JP2012077060A5

JP2012077060A5 -

Info

Publication number: JP2012077060A5
Application number: JP2010226734A
Authority: JP
Filing date: 2010-10-06
Publication date: 2013-09-19
Anticipated expiration: 2030-10-06

Description

さらに、本発明者らは、Ｍ環部にヒドロキシ基を有するシガトキシン51-OH-CTX3CやCTX1Bを検出する抗体を作製するために、シガトキシン類のHIJKLM環部の構造を有する化合物をはじめて合成し、これをハプテンとして動物を免疫することにより受託番号FERM BP-11400として寄託されたハイブリドーマ8H4を作製し、該ハイブリドーマを用いてシガトキシン類51-OH-CTX3CやCTX1Bに特異的に結合する特異性の高いモノクローナル抗体8H4を調製することに成功した。また、本発明者らは、上記モノクローナル抗体8H4と10C9とを組み合わせることにより、検出特性を改善したサンドイッチ法によりシガトキシン51-OH-CTX3Cを検出するキットを作製した(特許文献４および非特許文献７)。 Furthermore, the present inventors synthesized for the first time a compound having the structure of the HIJKLM ring part of ciguatoxins in order to produce an antibody for detecting ciguatoxin 51-OH-CTX3C or CTX1B having a hydroxy group in the M ring part, This was prepared hybridomas 8H4 deposited as accession number FERM B P- 11400 by immunizing an animal as a hapten, specificity that bind specifically to ciguatoxins 51-OH-CTX3C and CTX1B using the hybridoma We succeeded in preparing high monoclonal antibody 8H4. In addition, the present inventors produced a kit for detecting ciguatoxin 51-OH-CTX3C by a sandwich method with improved detection characteristics by combining the monoclonal antibodies 8H4 and 10C9 (Patent Document 4 and Non-Patent Document 7). ).

本発明者らは、上記の課題を解決するために、Ａ環部にジヒドロキシブテニル基を有するシガトキシン類のABCDE環部の構造を有する化合物をはじめて合成し、これをハプテンとして用いることにより、上記の課題を解決できることを見出した。
よって、本発明は、Ａ環部にジヒドロキシブテニル基を有するシガトキシン類を特異的に認識するモノクローナル抗体を提供する。
また、本発明は、上記のモノクローナル抗体を産生する受託番号FERM BP-11401として寄託されたハイブリドーマ3G8（受託番号FERM BP-11401として独立行政法人産業技術総合研究所特許生物寄託センター(茨城県つくば市東1-1-1中央第６、〒305-8566、日本)に寄託された。受託日：平成22年9月15日、本明細書ではこのハイブリドーマを抗体と同じ名称3G8と表示する。）である。 In order to solve the above problems, the present inventors synthesized for the first time a compound having the ABCDE ring structure of ciguatoxins having a dihydroxybutenyl group in the A ring part, and using this as a hapten, I found that I could solve the problem.
Therefore, the present invention provides a monoclonal antibody that specifically recognizes ciguatoxins having a dihydroxybutenyl group in the A ring portion.
The present invention also consignment number FERM B hybridoma deposited as P- 11401 3G8 producing the monoclonal antibodies described above (consignment number FERM B P- 11401 as AIST Patent Organism Depositary ( Higashi, Tsukuba, Ibaraki, 1-1-1 central 6, Yubinbango305-8566, has been deposited in Japan) entrusted Date:., 2010 September 15, in the present specification, the same name 3G8 this hybridoma and antibody Display.)

また、本発明は、上記のモノクローナル抗体と、受託番号FERM BP-11400として寄託されたハイブリドーマ8H4（平成16年12月22日に独立行政法人産業技術総合研究所特許生物寄託センター(茨城県つくば市東1-1-1中央第６、〒305-8566、日本)に寄託）により産生されるモノクローナル抗体またはFERM BP-8298として受託されたハイブリドーマ3D11(平成14年3月5日に独立行政法人産業技術総合研究所特許生物寄託センター(茨城県つくば市東1-1-1中央第６、〒305-8566、日本)に寄託）により産生されるモノクローナル抗体とを含むシガトキシン類検出キットである。 In addition, the present invention, the above monoclonal antibody, accession number FERM B P- 11400 hybridoma deposited as 8H4 (independent in 2004 December 22, National Institute of Advanced Industrial Science and Technology Patent Organism Depositary (Tsukuba, Ibaraki Prefecture Monoclonal antibody produced by 1-1-1, Chuo 6-Chito, Japan 305-8566, Japan) or hybridoma 3D11 (FERM BP-8298) on 5th March 2002 This is a kit for detecting ciguatoxins comprising a monoclonal antibody produced by the Research Center for Biological Biology (deposited at Higashi 1-1-1 Higashi 1-1-1, Tsukuba, Ibaraki, Japan, 305-8566, Japan).

その他のモノクローナル抗体としては、受託番号FERM BP-11400として平成16年12月22日に独立行政法人産業技術総合研究所特許微生物寄託センターに寄託されたハイブリドーマ8H4により産生されるモノクローナル抗体8H4、または受託番号FERM BP-8293として平成14年３月５日に独立行政法人産業技術総合研究所特許微生物寄託センターに原寄託されたハイブリドーマ3D11により産生されるモノクローナル抗体3D11を好ましく用いることができる。
モノクローナル抗体8H4は、本発明者らにより、Ｍ環部にヒドロキシ基を有するシガトキシン類のHIJKLM環部を合成ハプテンとして用いて、Ｍ環部にヒドロキシ基を有するシガトキシン類に特異的に結合し得るモノクローナル抗体として作製された（特開２００６−１９３４８５号公報参照）。また、モノクローナル抗体3D11は、本発明者らにより、Ｍ環部にヒドロキシ基を有しないシガトキシン類のIJKLM環部を合成ハプテンとして用いて、Ｍ環部にヒドロキシ基を有しないシガトキシン類に特異的に結合し得るモノクローナル抗体として作製された（特開２００３−５５４００号公報参照）。 Other monoclonal antibodies include the monoclonal antibody 8H4 produced by the hybridoma 8H4 deposited at the Patent Microbiology Depositary Center of the National Institute of Advanced Industrial Science and Technology on December 22, 2004 as the accession number FERM B P- 11400 , or As the accession number FERM BP-8293, the monoclonal antibody 3D11 produced by the hybridoma 3D11 originally deposited at the Patent Microorganism Depositary, National Institute of Advanced Industrial Science and Technology on March 5, 2002 can be preferably used.
Monoclonal antibody 8H4 is a monoclonal antibody that can specifically bind to ciguatoxins having a hydroxy group in the M ring portion by using the HIJKLM ring portion of a ciguatoxin having a hydroxy group in the M ring portion as a synthetic hapten. It was produced as an antibody (see JP 2006-193485 A). In addition, the monoclonal antibody 3D11 was specifically developed by the present inventors using the IJKLM ring portion of a ciguatoxin having no hydroxy group in the M ring portion as a synthetic hapten, and specifically for ciguatoxins having no hydroxy group in the M ring portion. It was produced as a monoclonal antibody capable of binding (see JP 2003-55400 A).

得られたハイブリドーマ細胞 (Mouse-Mouse hybridoma)をHAT培地[無血清培地144 mLにFCS 40 mL、L-グルタミン(200 mM、GIBCO 社製25030-081) 2 mL、ブライクローン(BioResearch Ireland社製BR-001)10 mL、HAT (GIBCO社製31062-011)4 mLを加えたもの] 60 mLに浮遊させ、100μL/ウェルずつ96ウェルプレート６枚に移した。炭酸ガス培養装置中、約２週間培養し、式(II)の化合物とBSAとのコンジュゲート（すなわち、式(I)におけるタンパク質がBSAの化合物）に結合する抗体を産生するハイブリドーマを、該BSAコンジュゲートを用いてELISA法でスクリーニングした。陽性のウェルを選択し、２回クローニング後に、再度ELISA陽性となったモノクローナル抗体を産生するハイブリドーマを順次培養し、それぞれ約200 mLまで増殖させた。これにより、高い抗体価でモノクローナル抗体を産生するハイブリドーマ3G8を得て、これを、平成22年9月15日に、独立行政法人産業技術総合研究所特許生物寄託センターに、受託番号FERM BP-11401として寄託した(このハイブリドーマを抗体と同じ名称3G8と表示する)。 The resulting hybridoma cells (Mouse-Mouse hybridoma) were added to HAT medium (144 mL of serum-free medium, 40 mL of FCS, L-glutamine (200 mM, GIBCO 25030-081) 2 mL, Blycron (BioResearch Ireland BR -001) 10 mL and HAT (GIBCO 31062-011) plus 4 mL] were suspended in 60 mL and transferred to 6 96-well plates at 100 μL / well. A hybridoma that is cultured in a carbon dioxide culture apparatus for about 2 weeks and produces an antibody that binds to a conjugate of the compound of formula (II) and BSA (ie, the protein of formula (I) is a compound of BSA), The conjugate was screened by ELISA. Positive wells were selected, and after cloning twice, hybridomas producing monoclonal antibodies that became ELISA positive again were sequentially cultured and grown to about 200 mL each. Thus, to obtain a hybridoma 3G8 to produce a monoclonal antibody with a high antibody titer, this, on September 15, 2010, the National Institute of Advanced Industrial Science and Technology, International Patent Organism Depositary, consignment number FERM B P -Deposited as 11401 (this hybridoma is labeled 3G8 with the same name as the antibody).

F. サンドイッチ法によるシガトキシン類検出キット
8H4抗体の酵素標識（サンドイッチ法に用いる8H4-HRPの合成）
受託番号FERM BP-11400として寄託されたハイブリドーマ8H4により産生される抗体8H4（Ｍ環部にOH基を有するシガトキシン類を認識するモノクローナル抗体、特開２００６−１９３４８５号公報を参照）を、HRP-Antibody All-in-One^TM Conjugationキット(solulink社製)を同社説明書に従って用いて、HRP標識した。8H4抗体(100μg)を用いて8H4-HRP溶液約150μLが得られた。この溶液を500μLチューブに入れ、４℃で保存した。 F. Detection kit for ciguatoxins by sandwich method
Enzyme labeling of 8H4 antibody (synthesis of 8H4-HRP used in sandwich method)
Antibody 8H4 produced by hybridoma 8H4 deposited under accession number FERM B P- 11400 (monoclonal antibody recognizing ciguatoxins having an OH group in the M ring, see JP 2006-193485 A), HRP- Antibody All-in-One ^™ Conjugation kit (manufactured by solulink) was used according to the manufacturer's instructions for HRP labeling. About 150 μL of 8H4-HRP solution was obtained using 8H4 antibody (100 μg). This solution was placed in a 500 μL tube and stored at 4 ° C.

Claims

The following formula (I):

(Where n represents an integer)
Which produces a monoclonal antibody specifically recognizing ciguatoxins having a dihydroxybutenyl group in the A ring part, which is obtained by immunizing a mouse with the protein conjugate represented by (2010) as accession number FERM BP-11401. Hybridoma 3G8 deposited on May 15 at the National Institute of Advanced Industrial Science and Technology Patent Biological Deposit Center (postal code 305-8566, 1st East, 1st Street, Tsukuba, Ibaraki, Japan, 1st 6th).

The hybridoma 3G8 according to claim 1, wherein the ciguatoxins having a dihydroxybutenyl group in the A ring portion is ciguatoxin CTX1B or 54-deoxy-CTX1B.

A monoclonal antibody that specifically recognizes ciguatoxins having a dihydroxybutenyl group in the A ring part, produced by the hybridoma 3G8 according to claim 1 or 2.

The monoclonal antibody according to claim 3 and the Patent Organism Depositary, National Institute of Advanced Industrial Science and Technology (Postal Code 305-8566, Tsukuba City, Ibaraki, Japan) on December 22, 2004 as the accession number FERM B P- 11400 National Institute of Advanced industrial Science and technology Patent organism Depositary Center in central 6, 1-1-1) to the nearest entrusted the 2002 as hybridoma 8H4 or accession number FERM BP-8293 (2002 years) March 5 (postal number 305-8566, and a monoclonal antibody produced by the nearest committed the hybridoma 3D11 in Japan Higashi, Tsukuba, Ibaraki central 6, 1-1-1), ciguatoxins detection kit.

The monoclonal antibody according to claim 3 or the accession number FERM B P- 11400 on December 22, 2004, National Institute of Advanced Industrial Science and Technology, Patent Biological Deposit Center (Zip code 305-8566, Tsukuba City East, Ibaraki, Japan) National Institute of Advanced industrial Science and technology in central 6, 1-1-1) to the nearest entrusted monoclonal antibody or accession number produced by hybridoma 8H4 FERM BP-8293 as a 2002 (2002) March 5 days Patent organism Depositary Center (zip code 305-8566, Japan Higashi, Tsukuba, Ibaraki central 6, 1-1-1) monoclonal antibody produced by the nearest committed the hybridoma 3D11 in is according to claim 4 which is labeled Ciguatoxin detection kit.

The kit for detecting ciguatoxins according to claim 4 or 5 , wherein the label is an enzyme label.

The following formula (II):

A compound represented by

The compound according to claim 7 , wherein the compound is bound to a carrier protein and has the following formula (I)

(Where n represents an integer)
A protein conjugate represented by

The protein conjugate according to claim 8 , wherein the carrier protein is bovine serum albumin, keyhole limpet hemocyanin or egg albumin.