detection step).
前処理工程は、被験者から採取されたサンプルと前処理液とを混合することによって行われてよい。サンプル及び前処理液は、予め、洗浄液、水、又は緩衝液によって希釈されていてもよい。被験者から採取されたサンプルの代わりに陽性対照サンプル及び陰性対照サンプルを使用する場合は、これらの対照サンプルを前処理液と混合することで前処理してよい。
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. When using a positive control sample and a negative control sample instead of a sample collected from a subject, these control samples may be pretreated by mixing with a pretreatment solution.
抗原抗体複合体形成工程は、前処理したサンプルを、ウェル内に抗原が保持されたマイクロタイタプレートに分注し、マイクロタイタプレートをインキュベートすることによって行われてよい。インキュベート後、ウェル内の試薬を除去し、ウェルを、洗浄液、水、又は緩衝液で洗浄してもよい。
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.
標識工程は、ウェル内に抗原抗体複合体が形成されたマイクロタイタプレートに、標識二次抗体を含む溶液を分注し、マイクロタイタプレートをインキュベートすることによって行われてもよい。インキュベート後、ウェル内の試薬を除去し、ウェルを、洗浄液、水、又は緩衝液で洗浄してもよい。この工程により、ウェル内に抗原－標的抗体－標識二次抗体複合体が形成される。
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.
検出工程は、標識二次抗体の標識物を、各標識物に適した公知の方法で検出することによって行われてよい。マイクロタイタプレートにおける標識物の量は標的抗体の量に依存するため、標識物の量を定量的に解析することで、標的抗体を定量的に検出できる。例えば、標識物がアルカリフォスファターゼの場合、まず、抗原－標的抗体－標識二次抗体複合体が形成されたウェル内に、アルカリフォスファターゼの基質であるｐ－ニトロフェニルリン酸を加える。アルカリフォスファターゼとｐ－ニトロフェニルリン酸との反応は、反応停止液を加えて停止してもよい。生成したｐ－ニトロフェノールの吸光度を測定することで、標的抗体を定量的に検出することができる。
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.
（実施例１）
抗クラミジア・ニューモニエＩｇＭ抗体検出キット「ヒタザイム Ｃ．ニューモニエ Ａｂ－ＩｇＭ」（日立化成株式会社製）を使用して、健常人８人（健常人Ａ～Ｈ）及び呼吸器疾患の患者８人（患者Ｉ～Ｐ）から採取された血清サンプルについて、抗クラミジア・ニューモニエＩｇＭ抗体の抗体検査を行った。ただし、上記キットにおける「ラテックス液」の代わりに、前処理液として、抗ヒトＩｇＧ（Ｆｃ）ヤギ血清（カタログ番号：１００Ｇ、インターナショナルイムノロジー社製）、又は、これと抗ヒトＩｇＧ（Ｈ＋Ｌ）ヤギ血清（カタログ番号：ＩＧＧ－０００－１００１５、ＣＡＰＲＩＣＯＲＮ社製）との混合液を使用した。前処理液における、抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体と抗ヒトＩｇＧ（Ｆｃ）抗体との質量の比は、表１に示すとおりである。表１のその他の数値は、キットのプロトコルに従って算出したインデックスである。インデックスの値と抗体検査の判定結果との関係は表２に示すとおりである。 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. However, instead of the “latex solution” in the above kit, as a pretreatment solution, anti-human IgG (Fc) goat serum (catalog number: 100G, manufactured by International Immunology), or this and anti-human IgG (H + L) goat A mixed solution with serum (catalog number: IGG-000-10015, manufactured by CAPRICORN) was used. 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.
抗クラミジア・ニューモニエＩｇＭ抗体検出キット「ヒタザイム Ｃ．ニューモニエ Ａｂ－ＩｇＭ」（日立化成株式会社製）を使用して、健常人８人（健常人Ａ～Ｈ）及び呼吸器疾患の患者８人（患者Ｉ～Ｐ）から採取された血清サンプルについて、抗クラミジア・ニューモニエＩｇＭ抗体の抗体検査を行った。ただし、上記キットにおける「ラテックス液」の代わりに、前処理液として、抗ヒトＩｇＧ（Ｆｃ）ヤギ血清（カタログ番号：１００Ｇ、インターナショナルイムノロジー社製）、又は、これと抗ヒトＩｇＧ（Ｈ＋Ｌ）ヤギ血清（カタログ番号：ＩＧＧ－０００－１００１５、ＣＡＰＲＩＣＯＲＮ社製）との混合液を使用した。前処理液における、抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体と抗ヒトＩｇＧ（Ｆｃ）抗体との質量の比は、表１に示すとおりである。表１のその他の数値は、キットのプロトコルに従って算出したインデックスである。インデックスの値と抗体検査の判定結果との関係は表２に示すとおりである。 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. However, instead of the “latex solution” in the above kit, as a pretreatment solution, anti-human IgG (Fc) goat serum (catalog number: 100G, manufactured by International Immunology), or this and anti-human IgG (H + L) goat A mixed solution with serum (catalog number: IGG-000-10015, manufactured by CAPRICORN) was used. 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.
抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体と抗ヒトＩｇＧ（Ｆｃ）抗体とを含む前処理液を使用した場合、抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体を含まない前処理液を使用した場合に比べて、偽陽性率が低減した。また、前処理液における抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体と抗ヒトＩｇＧ（Ｆｃ）抗体との質量の比が所定の値以下である場合、偽陰性率の上昇が抑えられた。
When using a pretreatment solution containing an anti-human IgG (H + L) antibody and an anti-human IgG (Fc) antibody, a false positive rate compared to using a pretreatment solution containing no anti-human IgG (H + L) antibody Reduced. In addition, when the mass ratio of the anti-human IgG (H + L) antibody to the anti-human IgG (Fc) antibody in the pretreatment solution is a predetermined value or less, an increase in the false negative rate was suppressed.
（実施例２）
健常人Ｑ～Ｚについて、実施例１と同様にして抗クラミジア・ニューモニエＩｇＭ抗体の抗体検査を行った。前処理液の組成及び算出したインデックスは表３に示すとおりである。 (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.
健常人Ｑ～Ｚについて、実施例１と同様にして抗クラミジア・ニューモニエＩｇＭ抗体の抗体検査を行った。前処理液の組成及び算出したインデックスは表３に示すとおりである。 (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.
抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体を含まない前処理液を使用した場合は偽陽性が生じたが、抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体と抗ヒトＩｇＧ（Ｆｃ）抗体とを含む前処理液を使用した場合には、偽陽性が生じなかった。
When using a pretreatment solution that does not contain anti-human IgG (H + L) antibody, a false positive occurred, but when a pretreatment solution containing anti-human IgG (H + L) antibody and anti-human IgG (Fc) antibody was used There were no false positives.
（実施例３）
陽性管理血清ａ及びｂについて、各血清につき２回ずつ、実施例１と同様にして抗クラミジア・ニューモニエＩｇＭ抗体の抗体検査を行った。陽性管理血清ａ及びｂは、マイクロ蛍光抗体法（Ｍｉｃｒｏ－ＩＦ法）によるＩｇＭ抗体価が、それぞれ１２８倍及び１６倍の血清である。前処理液の組成、及び算出したインデックスの平均値を表４に示す。 (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.
陽性管理血清ａ及びｂについて、各血清につき２回ずつ、実施例１と同様にして抗クラミジア・ニューモニエＩｇＭ抗体の抗体検査を行った。陽性管理血清ａ及びｂは、マイクロ蛍光抗体法（Ｍｉｃｒｏ－ＩＦ法）によるＩｇＭ抗体価が、それぞれ１２８倍及び１６倍の血清である。前処理液の組成、及び算出したインデックスの平均値を表４に示す。 (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.
前処理液における、抗ヒトＩｇＧ（Ｈ＋Ｌ）抗体と抗ヒトＩｇＧ（Ｆｃ）抗体との質量の比が所定の値以下である場合、偽陰性率の上昇が抑えられた。
When the mass ratio of the anti-human IgG (H + L) antibody to the anti-human IgG (Fc) antibody in the pretreatment solution is not more than a predetermined value, an increase in the false negative rate was suppressed.
Claims (4)
- 抗体検査のための、被験者から採取されたサンプルの前処理液であって、
非標的抗体の重鎖及び軽鎖を認識する抗体と、
非標的抗体のＦｃ重鎖を認識する抗体と、を含む、
前処理液。 A pretreatment solution for a sample collected from a subject for antibody testing,
An antibody that recognizes the heavy and light chains of the non-target antibody;
An antibody that recognizes the Fc heavy chain of a non-target antibody,
Pretreatment liquid. - 前処理液における、非標的抗体の重鎖及び軽鎖を認識する抗体と非標的抗体のＦｃ重鎖を認識する抗体との質量の比が、０．０５～０．７５である、請求項１に記載の前処理液。 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 is 0.05 to 0.75. The pretreatment liquid described in 1.
- 標的抗体によって認識される抗原が保持された担体と、
標的抗体を認識する標識二次抗体と、
請求項１又は２に記載の前処理液と、を備える、
抗体検査キット。 A carrier carrying an antigen recognized by the target antibody;
A labeled secondary antibody that recognizes the target antibody;
The pretreatment liquid according to claim 1 or 2.
Antibody test kit. - 被験者から採取されたサンプルを請求項１又は２に記載の前処理液で前処理する工程と、
前処理したサンプルと担体上に保持された抗原とを反応させて、担体上に抗原と標的抗体との抗原抗体複合体を形成させる工程と、
形成された抗原抗体複合体を標識二次抗体と反応させる工程と、
標識二次抗体の標識物を検出する工程と、を含む、
抗体検査方法。 Pre-treating a sample collected from a subject with the pre-treatment liquid according to claim 1 or 2, and
Reacting the pretreated sample with the antigen retained on the carrier to form an antigen-antibody complex of the antigen and the target antibody on the carrier;
Reacting the formed antigen-antibody complex with a labeled secondary antibody;
Detecting a label of the labeled secondary antibody,
Antibody testing method.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP2016/066456 WO2017208425A1 (en) | 2016-06-02 | 2016-06-02 | Pretreatment liquid for antibody test, antibody test kit, and antibody test method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| PCT/JP2016/066456 WO2017208425A1 (en) | 2016-06-02 | 2016-06-02 | Pretreatment liquid for antibody test, antibody test kit, and antibody test method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| WO2017208425A1 true WO2017208425A1 (en) | 2017-12-07 |
Family
ID=60478129
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/JP2016/066456 WO2017208425A1 (en) | 2016-06-02 | 2016-06-02 | Pretreatment liquid for antibody test, antibody test kit, and antibody test method |
Country Status (1)
| Country | Link |
|---|---|
| WO (1) | WO2017208425A1 (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02152999A (en) * | 1988-07-25 | 1990-06-12 | Nippon Kayaku Co Ltd | Monoclonal antibody-originated substance for removal and suppression of non-specific reaction in immunoassay, its production and use |
| JPH09511569A (en) * | 1993-12-20 | 1997-11-18 | エンテロン， エル．ピー． | Methods and compositions for the detection and treatment of diseases associated with microbial antigens |
| JP2004301684A (en) * | 2003-03-31 | 2004-10-28 | Denka Seiken Co Ltd | Dilution liquid for norovirus or sapovirus specimen, and virus detection reagent |
-
2016
- 2016-06-02 WO PCT/JP2016/066456 patent/WO2017208425A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH02152999A (en) * | 1988-07-25 | 1990-06-12 | Nippon Kayaku Co Ltd | Monoclonal antibody-originated substance for removal and suppression of non-specific reaction in immunoassay, its production and use |
| JPH09511569A (en) * | 1993-12-20 | 1997-11-18 | エンテロン， エル．ピー． | Methods and compositions for the detection and treatment of diseases associated with microbial antigens |
| JP2004301684A (en) * | 2003-03-31 | 2004-10-28 | Denka Seiken Co Ltd | Dilution liquid for norovirus or sapovirus specimen, and virus detection reagent |
Non-Patent Citations (1)
| Title |
|---|
| TATE, JILL ET AL.: "Interferences in Immunoassay", THE CLINICAL BIOCHEMIST REVIEWS, vol. 25, 2004, pages 105 - 120, XP055268650 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| AU2012340722B2 (en) | Signal amplification in lateral flow and related immunoassays | |
| Hayrapetyan et al. | Enzyme-linked immunosorbent assay: types and applications | |
| US11714084B2 (en) | Method and associated device for rapid detection of target biomolecules with enhanced sensitivity | |
| Verma et al. | ELISA-based identification and detection of microbes | |
| US20210364515A1 (en) | Optimizing diagnostics for galactofuranose containing antigens | |
| Betts Carpenter | Immunoassays for the diagnosis of infectious diseases | |
| US9482674B2 (en) | Serological methods and diagnostic tests for syphilis antibodies | |
| Theel et al. | Immunoassays for diagnosis of infectious diseases | |
| Soroka et al. | Modification of rapid human immunodeficiency virus (HIV) antibody assay protocols for detecting recent HIV seroconversion | |
| Binnicker et al. | Evaluation of three multiplex flow immunoassays compared to an enzyme immunoassay for the detection and differentiation of IgG class antibodies to herpes simplex virus types 1 and 2 | |
| Gharavi et al. | A comparative study on sensitivity and specificity of conventional and unconventional IgG and IgM assays for diagnosis of toxoplasmosis | |
| RU181920U1 (en) | IMMUNOCHIP FOR TREPONEM-SPECIFIC SEROLOGICAL DIAGNOSTICS OF SYPHILIS | |
| US11703507B2 (en) | Immunoassay for SARS-CoV-2 antibodies | |
| Premjeet et al. | Enzyme-Linked Immuno-Sorbent Assay (ELISA), basics and it’s application: A comprehensive review | |
| US20220050106A1 (en) | Methods and kits for detecting sars-coronavirus-2 antigen | |
| WO2017208425A1 (en) | Pretreatment liquid for antibody test, antibody test kit, and antibody test method | |
| KR102330837B1 (en) | Biosensor and analyzing method using the same | |
| Wu et al. | Evaluation of a novel array-based toxoplasma, rubella, cytomegalovirus, and herpes simplex virus IgG enzyme linked immunosorbent assay and its comparison with Virion/Serion enzyme linked immunosorbent assays | |
| Gouriet et al. | Comparison of the new InoDiag automated fluorescence multiplexed antigen microarray to the reference technique in the serodiagnosis of atypical bacterial pneumonia | |
| Cremer et al. | Comparison of the hemagglutination inhibition test and an indirect fluorescent-antibody test for detection of antibody to rubella virus in human sera | |
| Surendran et al. | Dot-immunobinding assay (Dot-Iba) | |
| US20220034904A1 (en) | IMMUNOASSAY FOR SARS-CoV-2 ANTIBODIES | |
| US20230358746A1 (en) | METHODS AND KITS FOR DETERMINING SEROSTATUS OF SARS-CoV-2 | |
| CN105954507A (en) | ELISA kit | |
| WO2023028565A1 (en) | Lateral flow assay by using carboxyl latex beads and biotin-polystreptavidin for the detection of covid-19 infection and diagnostic kit using the lateral flow assay |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| 121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 16904050 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: DE |
|
| 122 | Ep: pct application non-entry in european phase |
Ref document number: 16904050 Country of ref document: EP Kind code of ref document: A1 |
|
| NENP | Non-entry into the national phase |
Ref country code: JP |