JPH07294530A - Detection of antibody in body fluids against pathogn - Google Patents
Detection of antibody in body fluids against pathognInfo
- Publication number
- JPH07294530A JPH07294530A JP12085394A JP12085394A JPH07294530A JP H07294530 A JPH07294530 A JP H07294530A JP 12085394 A JP12085394 A JP 12085394A JP 12085394 A JP12085394 A JP 12085394A JP H07294530 A JPH07294530 A JP H07294530A
- Authority
- JP
- Japan
- Prior art keywords
- pathogen
- antibody
- antigen
- pylori
- soluble
- 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
Landscapes
- Peptides Or Proteins (AREA)
Abstract
Description
【０００１】[0001]
【産業上の利用分野】本発明は、体液中の病原体抗体検
出用試薬に関する。TECHNICAL FIELD The present invention relates to a reagent for detecting pathogen antibodies in body fluids.
【０００２】[0002]
【従来の技術】病原体に関して、過去（場合によっては
現在も継続している）に既往症が有るかどうかを判定す
る手法として、体液中の抗体検出が行われることは公知
のことである。その方法としては病原体そのもの、もし
くは部分成分を固相に感作し、体液と接触させ、その後
固相に吸着した免疫グロブリンを検出する、いわゆる免
疫学的操作法により行われることが一般的である。とこ
ろが、種族の近い病原体には種が異なるにもかかわらず
類似した成分から構成されることがよくある。この場
合、一般の抗体測定法では、目的とする病原体に対する
抗体が存在しなくても、類似の病原体に対する抗体が存
在すれば、この類似抗体を検出して陽性と判定される
（一般的に偽陽性と言われている）。これは、医師が医
療行為を行う上で、診断や治療方針を誤る結果となり、
大きな問題となっている。これを解決する方法として、
種々の方法が試みられているが、以下に病原体Ｈｅｌｉ
ｃｏｂａｃｔｅｒ ｐｙｌｏｒｉを例として説明する。
ＷａｒｒｅｎとＭａｒｓｈａｌｌ（Ｍａｒｓｈａｌｌ
ＢＪ ＆ Ｗａｒｒｅｎ ＪＲ：Ｕｎｉｔｅｎｔｉｆｉ
ｅｄ ｃｕｒｖｅｄ ｂａｃｉｌｌｉ ｉｎｔｈｅ ｓ
ｔｏｍａｃｈ ｏｆ ｐａｔｉｅｎｔｓ ｗｉｔｈ ｇ
ａｓｔｒｉｔｉｓ ｐｅｐｔｉｃ ｕｌｃｅｒａｔｉｏ
ｎ．Ｌａｎｃｅｔ ｉ：１３１１−１３１５，１９８
４）らによって初めてヒト胃前庭部より分離、培養され
たＨｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉは、現在で
は胃粘膜病変の主原因の一つと考えられている。このた
め、ヒト胃内部にＨｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏ
ｒｉが存在するかどうかを知ることは、胃・十二指腸疾
患の診断に大いに役立つものである。さて、Ｈｅｌｉｃ
ｏｂａｃｔｅｒ ｐｙｌｏｒｉ感染を調べる方法とし
て、内視鏡施行時にＨｅｌｉｃｏｂａｃｔｅｒ ｐｙｌ
ｏｒｉ菌を分離同定すること、もしくは１３Ｃ−Ｕｒｅ
ａ等を用いたＢｒｅａｔｈテスト（Ｇｒａｈａｍ Ｄ
Ｙ，ｅｔ ａｌ：Ｃａｍｐｙｌｏｂａｃｔｅｒ ｐｙｌ
ｏｒｉｄｉｓ ｄｅｔｅｃｔｅｄ ｎｏｎｉｎｖａｓｉ
ｖｅｌｙ ｂｙ ｔｈｅ １３Ｃ−ｕｒｅａ ｂｒｅａ
ｔｈ ｔｅｓｔ．Ｌａｎｃｅｔ ｉ：１１７４−１１７
７，１９８７）が最も確実な方法である。しかし、患者
に与える負担も大きく、培養施設等の問題も有り簡便と
はいえない。これに対し、ＥＩＡ法による血清中のＨｅ
ｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ抗体検出が報告
（Ｒａｔｈｂｏｎｅ ＢＪ，ｅｔ ａｌ：Ｉｍｍｕｎｅ
ｒｅｓｐｏｎｓｅ ｔｏ Ｃａｍｐｙｌｏｂａｃｔｅ
ｒ ｐｙｌｏｒｉｄｉｓ．ｌａｎｃｅｔ １：１２１
７，１９８５）されている。しかしＨｅｌｉｃｏｂａｃ
ｔｅｒ ｐｙｌｏｒｉ抗原とＣａｍｐｙｌｏｂａｃｔｅ
ｒ属との共通抗原性が指摘されており、Ｃａｍｐｙｌｏ
ｂａｃｔｅｒ属の菌体を用いた吸収による免疫反応も報
告されている（Ｐｅｒｅｚ−ｐｅｒｅｚ ＧＩ，ｅｔ
ａｌ：Ｃａｍｐｙｌｏｂａｃｔｅｒ ｐｙｌｏｒｉ ａ
ｎｔｉｂｏｄｉｅｓ ｉｎ ｈｕｍａｎｓ．Ａｎｎ Ｉ
ｎｔｅｎ Ｍｅｄ １０９：１１−１７，１９８８）。
しかし、この吸収操作には、菌体すべてを用いるため、
吸収後の遠心操作が必要であり、大量検体を扱うには煩
雑なものであった。また、ゲル濾過により得たＵｒｅａ
ｓｅ活性を含む高分子分画を抗原としたＥＬＩＳＡ法も
報告されている（Ｅｖａｎｓ ＪＤ，ｅｔ ａｌ：Ａ
ｓｅｎｓｉｔｉｖｅ ａｎｄ ｓｐｅｃｉｆｉｃ ｓｅ
ｒｏｌｏｇｉｃ ｔｅｓｔ ｆｏｒ ｄｅｔｅｃｔｉｏ
ｎ ｏｆ Ｃａｍｐｙｌｏｂａｃｔｅｒ ｐｙｌｏｒｉ
ｉｎｆｅｃｔｉｏｎ．Ｇａｓｔｒｏｅｎｔｅｒｏｌｏ
ｇｙ ９６：１００４−１００８，１９８９）。同法
は、特異性、感度共優れた方法である。とこらが同法
は、Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ菌の一部
の蛋白を抗原としているため、その用いた一部の抗原に
たいする抗体の少ない患者は偽陰性となることが指摘さ
れていた。2. Description of the Related Art It is well known that antibody detection in body fluids is performed as a method for determining whether or not a pathogen has a past condition (which is still present in some cases). The method is generally performed by a so-called immunological operation method in which the pathogen itself or a partial component is sensitized to a solid phase, brought into contact with a body fluid, and then the immunoglobulin adsorbed on the solid phase is detected. . However, pathogens of close races are often composed of similar components, despite different species. In this case, in the general antibody measurement method, even if there is no antibody against the target pathogen, if there is an antibody against a similar pathogen, this similar antibody is detected and determined to be positive (generally false It is said to be positive). This is a result of the doctor making a mistake in diagnosis and treatment policy when performing medical treatment,
It's a big problem. As a way to solve this,
Various methods have been tried, but the following are pathogens Heli.
A description will be given by taking the case of P. coli pylori as an example.
Warren and Marshall (Marshall
BJ & Warren JR: Unitif
ed curved bacilli inthe s
tomach of patients with g
astritis peptic ulceratio
n. Lancet i: 1311-1315, 198.
Helicobacter pylori, which was first isolated and cultured from the human gastric antrum by 4) et al., Is now considered to be one of the main causes of gastric mucosal lesions. Therefore, the Helicobacter pyllo inside the human stomach
Knowing whether ri is present is of great help in the diagnosis of gastroduodenal disease. Well, Helic
As a method for investigating the infection of O. pylori, Helicobacter pyl at the time of performing endoscopy
ori bacterium, or 13 C-Ure
Breath test (Graham D
Y, et al: Campylobacter pyl
oridis detected noninvasi
very by the 13 C-urea breath
th test. Lancet i: 1174-117
7,1987) is the most reliable method. However, it is not easy because it imposes a heavy burden on patients and causes problems such as culture facilities. In contrast, He in serum by EIA method
Licobacterium pylori antibody detection reported (Rathbone BJ, et al: Immune
response to Campylobacter
r pyloridis. lancet 1: 121
7, 1985). But Helicobac
ter pylori antigen and Campylobacter
A common antigenicity with the genus r has been pointed out, and Campylo
An immune reaction by absorption using bacteria of the genus Bacter has also been reported (Perez-perez GI, et.
al: Campylobacter pylori a
ntibodies in humans. Ann I
Nten Med 109: 11-17, 1988).
However, since all the bacterial cells are used for this absorption operation,
Centrifugation after absorption is required, and handling a large amount of sample is complicated. Also, Urea obtained by gel filtration
An ELISA method using a high molecular weight fraction containing se activity as an antigen has also been reported (Evans JD, et al: A
sensitive and specific se
logic test for detection
no of Campylobacter pylori
injection. Gastroenterolo
gy 96: 1004-1008, 1989). This method has excellent specificity and sensitivity. It was pointed out that this method uses a partial protein of Helicobacter pylori as an antigen, so that a patient with a small amount of antibodies against the partial antigen used is false-negative.
【０００３】[0003]
【発明が解決しようとする課題】そこで本発明は、有用
な医薬の開発に応用できる、体液中の病原体抗体を高感
度かつ特異的に検出する検査用試薬を提供することを目
的とする。SUMMARY OF THE INVENTION It is therefore an object of the present invention to provide a test reagent which can be applied to the development of useful drugs and which can detect pathogen antibodies in body fluids with high sensitivity and specificity.
【０００４】[0004]
【課題を解決するための手段】本発明の目的は、体液中
の病原体抗体を検出する試薬を提供することであり、検
出に用いる抗原は、可能な限り多くの病原体由来の可溶
性抗原を使用する。続いて、複数濃度の病原体可溶性抗
原を用意して、それぞれを単体（ボール、マイクロプレ
ート等）に感作させる、そしてそれぞれの単体を体液と
接触させ単体感作可溶性抗原−免疫グロブリンの複合物
作成させ、その後固相に吸着した免疫グロブリンを標識
抗免疫グロブリン等を用いて測定する。測定検体中の抗
体が陰性もしくは陽性であるかの判定は、単体感作濃度
差間の測定値の差で判定する。測定値間データが複数で
存在する場合（つまり３チャンネル以上ある場合）は１
判定データが陽性（比較される陰性コントロールにて求
めた基準値を越える場合）である場合には陽性と判定す
る。一般に、病原体は蛋白質、糖類、脂質等を含む多く
の成分から構成されている。このため、生体体液中には
これら全てについて対応する抗体が存在する可能性があ
り、個体差により種々雑多である。ところが、これらの
抗原を単体に感作する場合、同じ条件でも感作されやす
い抗原と、されにくい抗原がある。また感作する単体に
は感作許容量があるため、高濃度で感作しても、感作さ
れるトータルの抗原量は小量しか増加しない。これに対
し感作される抗原の成分は、感作されやすい抗原量が増
加し、逆に感作されにくい抗原は減少する。低濃度で感
作する場合は、逆に感作溶液中の抗原量が小量のため、
単体に感作されにくい物質も単体に感作され、感作され
にくい物質が単体に感作される比率は増加することにな
る。つまり、病原体の可溶性抗原を、単体に高濃度で感
作する場合と低濃度で感作する場合で、感作される抗原
成分が変化する。このため本発明では、測定検体を複数
濃度で感作した単体を用い測定し、そのデータを複合す
ることにより測定感度をあげるものである。以下に、病
原体をＨｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉとして
説明する。本発明の目的は、体液中のＨｅｌｉｃｏｂａ
ｃｔｅｒ ｐｙｌｏｒｉ抗体を検出する試薬を提供する
ことであり、検出に用いる抗原は、可能な限り多くのＨ
ｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ由来の可溶性抗
原を使用することである。すなわち、全Ｈｅｌｉｃｏｂ
ａｃｔｅｒ ｐｙｌｏｒｉ菌体可溶性抗原を用いた抗体
検出用試薬を提供することであるが、この場合非特異反
応を示す、交差性物質をも測定することは公知のことで
ある。この非特異反応を除外するために、本発明では測
定チャンネルとは別にブランクチャンネルを設け、かつ
ブランクチャンネルには測定チャンネルに用いたＨｅｌ
ｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ可溶性抗原と同一の
成分を低濃度で感作することである。一般に、抗体検出
試薬の場合測定チャンネルとは別にブランクチャンネル
を設け検体ブランクを測定することは、よく行われるこ
とである。しかし、一般に行われているブランクチャン
ネルには、目的とする抗体を検出するための抗原成分は
なんら感作されておらず本発明とは異なるものである。
抗原に反応する抗体量は、抗原量に比例することは、公
知のことである。すなわち、抗体検出に用いる抗原量が
多くなればなるほど、検出される抗体量も増大する。と
ころが、我々はＨｅｌｉｃｏｂａｃｔｅｒｐｙｌｏｒｉ
抗原量の増加に対し、非特異反応と言われる交差性物質
（抗体）の増加は、目的とする特異抗体（Ｈｅｌｉｃｏ
ｂａｃｔｅｒ ｐｙｌｏｒｉに対する抗体）の増加に比
較し比較的緩やかな増加傾向を示すことを見いだした。
この為、高濃度Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒ
ｉ可溶性抗原で測定したデータから低濃度Ｈｅｌｉｃｏ
ｂａｃｔｅｒ ｐｙｌｏｒｉ可溶性抗原で測定したデー
タを差し引くことにより、非特異物質のデータが消去で
き、特異物質（Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒ
ｉに対する抗体）のみを検出することが可能であること
がわかった。本発明試薬を用いた場合、Ｈｅｌｉｃｏｂ
ａｃｔｅｒｐｙｌｏｒｉ抗原はすべての可溶性抗原を用
いているため、Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒ
ｉの一部分に対する抗体しか持たない患者についても偽
陰性を示すことはない。また、ブランクチャンネルに感
作する抗原は、Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒ
ｉ以外の菌体可溶性抗原でも可能である。この場合可溶
性抗原感作量は測定チャンネルのＨｅｌｉｃｏｂａｃｔ
ｅｒ ｐｙｌｏｒｉ可溶性抗原感作量と同程度が望まし
い。また、この場合菌種はＨｅｌｉｃｏｂａｃｔｅｒ
ｐｙｌｏｒｉに類似する菌種が好く、特にＣａｍｐｙｌ
ｏｂａｃｔｅｒ属が好ましい。さらに、ブランクチャン
ネルに用いる菌種は一種類でも良く、また二種類以上で
も良い。An object of the present invention is to provide a reagent for detecting pathogen antibodies in body fluids, wherein the antigen used for detection is soluble antigen derived from as many pathogens as possible. . Subsequently, multiple concentrations of pathogen-soluble antigens are prepared, and each is sensitized to a single substance (ball, microplate, etc.), and each single substance is brought into contact with body fluid to produce a single-sensitized soluble antigen-immunoglobulin complex. Then, the immunoglobulin adsorbed on the solid phase is measured using labeled anti-immunoglobulin or the like. Whether the antibody in the measurement sample is negative or positive is determined by the difference in the measured values between the differences in the sensitization concentrations of the single substances. 1 if there is more than one inter-measurement value data (that is, if there are 3 or more channels)
If the judgment data is positive (exceeds the standard value determined by the negative control to be compared), it is judged as positive. Generally, pathogens are composed of many components including proteins, sugars, lipids and the like. For this reason, there is a possibility that antibodies corresponding to all of them exist in the biological fluid, and there are various types due to individual differences. However, when sensitizing these antigens alone, there are antigens that are easily sensitized and antigens that are not easily sensitized even under the same conditions. Further, since the sensitized single body has an allowable sensitization amount, even if sensitization is performed at a high concentration, the total amount of sensitized antigen increases only by a small amount. On the other hand, in the component of the antigen to be sensitized, the amount of the antigen that is easily sensitized increases, and conversely, the amount of the antigen that is difficult to sensitize decreases. On the contrary, when sensitizing at a low concentration, since the amount of antigen in the sensitizing solution is small,
Substances that are difficult to sensitize alone are also sensitized to single substances, and the ratio of substances that are difficult to sensitize to single substances increases. That is, the antigen component to be sensitized changes depending on whether the soluble antigen of the pathogen is sensitized at a high concentration or at a low concentration. Therefore, in the present invention, the measurement sensitivity is increased by measuring the test sample using a single substance sensitized at a plurality of concentrations and combining the data. Hereinafter, the pathogen will be described as Helicobacter pylori. The object of the present invention is to identify Helicoba in body fluids.
The purpose of the present invention is to provide a reagent for detecting cter pylori antibody.
The use of soluble antigens from E.coli pylori. That is, all Helicob
The object of the present invention is to provide a reagent for antibody detection using a soluble antigen of Lactobacillus pylori, and in this case, it is well known to measure a cross-substance which shows a non-specific reaction. In order to exclude this non-specific reaction, in the present invention, a blank channel is provided separately from the measurement channel, and the blank channel is made of Hel used for the measurement channel.
It is to sensitize the same components as those of the Ecobacterium pylori soluble antigen at low concentrations. Generally, in the case of an antibody detection reagent, it is a common practice to provide a blank channel separately from the measurement channel and measure the sample blank. However, the generally used blank channel is different from the present invention in that no antigen component for detecting the target antibody is sensitized.
It is well known that the amount of antibody that reacts with the antigen is proportional to the amount of antigen. That is, the greater the amount of antigen used for antibody detection, the greater the amount of antibody detected. However, we are Helicobacterium pylori
An increase in the amount of cross-linking substances (antibodies), which is said to be a non-specific reaction, in response to an increase in the amount of the antigen
It was found that there is a relatively gradual increase tendency in comparison with the increase in the antibody against B. pylori.
Therefore, high-concentration Helicobacter pylor
Low-concentration Helico from data measured with i-soluble antigen
By subtracting the data measured with the Bacterial pylori soluble antigen, the data of the non-specific substance can be deleted, and the specific substance (Helicobacter pylor) can be deleted.
It was found that it is possible to detect only (antibody against i). When the reagent of the present invention is used, Helicob
Since all of the soluble antigens are used for the Actyl pylori antigen, the Helicobacter pylori antigen is used.
No false negatives are shown for patients who have only antibodies to a portion of i. The antigen sensitized to the blank channel is Helicobacter pylor.
Cell soluble antigens other than i are also possible. In this case, the soluble antigen sensitization amount is determined by the measurement channel Helicobacter
er pylori soluble antigen sensitization amount is desirable. Also, in this case, the bacterial species is Helicobacter
Bacteria species similar to pylori are preferred, especially Campyl
The genus Obacter is preferred. Further, the bacterial species used for the blank channel may be one type or two or more types.
【０００５】[0005]
（実施例１）Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ
を長田（ｐｏｔｅｎｔ ｉｎｈｉｂｉｔｏｒｙ ａｃｔ
ｉｏｎ ｏｆ ｔｈｅ ｇａｓｔｒｉｃ ｐｒｏｔｏｎ
ｐｕｍｐ ｉｎｈｉｂｉｔｏｒ ｌａｎｓｏｐｒａｚ
ｏｌｅ ａｇａｉｎｓｔ ｕｒｅａｓｅ ａｃｔｉｖｉ
ｔｙ ｏｆ ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒ
ｉ：ｕｎｉｑｕｅ ａｃｔｉｏｎ ｓｅｌｅｃｔｉｖｅ
ｆｏｒ Ｈ．ｐｙｌｏｒｉ ｃｅｌｌｓ．ａｎｔｉｍ
ｉｃｒｏｂ ａｇｅｎｔｓ ｃｈｅｍｏｔｈｅｒ３７：
７６９−７７４，１９９３）らの方法により培養した
後、Ｓｔａｃｅｙ（Ａｎｔｉｇｅｎｉｃｉｔｙ ｏｆ
ｆｒａｃｔｉｏｎｓ ｏｆ ｈｅｌｉｃｏｂａｃｔｅｒ
ｐｙｌｏｒｉ ｐｒｅｐａｒｅｄ ｂｙ ｆａｓｔ
ｐｒｏｔｅｉｎ ｌｉｑｕｉｄ ｃｈｒｏｍａｔｏｇｒ
ａｐｈｙ ａｎｄ ｕｒｅａｓｅ ｃａｐｔｕｒｅ ｂ
ｙ ｍｏｎｏｃｌｏｎａｌ ａｎｔｉｂｏｄｉｅｓ．Ｅ
ｕｒｊ ｃｌｉｎ ｍｉｃｒｏｂｉｏｌ ｉｎｆｅｃｔ
ｄｉｓ ９：７３２−７３７，１９９０）らの方法に
準じ可溶性抗原を作成した。この抗原を、ＥＬＩＳＡ用
９６穴マイクロプレートに、測定チャンネルには１μｇ
／ｍｌで、またブランクチャンネルには０．１μｇ／ｍ
ｌで分注し、４℃一夜静置した。プレートをＰＢＳ
（０．１％Ｔｗｅｅｎ２０含む）で洗浄後、ブロックエ
ースでブロッキングした。患者血清を１％の牛アルブミ
ンを含むＰＢＳで５００倍希釈して添加後１時間反応さ
せた。洗浄の後、ペルオキシダーゼ標識抗ヒトＩｇＧ抗
体を加え１時間反応させた。再度洗浄後、基質液（０．
０３％オルトフェニレンジアミン，０．００３％Ｈ２Ｏ
２）を加え３７℃、１０分反応させた。反応は２Ｎ硫酸
を加え停止し、各ウエルをマイクロプレートリーダー
（λ１＝４９２ｎｍ，λ２＝６６０ｎｍ）で比色した。
そして、測定チャンネル（固相Ｈｅｌｉｃｏｂａｃｔｅ
ｒｐｙｌｏｒｉ抗原１μｇ／ｍｌの測定値）からブラン
クチャンネル（固相Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌ
ｏｒｉ抗原０．１μｇ／ｍｌの測定値）を差し引いた値
を判定値とした。内視鏡診断で正常、かつ細菌培養検査
が陰性であったヒト血清（ｎ＝８）を測定し、そのＯ．
Ｄ．値のｘ＋２ＳＤ＝０．２００をカットオフ値と定め
それ以上を陽性と判定した。結果を表に示した。なお、
Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉの感染は内視
鏡時に採取した生検組織の培養検査によって陽性か陰性
を診断した。本発明による検出感度は８６．９％であり
特異性は７０．８％、そして正確度は８１．２％であっ
た。これらの結果は以下に記した比較例と比べ高感度で
あるのがわかる。 （比較例１） （実施例１）でブランクチャンネルには、何等抗原を吸
着させない場合の結果を表に示した。検出感度は６９．
２％であり特異性は７３．６％、そして正確度は７０．
６％であった。 （比較例２）Ｅｖａｎｓ（Ａ ｓｅｎｓｉｔｉｖｅ ａ
ｎｄ ｓｐｅｃｉｆｉｃ ｓｅｒｐｌｏｇｉｃ ｔｅｓ
ｔ ｆｏｒ ｄｅｔｅｃｔｉｏｎ ｏｆ Ｃａｍｐｙｌ
ｏｂａｃｔｅｒ ｐｙｌｏｒｉ ｉｎｆｅｃｔｉｏｎ．
Ｇａｓｔｒｏｅｎｔｅｒｏｌｏｇｙ ９６：１００４−
１００８，１９８９）の方法に従い測定した。すなわ
ち、界面活性剤（ｎ−オクチル−グルコシド）で可溶可
したＨｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ抗原をゲ
ルろ過かし、その第一ピークの抗原を、ＥＬＩＳＡ用９
６穴マイクロプレートに、１μｇ／ｍｌで、４℃一夜静
置した。プレートを、ＰＢＳ（０．１％Ｔｗｅｅｎ２０
含む）で洗浄後、ブロックエースでブロッキングした。
患者血清を１％の牛アルブミンを含むＰＢＳで５００倍
希釈にて添加後１時間反応させた。洗浄の後、ペルオキ
シダーゼ標識抗ヒトＩｇＧ抗体を加え１時間反応させ
た。再度洗浄後、基質液（０．０３％オルトフェニレン
ジアミン，０．００３％Ｈ２Ｏ２）を加え３７℃、１０
分反応させた。反応は２Ｎ硫酸を加え停止し、各ウェル
をマイクロプレートリーダー（λ１＝４９２ｎｍ，λ２
＝６６０ｎｍ）で比色した。内視鏡診断で正常、かつ培
養検査が陰性であったヒト血清（ｎ＝８）を各法にて測
定し、そのＯ．Ｄ．値のｘ＋２ＳＤ＝０．３００をカッ
トオフ値と定めそれ以上を陽性と判定した。その結果を
表に示した。検出感度は８２．３９％であり特異性は７
０．８％、そして正確度は７８．２％であった。 (Example 1) Helicobacter pylori
To Nagata (potent inhibitory act)
ion of the gastric protein
pump inhibitor lansopraz
ole again urease activ
ty of helicobacter pylor
i: unique action selective
for H. pylori cells. antim
ibrob agents chemother37:
769-774, 1993) followed by culturing, followed by Stagey (Antigenicity of).
fractions of helicobacter
pylori prepared by fast
protein liquid chromatogr
aphy and urease capture b
y monoclonal antibodies. E
urj clin microbiol infect
A soluble antigen was prepared according to the method of dis 9: 732-737, 1990). This antigen was put in a 96-well microplate for ELISA and 1 μg in the measurement channel.
/ Ml and 0.1 μg / m for blank channels
It was dispensed at 1 l and left at 4 ° C overnight. Plate the PBS
After washing with (containing 0.1% Tween 20), blocking was performed with Block Ace. The patient serum was diluted 500 times with PBS containing 1% bovine albumin, and after the addition, reaction was carried out for 1 hour. After washing, peroxidase-labeled anti-human IgG antibody was added and reacted for 1 hour. After washing again, the substrate solution (0.
03% orthophenylenediamine, 0.003% H 2 O
2 ) was added and reacted at 37 ° C. for 10 minutes. The reaction was stopped by adding 2N sulfuric acid, and each well was subjected to colorimetry with a microplate reader (λ 1 = 492 nm, λ 2 = 660 nm).
Then, the measurement channel (solid phase Helicobacte
blank channel (solid phase Helicobacter pyl) from rpylori antigen (measured value of 1 μg / ml)
The value obtained by subtracting the ori antigen (measured value of 0.1 μg / ml) was used as the judgment value. Human serum (n = 8), which was normal in endoscopic diagnosis and was negative in the bacterial culture test, was measured, and its O.
D. The value of x + 2SD = 0.200 was set as the cutoff value, and the value above that was determined to be positive. The results are shown in the table. In addition,
The infection with Helicobacter pylori was diagnosed as positive or negative by a culture test of biopsy tissue collected at the time of endoscopy. The detection sensitivity according to the invention was 86.9%, the specificity 70.8% and the accuracy 81.2%. It can be seen that these results have higher sensitivity than the comparative examples described below. (Comparative Example 1) The results when no antigen is adsorbed to the blank channel in (Example 1) are shown in the table. The detection sensitivity is 69.
2% with a specificity of 73.6% and an accuracy of 70.
It was 6%. (Comparative Example 2) Evans (A sensitive a)
nd specific serplogic tes
t for detection of Campyl
object pylori infection.
Gastroenterology 96: 1004-
1008, 1989). That is, the Helicobacter pylori antigen solubilized with a surfactant (n-octyl-glucoside) was subjected to gel filtration, and the first peak of the antigen was used for ELISA.
The plate was placed in a 6-well microplate at 1 μg / ml at 4 ° C. overnight. Plate the plate with PBS (0.1% Tween 20).
It was washed with Block Ace and then blocked.
Patient sera were diluted with PBS containing 1% bovine albumin at a 500-fold dilution, and reacted for 1 hour. After washing, peroxidase-labeled anti-human IgG antibody was added and reacted for 1 hour. After washing again, a substrate solution (0.03% orthophenylenediamine, 0.003% H 2 O 2 ) was added, and the mixture was incubated at 37 ° C. for 10 hours.
It was made to react for minutes. The reaction was stopped by adding 2N sulfuric acid, and each well was added to a microplate reader (λ 1 = 492 nm, λ 2
= 660 nm). Human serum (n = 8), which was normal in endoscopic diagnosis and negative in the culture test, was measured by each method, and the O. D. The value of x + 2SD = 0.300 was set as the cutoff value, and a value higher than that was determined to be positive. The results are shown in the table. The detection sensitivity is 82.39% and the specificity is 7
The accuracy was 0.8% and the accuracy was 78.2%.
【０００６】[0006]
【考案の効果】実施例１、比較例１および比較例２に示
される如く、本発明の方法によれば、高感度かつ特異的
に体液中の病原体（Ｈｅｌｉｃｏｂａｃｔｅｒｐｙｌｏ
ｒｉ）抗体を検出する事が可能であった。すなわち、簡
便に病原体（Ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒ
ｉ）感染を診断可能であった。EFFECTS OF THE INVENTION As shown in Example 1, Comparative Example 1 and Comparative Example 2, according to the method of the present invention, pathogens (Helicobacter pyllo) in body fluids are highly sensitive and specific.
ri) It was possible to detect the antibody. That is, a pathogen (Helicobacter pylor) is simply
i) The infection could be diagnosed.
【図１】図１は、酵素免疫測定法による血清Ｈｅｌｉｃ
ｏｂａｃｔｅｒ ｐｙｌｏｒｉ抗体測定結果を示すグラ
フである。（ａ）は陽性、（ｂ）は陰性そして（ｃ）は
陽性検体である。FIG. 1 shows serum Helic by enzyme immunoassay.
It is a graph which shows the result of an antibody pylori antibody measurement. (A) is a positive sample, (b) is a negative sample, and (c) is a positive sample.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 小西 登 奈良県橿原市白橿町７丁目８−17 (72)発明者 日浅 義雄 奈良県奈良市秋篠早月町９−１−301 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Noboru Konishi 7-8-17 Shirakashi-cho, Kashihara-shi, Nara Prefecture (72) Yoshio Hisasa 9-1-301 Akishino Hayatsuki-cho, Nara-shi, Nara Prefecture
Claims (9)
て、複数の測定チャンネルを持ち、それぞれが異なる濃
度の病原体由来可溶性抗原を含む組成物。1. A composition containing a soluble antigen of a pathogen, the composition having a plurality of measurement channels, each containing different concentrations of the pathogen-derived soluble antigen.
作を行い、各測定チャンネル間の測定データ差により、
体液中の病原体にたいする抗体を検出するための測定
法。2. The method according to claim 1, wherein the operation is performed according to the immunological assay method, and
Assay for detecting antibodies to pathogens in body fluids.
作を行い、各測定チャンネルにおいて１チャンネルでも
陽性があれば陽性と判定する、体液中の病原体にたいす
る抗体を検出するための測定法。3. A method for detecting an antibody against a pathogen in a body fluid, which is carried out according to the immunological assay method according to claim 1 and is positive if even one channel is positive in each measurement channel. .
の可溶性抗原を含む組成物であって、複数の測定チャン
ネルを持ち、それぞれが異なる濃度のＨｅｌｉｃｏｂａ
ｃｔｅｒ ｐｙｌｏｒｉ由来可溶性抗原を含む組成物。4. Helicobacter pylori
A soluble antigen of Helicoba that has multiple measurement channels, each of which has a different concentration.
A composition comprising a soluble antigen derived from cter pylori.
の可溶性抗原を含む組成物であって、ブランクチャンネ
ルにＨｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ以外の菌
体を含む組成物。5. Helicobacter pylori
A composition containing the soluble antigen of 1., wherein the blank channel contains cells other than Helicobacter pylori.
作を行い、各測定チャンネル間の測定データ差により、
体液中の病原体にたいする抗体を検出するための測定
法。6. The method according to claim 4, wherein an operation is performed according to an immunological measurement method,
Assay for detecting antibodies to pathogens in body fluids.
作を行い、各測定チャンネルにおいて１チャンネルでも
陽性があれば陽性と判定する、体液中の病原体にたいす
る抗体を検出するための測定法。7. An assay method for detecting an antibody against a pathogen in a body fluid, which is carried out according to the immunological assay method according to claim 4, and is positive if even one channel is positive in each measurement channel. .
測定法、ラジオイムノアッセイ法に従って検査すべき体
液と結合させることを含む病原体抗体検出のための測定
法。8. A method for detecting a pathogen antibody, which comprises binding to a body fluid to be tested according to the enzyme immunoassay method or the radioimmunoassay method according to claim 2 or 3.
測定法、ラジオイムノアッセイ法に従って検査すべき体
液と結合させることを含むＨｅｌｉｃｏｂａｃｔｅｒ
ｐｙｌｏｒｉ体抗体検出のための測定法。9. A Helicobacter according to claims 6 and 7, which comprises binding with a body fluid to be tested according to enzyme immunoassay, radioimmunoassay.
Assay for detection of pylori antibody.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12085394A JPH07294530A (en) | 1994-04-20 | 1994-04-20 | Detection of antibody in body fluids against pathogn |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP12085394A JPH07294530A (en) | 1994-04-20 | 1994-04-20 | Detection of antibody in body fluids against pathogn |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JPH07294530A true JPH07294530A (en) | 1995-11-10 |
Family
ID=14796571
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP12085394A Pending JPH07294530A (en) | 1994-04-20 | 1994-04-20 | Detection of antibody in body fluids against pathogn |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH07294530A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2008542703A (en) * | 2005-05-27 | 2008-11-27 | オーエヌシーイミューン リミテッド | Improved immunoassay method |
| US8574848B2 (en) | 2006-09-13 | 2013-11-05 | Oncimmune Ltd. | Immunoassay methods |
| US8592169B2 (en) | 2002-11-14 | 2013-11-26 | Oncimmune Limited | Tumour marker proteins and uses thereof |
| US9696319B2 (en) | 1998-05-11 | 2017-07-04 | Oncimmune Ltd. | Tumour markers |
| US9714938B2 (en) | 2005-05-27 | 2017-07-25 | Oncimmune Ltd. | Immunoassay methods |
-
1994
- 1994-04-20 JP JP12085394A patent/JPH07294530A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9696319B2 (en) | 1998-05-11 | 2017-07-04 | Oncimmune Ltd. | Tumour markers |
| US8592169B2 (en) | 2002-11-14 | 2013-11-26 | Oncimmune Limited | Tumour marker proteins and uses thereof |
| JP2008542703A (en) * | 2005-05-27 | 2008-11-27 | オーエヌシーイミューン リミテッド | Improved immunoassay method |
| JP4876127B2 (en) * | 2005-05-27 | 2012-02-15 | オーエヌシーイミューン リミテッド | Improved immunoassay method |
| US8722339B2 (en) | 2005-05-27 | 2014-05-13 | Oncimmune Ltd. | Immunoassay methods |
| US9714938B2 (en) | 2005-05-27 | 2017-07-25 | Oncimmune Ltd. | Immunoassay methods |
| US9719984B2 (en) | 2005-05-27 | 2017-08-01 | Oncimmune Ltd. | Immunoassay methods |
| US8574848B2 (en) | 2006-09-13 | 2013-11-05 | Oncimmune Ltd. | Immunoassay methods |
| US8927223B2 (en) | 2006-09-13 | 2015-01-06 | Oncimmune Ltd. | Immunoassay methods |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Alemohammad et al. | Detection of immunoglobulin G antibodies to Helicobacter pylori in urine by an enzyme immunoassay method | |
| Dominguez et al. | Evaluation of a rapid immunochromatographic assay for the detection of Legionella antigen in urine samples | |
| JP2901296B2 (en) | Preparation of Campylobacter pylori macromolecular cell-associated protein and its use for serological detection of Campylobacter pylori infection | |
| US5262156A (en) | Antigenic compositions and their use for the detection of Helicobacter pylori | |
| US5187065A (en) | Method and materials for detecting lyme disease | |
| Vaira et al. | invasive and non‐invasive tests for Helicobacter pylori infection | |
| AU2001243593B2 (en) | Method for diagnosing immunologic food sensitivity | |
| JP3969722B2 (en) | Candida detection | |
| Charpin et al. | Value of ELISA Using A60 Antigen in the Diagnosis of Active Pulmonary Tuberculosis1, 2 | |
| Stefano et al. | Non-invasive tests for the diagnosis of helicobacter pylori: state of the art | |
| De Klerk et al. | Comparative evaluation of the enzyme-linked immunosorbent assay in the laboratory diagnosis of brucellosis | |
| Rocha et al. | Immunoblot analysis of humoral immune response to Helicobacter pylori in children with and without duodenal ulcer | |
| Barrett et al. | Enzyme-linked immunosorbent assay for detection of Salmonella typhi Vi antigen in urine from typhoid patients | |
| Aumpan et al. | High efficacy of rapid urine test for diagnosis of Helicobacter pylori infection in Thai people | |
| Friis et al. | Screening for coeliac disease in adults by simultaneous determination of IgA and IgG gliadin antibodies | |
| Wunderlich et al. | A high-sensitivity enzyme-linked immunosorbent assay for serum thyroglobulin | |
| Boom et al. | New cause for false-positive results with the cryptococcal antigen test by latex agglutination | |
| Chiang et al. | A comparative study of three fecal occult blood tests in upper gastrointestinal bleeding | |
| Kiska et al. | Evaluation of new monoclonal antibody-based latex agglutination test for detection of cryptococcal polysaccharide antigen in serum and cerebrospinal fluid | |
| Sheehan et al. | Entamoeba histolytica: efficacy of microscopic, cultural, and serological techniques for laboratory diagnosis | |
| CA2134667C (en) | In vitro test for helicobacter pylori | |
| JPH07294530A (en) | Detection of antibody in body fluids against pathogn | |
| Wu et al. | Comparison of stool enzyme immunoassay and immunochromatographic method for detecting Helicobacter pylori antigens before and after eradication | |
| Karlsson et al. | Application and usefulness of enzyme immunoassay for diagnosis of Salmonella typhimurium infection | |
| Simm et al. | Storage conditions for serum for estimating prostate-specific antigen |