JP2004301646A - Nonspecific reaction absorbent for immunity inspection - Google Patents
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Abstract
Description
【０００１】
【発明が属する技術分野】
本発明は、各種細胞により産生された抗原または抗体を用いて免疫検査を行う場合の非特異反応吸収剤に関する。
【０００２】
【従来の技術】
臨床検査で用いられる免疫検査法としては、ラジオイムノアッセイ、酵素免疫測定法、免疫比濁法、ラテックス凝集法等の種々の検査方法が公知であるが、いずれの場合も測定の目的とする抗原又は抗体と、それに反応する抗体又は抗原を反応させることにより測定が行われる。検体中の抗原、抗体等を免疫測定する方法は、例えば対応する抗体、抗原等を免疫測定用の固相に結合させて感作し、試薬と検体とを反応させ、この固相での免疫反応により免疫複合体を形成させて結合する抗原、抗体等を測定する。例えば検体中の抗体を測定する場合には、抗原を結合した固相が用いられる。この抗原は、生体や培養液から得られた天然型蛋白質、遺伝子組換法によって製造した組換蛋白質、アミノ酸を縮合反応させて製造した合成蛋白質等が知られている。遺伝子組換技術が開発されて以来、所望の抗原をコードする遺伝子を組み込んだ宿主細胞を培養することによって、均一な性質を有する蛋白質を大量かつ安価に、また感染の危険を伴うことなく得ることができるようになった。さらに、遺伝子組換蛋白質は検体中の抗体との特異的な反応性が高いため、免疫検査において広く用いられるようになった。
【０００３】
例えば、肝炎ウイルス、ＨＩＶウイルス、ＨＴＬＶウイルス、ヘルペスウイルス、麻疹ウイルス、サイトメガロウイルス、インフルエンザウイルスなどの関連抗原が遺伝子組換技術によって製造され、実際に診断薬に利用されている。
【０００４】
抗原又は抗体産生細胞から目的とする抗原又は抗体を生成して得たものを固相に感作して免疫測定を行う場合に、所望の抗原又は抗体の他に産生細胞成分が固相に感作されることがある。このような場合に、測定試薬や測定検体中に産生細胞成分と反応しうる物質が混入していると、産生細胞成分と該反応しうる物質が免疫複合体を形成することで、所望の測定結果が得られない。このような非特異反応を回避する方法が検討されてきた。例えば、抗原産生に利用する宿主となる細菌と同種細菌成分を反応液中に添加することが行われている（特許文献１）。あるいは、抗原産生に用いられるベクターと同種であって、且つ上記抗原をコードする遺伝子を含まないベクターを組み込んだ宿主細胞の培養成分を用いた方法が知られている（特許文献２）。
【０００５】
【先行技術】
【特許文献１】
特公平３−５９３８２号公告公報
【特許文献２】
特開平８−４３３９２号公開公報
【０００６】
【発明が解決しようとする課題】
上記の方法は、非特異反応抑制には効果がみられた。しかしながら、遺伝子組み換え抗原を用いた抗体検出試薬を感染症の診断薬として実用化するにはまだその効果は十分とは言えなかった。本発明は、より効果的な非特異反応抑制効果が得られる非特異反応吸収剤を提供することを課題とする。
【０００７】
【課題を解決するための手段】
本発明の抗体検出用試薬の非特異反応吸収剤は、免疫検査に使用する抗原又は抗体の産生細胞と同種の細胞の水抽出物を加熱処理し、生成した不溶性画分を該水抽出物から除去することにより、効果的な非特異反応吸収剤が得られる。
【０００８】
すなわち本発明は、
１．免疫検査に使用する抗原又は抗体の産生細胞と同種の細胞で、前記抗原又は抗体を含まない細胞成分の水抽出物を加熱処理し、生成した不溶性画分を該水抽出物から除去してなる免疫検査用非特異反応吸収剤、
２．抗原又は抗体の産生細胞が大腸菌である前項１に記載の免疫検査用非特異反応抑制物質、
３．加熱処理温度が、８０℃〜１００℃である前項１又は２に記載の免疫検査用非特異反応吸収剤、
４．加熱処理時間が３分〜１０分である前項１〜３のいずれか１に記載の抗体検出用試薬の非特異反応吸収剤、
５．加熱処理が非加圧条件下で行われることを特徴とする前項１〜４のいずれか１に記載の抗体検出用試薬の非特異反応吸収剤、
６．前項１〜５のいずれか１に記載の非特異反応吸収剤を含む免疫検査用試薬、
７．前項１〜５のいずれか１に記載の非特異反応吸収剤を使用する免疫検査方法、からなる。
【０００９】
【発明の実施の形態】
本発明において、免疫検査に使用する抗原又は抗体の産生細胞は、抗原又は抗体を産生しうる細胞であれば真核細胞であっても原核細胞であっても良い。該細胞は、遺伝子組換技術や、モノクローナル抗体産生技術等の遺伝子や蛋白質の情報を人為的に加工することにより得られる抗原又は抗体を産生しうる細胞であることが好ましい。具体的には、宿主として大腸菌、酵母、枯草菌、昆虫細胞、動物細胞等の自体公知の宿主を本発明の産生細胞とすることができる。また、抗体産生細胞としては、マウスのＢ細胞とマウスの骨髄腫細胞とのハイブリドーマのように公知の細胞を本発明の産生細胞とすることができる。さらに具体的には、試薬用遺伝子組換え技術において汎用される大腸菌が抗原産生細胞として好適である。
【００１０】
本発明で使用される細胞は、抗原や抗体として使用される所望の遺伝子や蛋白質が含まれていない宿主細胞となりうる細胞である。例えば遺伝子組換え技術に使用される宿主では、遺伝子組み換え操作を行っていないものを適用することができ、抗原産生に用いられるベクターであって抗原をコードする遺伝子を含まないベクターを組み込んだ宿主細胞であっても良い。
【００１１】
上記産生細胞となりうる細胞の水抽出物は、例えば細胞培養後、培養液を超音波処理して細胞成分を破砕後、遠心分離を行って細胞成分を分離し上清分画を回収することによって得ることができる。超音波処理条件や遠心分離条件は、大腸菌等の菌体細胞内から遺伝子組み換え抗原を取り出すときと同様の条件で行うことができる。
【００１２】
次に、得られた水抽出物を加熱処理する。加熱処理にあたっては、加熱温度は８０℃以上で行うことが好ましく、具体的には８０〜１００℃、より具体的には９０〜１００℃の範囲で行うことが好ましい。加熱時間は３〜１０分であれば良く、４〜８分で行うことが好ましい。また、加熱処理は常圧下で行うことが望ましい。
【００１３】
水抽出物を加熱処理した後、生成した不溶性画分を除去したものを非特異反応吸収剤とすることができる。加熱処理後に生成する不溶性画分は、加熱処理による変性蛋白質などが主なものである。不溶性画分の除去はいかなる方法によっても良いが、例えば遠心分離やろ過膜を用いて処理する方法、これらを組み合わせた方法等を適用することができる。
【００１４】
本発明の非特異反応吸収剤は、抗原抗体反応を行うときに用いる水性溶媒（希釈液等）に添加して用いることができる。また、この水性溶媒には、緩衝剤、塩類やアルブミン、あるいはデキストランのような増感剤等を含有させることができる。本発明では、このような水性溶媒も免疫検査用試薬として取り扱う。また、本発明の非特異反応吸収剤それ自体も免疫反応用試薬として取り扱うことができる。
【００１５】
【実施例】
以下、本発明の内容を実施例を示して具体的に説明するが、本発明はこれらの実施例に何ら限定されるものではない。
【００１６】
（実施例１）非特異反応吸収剤の調製（本発明法）
１）大腸菌をＬＢ培地中で３７℃で一晩培養し、増殖した大腸菌を含む培養液を６ｌ採取し、遠心分離（３０００ｒｐｍ×１５分）した。
２）沈殿した菌体に１０ｍＭリン酸緩衝液（ｐＨ７．０）１１０ｍｌを加え、５分間超音波処理した。
３）超音波処理により破砕した溶液全量を遠心分離（１２０００ｒｐｍ×１０分）し、上清画分を回収した（大腸菌成分濃度は７．９０ｍｇ／ｍｌ）。
４）上記回収した上清画分を１００℃で５分間加熱処理し、さらに遠心分離（１２０００ｒｐｍ×１０分）により沈殿画分を除去し上清画分を得た。
加熱処理後の上清画分中には、大腸菌成分は１．８７ｍｇ／ｍｌ含まれる。この水溶液を非特異反応吸収剤とし、反応緩衝液に添加した。
また、上記３）で回収した上清画分を上清画分を１０ｍＭリン酸緩衝液（ｐＨ７．０）で希釈して上清中の大腸菌成分濃度を４ｍｇ／ｍｌに調製し、上記４）と同じように加熱処理して非特異反応吸収剤とし、反応緩衝液に添加した。
【００１７】
（比較例１）非特異反応吸収剤の調製（従来法）
実施例１の１）〜３）の処理を行った水溶液を従来法による非特異反応吸収剤とした。この非特異反応吸収剤には、大腸菌成分は７．９０ｍｇ／ｍｌ含まれる。該非特異反応吸収剤を反応用緩衝液に添加した。
【００１８】
（実験例１）免疫凝集試験
（材料）
１）ラテックス試薬：
大腸菌由来リコンビナントＨＩＶ抗原感作ラテックス試薬
ＨＩＶ抗原は、特開平８−４３３９２号の記載に従って調製し、抗原感作ラテックスは、特開２００２−２８６７２０号の記載に従って調製した。
２）検体：
陰性コントロール（５ｗ／ｖ％ウシ血清アルブミンを含む１０ｍＭ ＰＢＳ（ｐＨ７．２）水溶液）
正常血清（オーソ社製）
非特異反応検体（抗大腸菌ウサギ抗体（ＤＡＫＯ社製）
（１倍（×１）、２倍（×２）、５倍（×５）、１０倍（×１０）希釈試料）
３）反応緩衝液：
７．５ｗ／ｖ％ウシ血清アルブミンを含む０．１２ｗ／ｖ％Ｔｒｉｓ緩衝液（ｐＨ７．０）
４）非特異反応吸収剤：
従来法による非特異反応吸収剤 １００μｇ／ｍｌ
本発明の非特異反応吸収剤 １００μｇ／ｍｌ
【００１９】
（方法）
測定は、シスメックス社製測定装置（ＰＡＭＩＡ−５０）を用いてＳｙｓｍｅｘ Ｊｏｕｒｎａｌ Ｖｏｌ．２０ Ｎｏ．１， ｐ７７−８６（１９９７）に記載の方法に従った。
反応プレートのウェルに、ラテックス凝集反応用緩衝液を８０μｌ、検体を１０μｌ及び上記ラテックス粒子を含む溶液を１０μｌを添加し、４５℃で反応させた。反応を開始してから約２０秒後に、１９μｌの反応混合物を９５０μｌのシース液に加えて５１倍に希釈した。希釈された反応混合物を、ＰＡＭＩＡ−５０の光学検出部に導き、凝集度Ｐ／Ｔ（％）（Ｔ１）を測定した。
反応を開始してから約１５分後に、凝集度Ｐ／Ｔ（％）（Ｔ１）の測定と同様にして凝集度Ｐ／Ｔ（％）（Ｔ２）を測定した。なお、Ｔ１は反応初期の凝集度であり、試料が測定範囲内にあるかどうか確認する際に利用されるもので、通常はＴ２を試料の凝集度（凝集率）として採用する。ここで、凝集していないラテックス粒子のカウント数をＭ（Ｍｏｎｏｍｅｒ：モノマー）、２個以上のラテックス粒子が凝集したもののカウント数をＰ（Ｐｏｌｙｍｅｒ：ポリマー）、ＭとＰの和をＴ（Ｔｏｔａｌ）とする。
【００２０】
（結果）
その結果を表１に示した。加熱処理しない大腸菌水抽出物を添加した場合（従来法）は、対照である大腸菌水抽出物を添加しない場合と比較して抗大腸菌抗体に対する反応性が低下すること確認された。一方、水抽出物を熱処理した場合、加熱前の大腸菌水抽出物の濃度に関わらず、抗大腸菌抗体に対する反応性が更に低下することが確認された。このこから、従来法によってもある程度の非特異反応が抑制されたが、加熱処理によりさらに効果的に非特異反応が抑制されたものと考えられた。
【００２１】
【表１】
【発明の効果】
以上説明したように、本発明の非特異反応吸収剤を使用して免疫検査を行うと、従来法の非特異反応吸収剤を使用した場合に比べて非特異反応が抑制され、正しい測定結果が得られることとなった。[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a non-specific reaction absorbent for performing an immunological test using an antigen or an antibody produced by various cells.
[0002]
[Prior art]
As immunoassays used in clinical tests, various tests such as radioimmunoassay, enzyme immunoassay, immunoturbidimetry, latex agglutination, etc. are known. The measurement is performed by reacting the antibody with an antibody or antigen that reacts with the antibody. A method for immunoassaying an antigen, an antibody, etc. in a sample is, for example, binding the corresponding antibody, antigen, etc., to a solid phase for immunoassay, sensitizing, reacting a reagent with a sample, and immunizing with this solid phase. An antigen, an antibody and the like which form an immune complex and bind by reaction are measured. For example, when measuring an antibody in a sample, a solid phase to which an antigen is bound is used. As the antigen, a natural protein obtained from a living body or a culture solution, a recombinant protein produced by a gene recombination method, a synthetic protein produced by a condensation reaction of amino acids, and the like are known. Since the development of gene recombination technology, culturing host cells into which a gene encoding a desired antigen has been incorporated to obtain a protein having uniform properties in large quantities, at low cost, and without risk of infection Is now available. Further, the recombinant protein has a high specific reactivity with an antibody in a sample, and thus has been widely used in immunoassays.
[0003]
For example, related antigens such as hepatitis virus, HIV virus, HTLV virus, herpes virus, measles virus, cytomegalovirus, and influenza virus have been produced by gene recombination techniques and are actually used as diagnostic agents.
[0004]
In the case where a target antigen or antibody produced from an antigen or antibody-producing cell is produced and sensitized to a solid phase to perform immunoassay, in addition to a desired antigen or antibody, a producer cell component is sensitized to the solid phase. May be made. In such a case, if a substance capable of reacting with the production cell component is mixed in the measurement reagent or the measurement specimen, the substance capable of reacting with the production cell component forms an immune complex, whereby the desired measurement is performed. No result is obtained. Methods for avoiding such non-specific reactions have been studied. For example, a bacterial component which is the same as a bacterium used as a host for antigen production is added to a reaction solution (Patent Document 1). Alternatively, a method using a host cell culture component in which a vector that is the same species as the vector used for antigen production and does not contain the gene encoding the antigen is incorporated is known (Patent Document 2).
[0005]
[Prior art]
[Patent Document 1]
Japanese Patent Publication No. 3-59382 [Patent Document 2]
Japanese Patent Application Laid-Open Publication No. Hei 8-43392
[Problems to be solved by the invention]
The above method was effective in suppressing non-specific reactions. However, the effect has not been sufficient for practical use of an antibody detection reagent using a recombinant antigen as a diagnostic agent for infectious diseases. An object of the present invention is to provide a non-specific reaction absorbent that can obtain a more effective non-specific reaction inhibitory effect.
[0007]
[Means for Solving the Problems]
The non-specific reaction absorbent of the antibody detection reagent of the present invention is obtained by subjecting an aqueous extract of cells of the same species as the antigen- or antibody-producing cells used for immunoassay to a heat treatment, and separating the resulting insoluble fraction from the aqueous extract. By removing, an effective non-specific reaction absorbent is obtained.
[0008]
That is, the present invention
1. A cell extract of the same kind as the antigen- or antibody-producing cells used for the immunoassay is subjected to a heat treatment with an aqueous extract of the cell component not containing the antigen or antibody, and the generated insoluble fraction is removed from the aqueous extract. Nonspecific reaction absorbent for immunoassay,
2. The non-specific reaction-suppressing substance for immunological tests according to the above 1, wherein the antigen- or antibody-producing cells are Escherichia coli
3. The non-specific reaction absorbent for immunoassay according to the above 1 or 2, wherein the heat treatment temperature is 80 ° C to 100 ° C,
4. The non-specific reaction absorbent of the antibody detection reagent according to any one of Items 1 to 3, wherein the heat treatment time is 3 minutes to 10 minutes,
5. The nonspecific reaction absorbent of the antibody detection reagent according to any one of the above items 1 to 4, wherein the heat treatment is performed under non-pressurized conditions,
6. An immunoassay reagent comprising the nonspecific reaction absorbent according to any one of the above items 1 to 5,
7. An immunoassay method using the nonspecific reaction absorbent according to any one of the above items 1 to 5.
[0009]
BEST MODE FOR CARRYING OUT THE INVENTION
In the present invention, the antigen- or antibody-producing cells used for the immunoassay may be eukaryotic cells or prokaryotic cells as long as they can produce the antigen or antibody. The cell is preferably a cell capable of producing an antigen or an antibody obtained by artificially processing gene or protein information such as a gene recombination technique or a monoclonal antibody production technique. Specifically, known hosts such as Escherichia coli, yeast, Bacillus subtilis, insect cells, and animal cells can be used as the production cells of the present invention. In addition, as the antibody-producing cells, known cells such as hybridomas of mouse B cells and mouse myeloma cells can be used as the production cells of the present invention. More specifically, Escherichia coli commonly used in gene recombination technology for reagents is suitable as an antigen-producing cell.
[0010]
The cell used in the present invention is a cell that can be a host cell that does not contain a desired gene or protein used as an antigen or antibody. For example, as a host used for gene recombination technology, those that have not been subjected to genetic recombination can be applied, and host cells incorporating a vector that is used for antigen production and does not contain a gene encoding an antigen. It may be.
[0011]
The aqueous extract of cells that can be the above-mentioned producer cells is, for example, after cell culture, after crushing the cell components by sonication of the culture solution, centrifuging to separate the cell components, and collecting the supernatant fraction. Obtainable. Ultrasonic treatment conditions and centrifugation conditions can be performed under the same conditions as when genetically engineered antigens are extracted from bacterial cells such as Escherichia coli.
[0012]
Next, the obtained water extract is subjected to a heat treatment. The heat treatment is preferably performed at a heating temperature of 80 ° C. or higher, specifically, in the range of 80 to 100 ° C., more specifically, in the range of 90 to 100 ° C. The heating time may be 3 to 10 minutes, and preferably 4 to 8 minutes. It is desirable that the heat treatment be performed under normal pressure.
[0013]
After the water extract has been subjected to heat treatment, the resulting insoluble fraction can be removed and used as the non-specific reaction absorbent. The insoluble fraction generated after the heat treatment is mainly a denatured protein or the like by the heat treatment. The insoluble fraction may be removed by any method. For example, a method of processing using a centrifugal separation or a filtration membrane, a method combining these methods, and the like can be applied.
[0014]
The non-specific reaction absorbent of the present invention can be used by adding it to an aqueous solvent (diluent or the like) used when performing an antigen-antibody reaction. In addition, the aqueous solvent may contain a buffer, a salt, albumin, or a sensitizer such as dextran. In the present invention, such an aqueous solvent is also handled as an immunoassay reagent. In addition, the nonspecific reaction absorbent of the present invention itself can be handled as a reagent for an immune reaction.
[0015]
【Example】
Hereinafter, the content of the present invention will be specifically described with reference to examples, but the present invention is not limited to these examples.
[0016]
Example 1 Preparation of Nonspecific Reaction Absorbent (Method of the Present Invention)
1) Escherichia coli was cultured in an LB medium at 37 ° C. overnight, and 6 l of a culture solution containing the grown Escherichia coli was collected and centrifuged (3000 rpm × 15 minutes).
2) To the precipitated cells, 110 ml of 10 mM phosphate buffer (pH 7.0) was added, and sonicated for 5 minutes.
3) The total amount of the solution crushed by the ultrasonic treatment was centrifuged (12000 rpm × 10 minutes), and the supernatant fraction was collected (the concentration of Escherichia coli was 7.90 mg / ml).
4) The collected supernatant fraction was heat-treated at 100 ° C. for 5 minutes, and the precipitate fraction was removed by centrifugation (12000 rpm × 10 minutes) to obtain a supernatant fraction.
The supernatant fraction after the heat treatment contains 1.87 mg / ml of the E. coli component. This aqueous solution was used as a non-specific reaction absorbent and added to the reaction buffer.
Further, the supernatant fraction collected in the above 3) was diluted with a 10 mM phosphate buffer (pH 7.0) to adjust the E. coli component concentration in the supernatant to 4 mg / ml. Heat treatment was performed in the same manner as in Example 1 to obtain a non-specific reaction absorbent, which was added to the reaction buffer.
[0017]
Comparative Example 1 Preparation of Nonspecific Reaction Absorbent (Conventional Method)
The aqueous solution subjected to the treatments 1) to 3) of Example 1 was used as a nonspecific reaction absorbent according to a conventional method. This non-specific reaction absorbent contains 7.90 mg / ml of the E. coli component. The non-specific reaction absorbent was added to the reaction buffer.
[0018]
(Experimental example 1) Immunoagglutination test (material)
1) Latex reagent:
Escherichia coli-derived recombinant HIV antigen-sensitized latex reagent HIV antigen was prepared according to the description in JP-A-8-43392, and antigen-sensitized latex was prepared according to the description in JP-A-2002-286720.
2) Sample:
Negative control (10 mM PBS (pH 7.2) aqueous solution containing 5 w / v% bovine serum albumin)
Normal serum (Ortho)
Non-specific reaction sample (anti-Escherichia coli rabbit antibody (DAKO))
(1x (x1), 2x (x2), 5x (x5), 10x (x10) diluted sample)
3) Reaction buffer:
0.12 w / v% Tris buffer (pH 7.0) containing 7.5 w / v% bovine serum albumin
4) Non-specific reaction absorbent:
Non-specific reaction absorbent by conventional method 100μg / ml
Non-specific reaction absorbent of the present invention 100 μg / ml
[0019]
(Method)
The measurement was performed using Sysmex Journal Vol. 10 using a measuring device (PAMIA-50) manufactured by Sysmex Corporation. 20 No. 1, p77-86 (1997).
To the wells of the reaction plate, 80 μl of a buffer for latex agglutination reaction, 10 μl of a sample, and 10 μl of a solution containing the above latex particles were added, and reacted at 45 ° C. About 20 seconds after the start of the reaction, 19 μl of the reaction mixture was added to 950 μl of the sheath liquid and diluted 51-fold. The diluted reaction mixture was led to an optical detection unit of PAMIA-50, and the degree of aggregation P / T (%) (T1) was measured.
About 15 minutes after the start of the reaction, the aggregation degree P / T (%) (T2) was measured in the same manner as the measurement of the aggregation degree P / T (%) (T1). T1 is the degree of agglomeration at the beginning of the reaction, and is used when confirming whether the sample is within the measurement range. Usually, T2 is adopted as the degree of agglutination (aggregation rate) of the sample. Here, the count number of latex particles that are not aggregated is M (Monomer: monomer), the count number of aggregates of two or more latex particles is P (Polymer: polymer), and the sum of M and P is T (Total). And
[0020]
(result)
The results are shown in Table 1. It was confirmed that the reactivity with the anti-Escherichia coli antibody was reduced when the E. coli water extract without heat treatment was added (conventional method) as compared with the case where the E. coli water extract as a control was not added. On the other hand, when the water extract was heat-treated, it was confirmed that the reactivity with the anti-Escherichia coli antibody was further reduced regardless of the concentration of the E. coli water extract before heating. From this, it was considered that the non-specific reaction was suppressed to some extent by the conventional method, but the non-specific reaction was more effectively suppressed by the heat treatment.
[0021]
[Table 1]
【The invention's effect】
As described above, when an immunological test is performed using the non-specific reaction absorbent of the present invention, non-specific reactions are suppressed as compared with the case where the conventional non-specific reaction absorbent is used, and a correct measurement result is obtained. It was obtained.
Claims (7)
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