JP3033774B2 - Indirect agglutination reagent for detection of adult T-cell leukemia virus antibody - Google Patents
Indirect agglutination reagent for detection of adult T-cell leukemia virus antibodyInfo
- Publication number
- JP3033774B2 JP3033774B2 JP2169742A JP16974290A JP3033774B2 JP 3033774 B2 JP3033774 B2 JP 3033774B2 JP 2169742 A JP2169742 A JP 2169742A JP 16974290 A JP16974290 A JP 16974290A JP 3033774 B2 JP3033774 B2 JP 3033774B2
- Authority
- JP
- Japan
- Prior art keywords
- htlv
- adult
- cell leukemia
- leukemia virus
- reagent
- 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.)
- Expired - Fee Related
Links
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は成人T細胞白血病ウィルス(以下HTLV−Iと
記す)を界面活性剤で処理して得られる成分とHTLV−I
を構成するenv gp−21蛋白質を感作した担体粒子を用い
るHTLV−I抗体検出用間接凝集反応試薬に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] The present invention relates to a component obtained by treating adult T-cell leukemia virus (hereinafter referred to as HTLV-I) with a surfactant and HTLV-I.
The present invention relates to an indirect agglutination reagent for detecting an HTLV-I antibody using carrier particles sensitized with an env gp-21 protein.
HTLV−Iは白血病の一種であるT細胞リンパ腫をひき
おこす原因ウィルスとして知られており、このウィルス
感染の有無を検査することは臨床上、疫学上及び輸血の
際において重要である。HTLV-I is known as a causative virus that causes T-cell lymphoma, a type of leukemia, and testing for the presence of this virus is important in clinical, epidemiological and transfusion situations.
HTLV−I感染の有無を検査する方法としては、間接凝
集免疫測定法、螢光抗体法、酵素免疫測定法、放射免疫
測定法などが開発されており、その中でも間接凝集免疫
測定法は、測定を簡便かつ安価に測定することができる
点で優れている。HTLV−Iの測定に用いる間接凝集反応
試薬として、HTLV−Iを界面活性剤で処理し、得られる
成分を感作した担体粒子を用いる検査試薬(特開昭60−
44870号)がすでに知られている。As a method for testing for the presence or absence of HTLV-I infection, an indirect agglutination immunoassay, a fluorescent antibody method, an enzyme immunoassay, a radioimmunoassay, and the like have been developed. Is easy and inexpensive to measure. As an indirect agglutination reagent used for the measurement of HTLV-I, a test reagent using carrier particles treated with HTLV-I with a surfactant and sensitizing the resulting component (JP-A-60-1985)
44870) is already known.
しかしながら。従来の試薬は、測定に際し、検体が抗
体過剰である場合に、陽性検体であっても凝集が起こら
ないプロゾーン現象を生じることがあった。そのため、
陽性検体であっても陰性と判定されてしまう偽陰性の問
題を生じていた。However. In the case of a conventional reagent, a prozone phenomenon in which aggregation does not occur even in the case of a positive sample sometimes occurs when the sample is in excess of an antibody during measurement. for that reason,
The problem of a false negative in which even a positive sample is determined to be negative has occurred.
本発明者等は、従来の欠点を克服すべく検討した結
果、HTLV−Iを構成する蛋白質の1つであるenv gp−21
蛋白質とHTLV−Iを界面活性剤で処理して得られる成分
を感作した担体粒子を用いるHTLV−I抗体検出用間接凝
集反応試薬がプロゾーン現象を抑制しうることを見出し
本発明を完成した。The present inventors have studied to overcome the conventional disadvantages, and as a result, have found that one of the proteins constituting HTLV-I, env gp-21,
The present inventors have found that an indirect agglutination reagent for detecting HTLV-I antibodies using carrier particles sensitized with a component obtained by treating a protein and HTLV-I with a surfactant can suppress the prozone phenomenon, and completed the present invention. .
本発明はHTLV−Iを界面活性剤で処理して得られる成
分とHTLV−Iを構成するenv gp−21蛋白質を感作した担
体粒子を用いるHTLV−I抗体検出用間接凝集反応試薬で
ある。The present invention is an indirect agglutination reagent for detecting HTLV-I antibody using a component obtained by treating HTLV-I with a surfactant and carrier particles sensitized with env gp-21 protein constituting HTLV-I.
HTLV−Iを界面活性剤で処理して得られる成分を製造
するにあたっては、まずHTLV−I抗原を取得する必要が
あるが、HTLV−I抗原の取得するには、株化細胞を培養
し、培養液から細胞を分離してこの細胞から分離するか
又は培養上清からHTLV−Iを分離することにより取得す
ることができる。被化細胞としては、例えば、MT−2、
HUT−102等を用いることができる。In producing a component obtained by treating HTLV-I with a surfactant, it is necessary to first obtain an HTLV-I antigen.To obtain the HTLV-I antigen, a cell line is cultured, It can be obtained by separating cells from the culture solution and separating from the cells, or by separating HTLV-I from the culture supernatant. As the cells to be transformed, for example, MT-2,
HUT-102 or the like can be used.
培養液から細胞を分離してこの細胞から分離するに
は、培養後の培養液を遠心して集めた沈渣をトリス−塩
酸緩衝液−NaCl−EDTA、リン酸緩衝生理食塩溶液などで
洗浄した後、界面活性剤で処理することにより細胞及び
ウィルスを破壊して、この破壊した細胞及びウィルスの
遠心による沈殿物を除いた上清をウィルス抗原として用
いることができる。In order to separate the cells from the culture solution and separate them from the cells, the precipitate obtained by centrifuging the culture solution after culturing is washed with Tris-HCl buffer-NaCl-EDTA, a phosphate buffered saline solution, and the like. The cells and the virus are destroyed by treating with a surfactant, and the supernatant obtained by removing the precipitate of the disrupted cells and the virus by centrifugation can be used as a virus antigen.
また、培養上清からウィルスを取得する場合には、超
遠心密度勾配法によってHTLV−I画分を分離すればHTLV
−Iのみを分離することができ、この方法によればHTLV
−Iを高純度で取得することができる。分離したウィル
スは、通常、界面活性剤で破壊して抗原として用いる
が、ウィルスの増殖機能を失わせるだけでウィルス自体
を破壊することなく抗原として使用することもできる。When obtaining a virus from a culture supernatant, if the HTLV-I fraction is separated by ultracentrifugation density gradient method, HTLV
-I alone, and according to this method HTLV
-I can be obtained with high purity. The isolated virus is usually destroyed with a surfactant and used as an antigen. However, the virus can be used as an antigen without destroying the virus itself only by losing the growth function of the virus.
また、HTLV−Iを構成するenv gp−21蛋白質を取得す
るには、前記HTLV−Iを界面活性剤により処理して得ら
れる成分をレクチンクロマトグラフィーで精製すること
により得ることができる。The env gp-21 protein constituting HTLV-I can be obtained by treating the HTLV-I with a surfactant and purifying a component obtained by lectin chromatography.
上記抗原取得の際、使用することのできる界面活性剤
としては、ノニデットP40、トライトンΧ100等を挙げる
ことができる。Surfactants that can be used in obtaining the antigen include Nonidet P40 and Triton # 100.
HTLV−Iを界面活性剤で処理して得られる成分及びen
v gp−21蛋白質を担体粒子に感作する方法は、化学結合
を生成させることにより行なう方法又は物理吸着によっ
て行なう方法を挙げることができ、例えば、タンニン
酸、グルタールアルデヒド、ビスジアゾベンジジン、ト
リレンジイソシアナート、ジフロロジニトロベンゼン、
カルボジイミド類、キノン類、塩化クロム等のいわゆる
カップリング剤を使用する方法により達成することがで
きる。Components obtained by treating HTLV-I with a surfactant and en
v The method of sensitizing the carrier particles to the gp-21 protein can be a method by forming a chemical bond or a method by physical adsorption, such as tannic acid, glutaraldehyde, bisdiazobenzidine, Range isocyanate, difluorodinitrobenzene,
It can be achieved by a method using a so-called coupling agent such as carbodiimides, quinones, and chromium chloride.
試薬を調製するにあたっては、HTLV−Iを界面活性剤
で処理して得られる成分とenv gp−21蛋白質を所定量混
合し、上記方法を用いて同時に感作することにより行な
うことができる他、まず、HTLV−Iを界面活性剤で処理
して得られる成分又はenv gp−21蛋白質のいずれか一方
を感作し、次いで他方を感作するというように別々に感
作することにより行なうこともできる。さらに、それぞ
れの抗原成分のみからなる担体粒子を調製し、両担体粒
子を混合することにより本願発明の試薬とすることもで
きる。In preparing the reagent, a predetermined amount of the component obtained by treating HTLV-I with a surfactant and env gp-21 protein are mixed, and sensitization can be performed simultaneously using the above method. First, one of the components obtained by treating HTLV-I with a surfactant or the env gp-21 protein is sensitized, and then the other is sensitized to separately sensitize the components. it can. Furthermore, the reagent of the present invention can also be prepared by preparing carrier particles comprising only the respective antigen components and mixing both carrier particles.
担体粒子としては、ヒト、羊、ニワトリ等の動物の赤
血球、セラチア菌などの微生物菌体、ゼラチン粒子(特
開昭57−153658号)、ポリスチレンラテックス、カオリ
ン、炭末など、間接凝集免疫測定で用いられる粒子を使
用することができる。Examples of the carrier particles include erythrocytes of animals such as humans, sheep and chickens, microbial cells such as Serratia, gelatin particles (JP-A-57-153658), polystyrene latex, kaolin, charcoal powder, and the like. The particles used can be used.
実施例1 HTLV−I産生株化細胞であるHUT−102細胞を、ウシ胎
児血清を10%含有するRPMI−1640培地に接種し、5%炭
酸ガスを含む空気中で37℃、4日間培養した。この培養
液を20を3000rpmで10分間遠心して細胞を除去して上
清液を得た。この上清液を予め連続ゾーナルローター内
に作成した20〜55%の蔗糖密度勾配に重層し、循環しな
がら30000rpmで超遠心分離を行った。得られたウィルス
画分を再び遠心チューブ内に作成した20〜55%の連続密
度勾配に重層し、スイングローターにより25000rpmにて
4時間超遠心分離を行った。得られたウィルス画分を45
000rpmにて60分間遠心してウィルスを沈殿させ、この沈
殿に0.5%ノニデット−P40含リン酸緩衝生理食塩液を加
えた。沈殿を浮遊させ、氷水中で30分間攪拌して溶解さ
せ、13000rpm30分間遠心分離して得られた上清をHTLV−
I抗原とした。Example 1 HUT-102 cells, which are HTLV-I producing cell lines, were inoculated into RPMI-1640 medium containing 10% fetal bovine serum, and cultured at 37 ° C. for 4 days in air containing 5% carbon dioxide. . This culture was centrifuged at 3000 rpm for 10 minutes to remove cells, and a supernatant was obtained. This supernatant was layered on a 20-55% sucrose gradient previously prepared in a continuous zonal rotor, and ultracentrifuged at 30,000 rpm while circulating. The obtained virus fraction was again layered on a continuous density gradient of 20 to 55% prepared in a centrifuge tube, and ultracentrifuged at 25,000 rpm for 4 hours by a swing rotor. 45 of the virus fraction obtained
The virus was precipitated by centrifugation at 000 rpm for 60 minutes, and 0.5% Nonidet-P40-containing phosphate buffered saline was added to the precipitate. The precipitate is suspended, dissolved in ice water by stirring for 30 minutes, and the supernatant obtained by centrifugation at 13000 rpm for 30 minutes is subjected to HTLV-
I antigen.
調製したHTLV−I抗原350ml(400μg/ml)を10mlトリ
ス−塩酸緩衝液(pH8.3)で透析し、10000rpmで10分間
高速遠心分離して得られた上清をトリス−塩酸緩衝液
(pH8.3)で平衡化したレンチル−レクチンアフィニテ
ィーカラムに充填し、前記緩衝液で洗浄した後、メチル
αDマンノシドで溶出し、吸光度280nmの吸収を示す部
分を分取した。更に、得られた溶画分を10mMトリス−塩
酸緩衝液(pH8.3)で透析し、精製されたenv gp−21蛋
白質9mgを得た。The prepared HTLV-I antigen (350 ml, 400 μg / ml) was dialyzed against 10 ml Tris-HCl buffer (pH 8.3), and subjected to high-speed centrifugation at 10,000 rpm for 10 minutes. The column was packed in a lentil-lectin affinity column equilibrated in .3), washed with the above buffer solution, eluted with methyl αD mannoside, and a portion exhibiting an absorbance of 280 nm was collected. Further, the obtained fraction was dialyzed against 10 mM Tris-HCl buffer (pH 8.3) to obtain 9 mg of purified env gp-21 protein.
実施例2 ゼラチン粒子を、タンニン酸5ppmを含むpH7.2のリン
酸緩衝生理食塩水(以下、PBSと略記する。)中に粒子
濃度が2.5%になるように分散し、37℃で15分間加温し
た。次いで、粒子を遠心分離して生理食塩0で充分洗浄
した。得られた粒子をPBSに5%の濃度で分散し、この
分散液を実施例1で得られたenv gp−21蛋白質と混合
し、混合液を37℃で40分間加温して感作ゼラチン粒子を
調製した。同様の方法により、HTLV−I抗原のみが感作
されたゼラチン粒子を調製した。調製した感作粒子のそ
れぞれを1:1の比率で混合し、HTLV−I抗体検出用試薬
とした。Example 2 Gelatin particles were dispersed in phosphate-buffered saline (pH: 7.2) containing 5 ppm of tannic acid so as to have a particle concentration of 2.5%, and then dispersed at 37 ° C. for 15 minutes. Heated. The particles were then centrifuged and washed thoroughly with saline 0. The obtained particles were dispersed in PBS at a concentration of 5%, and this dispersion was mixed with the env gp-21 protein obtained in Example 1, and the mixture was heated at 37 ° C. for 40 minutes to give sensitized gelatin. Particles were prepared. In a similar manner, gelatin particles sensitized only with the HTLV-I antigen were prepared. Each of the prepared sensitized particles was mixed at a ratio of 1: 1 to obtain a reagent for detecting HTLV-I antibody.
実施例3 実施例1の方法により得られたenv gp−21蛋白質及び
HTLV−I抗原を5:1の比率で混合し、該混合物を実施例
2と同じ方法によりゼラチン粒子に感作し、HTLV−I抗
体検出用試薬とした。Example 3 env gp-21 protein obtained by the method of Example 1 and
The HTLV-I antigen was mixed at a ratio of 5: 1, and the mixture was sensitized to gelatin particles in the same manner as in Example 2 to obtain a reagent for detecting an HTLV-I antibody.
比較例1 実施例2で調製された試薬及び従来法により調製した
試薬を用い、HTLV−I抗体の偽陰性検体について間接凝
集反応テストを行った。得られた結果を下表に示す。Comparative Example 1 Using the reagent prepared in Example 2 and a reagent prepared by a conventional method, an indirect agglutination test was performed on a false-negative specimen of an HTLV-I antibody. The results obtained are shown in the table below.
比較例2 実施例2で調製された粒子及び従来法により調製した
粒子を用い、抗体過剰のHTLV−I抗体について間接凝集
反応テストを行った。 Comparative Example 2 Using the particles prepared in Example 2 and the particles prepared by the conventional method, an indirect agglutination test was performed on the antibody-rich HTLV-I antibody.
得られた結果を下表に示す。 The results obtained are shown in the table below.
〔発明の効果〕 本願発明の間接凝集反応試薬は、HTLV−I抗体の検査
において、従来の間接凝集反応試薬ではプロゾーン現象
により陰性と判定される可能性のある検体についても陽
性として適正に判定することができ、加えて測定感度も
高いという特徴を有するものである。従って、本願発明
により、HTLV−I抗体検査の検査制度を大幅に向上する
ことができる。 [Effects of the Invention] The indirect agglutination reagent of the present invention is properly determined as positive even in a test of an HTLV-I antibody, even if a conventional indirect agglutination reagent may be determined to be negative due to the prozone phenomenon. In addition, it is characterized by high measurement sensitivity. Therefore, according to the present invention, the inspection system of the HTLV-I antibody inspection can be significantly improved.
フロントページの続き (56)参考文献 特開 昭60−44870(JP,A) 特開 昭59−35143(JP,A) 特開 昭63−277970(JP,A) 特開 平2−1559(JP,A) J.Virol.,63(11)(1989) p4952−4957 J.Immunol.,142(3) (1989)p971−978Continuation of front page (56) References JP-A-60-44870 (JP, A) JP-A-59-35143 (JP, A) JP-A-63-277970 (JP, A) JP-A-2-1559 (JP) , A) J. Virol. , 63 (11) (1989) pp. 4952-4957. Immunol. , 142 (3) (1989) p971-978.
Claims (2)
処理して得られる成分と成人T細胞白血病ウィルスのen
v gp−21蛋白質を感作した担体粒子を用いる成人T細胞
白血病ウィルス抗体検出用間接凝集反応試薬。1. A component obtained by treating an adult T-cell leukemia virus with a surfactant, and a component obtained by treating the adult T-cell leukemia virus.
v Indirect agglutination reagent for detecting adult T-cell leukemia virus antibody using carrier particles sensitized with gp-21 protein.
試薬。2. The reagent according to claim 1, wherein the carrier particles are gelatin.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2169742A JP3033774B2 (en) | 1990-06-29 | 1990-06-29 | Indirect agglutination reagent for detection of adult T-cell leukemia virus antibody |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2169742A JP3033774B2 (en) | 1990-06-29 | 1990-06-29 | Indirect agglutination reagent for detection of adult T-cell leukemia virus antibody |
Publications (2)
Publication Number | Publication Date |
---|---|
JPH0460458A JPH0460458A (en) | 1992-02-26 |
JP3033774B2 true JP3033774B2 (en) | 2000-04-17 |
Family
ID=15892007
Family Applications (1)
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JP2169742A Expired - Fee Related JP3033774B2 (en) | 1990-06-29 | 1990-06-29 | Indirect agglutination reagent for detection of adult T-cell leukemia virus antibody |
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2752355C1 (en) * | 2020-08-18 | 2021-07-26 | Михаил Сергеевич Беллавин | Snow removal apparatus |
-
1990
- 1990-06-29 JP JP2169742A patent/JP3033774B2/en not_active Expired - Fee Related
Non-Patent Citations (2)
Title |
---|
J.Immunol.,142(3)(1989)p971−978 |
J.Virol.,63(11)(1989)p4952−4957 |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
RU2752355C1 (en) * | 2020-08-18 | 2021-07-26 | Михаил Сергеевич Беллавин | Snow removal apparatus |
Also Published As
Publication number | Publication date |
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JPH0460458A (en) | 1992-02-26 |
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