JP2008191135A

JP2008191135A - Nephritis detection reagent

Info

Publication number: JP2008191135A
Application number: JP2007056326A
Authority: JP
Inventors: Morisuke Yokoyama; 司甫横山
Original assignee: Individual
Current assignee: Individual
Priority date: 2007-02-06
Filing date: 2007-02-06
Publication date: 2008-08-21

Abstract

<P>PROBLEM TO BE SOLVED: To provide an early stage detection method as to a nephritis detection reagent for a kidney disease. <P>SOLUTION: The present invention is constituted as follows. 1. A method and a reagent for detecting an anti-7s-autoantibody characteristic to a nephritis, using the 7s of a type IV collagen α3 chain, α4 chain, α5 chain and/or α6 chain of a main component in a glomerule basement membrane (GBM) as an antigen, and/or a peptide thereof (7s hereafter) as the antigen. 2. A method and a reagent for detecting the 7s characteristic to the nephritis, using an antibody reacting using the 7s of the type IV collagen α3 chain, α4 chain, α5 chain and/or α6 chain of the main component in the glomerule basement membrane (GBM) as the antigen, and/or a peptide thereof (7s hereafter) as the antigen. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、腎炎検出試薬に関する。The present invention relates to a nephritis detection reagent.

タイプ４コラーゲン関連の腎炎検出には体外診断薬として尿中タイプ４コラーゲン測定試薬がある。同キットは当初、糖尿病性腎炎の早期診断を標榜して使用されていたが、現在は腎炎検出試薬に適用拡大している。
しかし、臨床の場では、より優れた腎炎検出試薬を求めていた。For detecting type 4 collagen-related nephritis, there is a reagent for measuring type 4 collagen in urine as an in-vitro diagnostic agent. The kit was initially used for the early diagnosis of diabetic nephritis, but is now expanding to use as a nephritis detection reagent.
However, in the clinical field, a better nephritis detection reagent has been sought.

Problems to be solved by the invention

本発明者は、長い間、腎疾患の早期検出方法の開発に努めてきた。The inventor has long been trying to develop a method for early detection of kidney disease.

Means for Solving the Invention

その結果、発明者は、次のように本発明を成した。
タイプ４コラーゲンは基底膜コラーゲンとも呼ばれ、生体組織基底膜の主成分である。タイプ４コラーゲンはα１鎖〜α６鎖までの６種の同位体（ｉｓｏｍｅｒ）が知られている。α１鎖、α２鎖は胎盤で富み、全身に分布するが、腎基底膜ではα３鎖〜α６鎖が特徴的に存在する。As a result, the inventor made the present invention as follows.
Type 4 collagen is also called basement membrane collagen and is the main component of biological tissue basement membrane. As for type 4 collagen, six types of isotopes from α1 chain to α6 chain are known. The α1 chain and α2 chain are rich in the placenta and distributed throughout the body, but the α3 chain to α6 chain are characteristically present in the renal basement membrane.

又、基底膜の由来組織をペプシン処理するとタイプ４コラーゲンは、主にＮ端の７Ｓと三本鎖中央領域（ＴＨ）が得られる（通常タイプ４コラーゲンと呼ばれる）。ペプシン処理したタイプ４コラーゲンは電気泳動すると細断片の混在像で各社市販品の泳動像でも濃淡に違いがあり、一致しない。
この点は、標準物質として用いるのに大きな課題があった。Moreover, when the tissue derived from the basement membrane is treated with pepsin, type 4 collagen mainly has N-terminal 7S and a triple-stranded central region (TH) (usually called type 4 collagen). When pepsin-treated type 4 collagen is electrophoresed, it is a mixed image of fine fragments, and the electrophoretic images of the commercial products of each company are different in density and do not match.
This point has a big problem to use as a standard substance.

又、この標準物質を抗原として抗体を作製すると抗体間に認識部位の違いが生じやすく、免疫反応を論じる時に食い違いを生じる懸念があった。In addition, when an antibody is prepared using this standard substance as an antigen, a difference in recognition site tends to occur between antibodies, and there is a concern that a discrepancy may occur when discussing an immune reaction.

更に、腎炎の体外診断薬として知られ唯一販売されている尿中タイプ４コラーゲン測定試薬キットは、標準がヒト胎盤由来タイプ４コラーゲンで、使用モノクロ−ナル抗体作製時の抗原もペプシン処理で得られた胎盤由来タイプ４コラーゲンである。
しかし、腎炎で尿中に出現するタイプ４コラ−ゲン（７ＳやＮＣ１を含めて）は腎臓由来である。胎盤由来でペプシン処理した現行のタイプ４コラ−ゲン測定キットは、腎炎の検出に用いるには充分なもので無かった。
又、症例によっては腎臓由来のタイプ４コラーゲン（７ＳやＮＣ１を含めて）が血流中にも出現するが、肝臓の線維化などのタイプ４コラーゲン（７ＳやＮＣ１を含めて）との識別が必要であった。Furthermore, the urinary type 4 collagen measurement reagent kit known and sold as an in-vitro diagnostic agent for nephritis is based on human placenta-derived type 4 collagen, and the antigen used to produce the monoclonal antibody is also obtained by pepsin treatment. Placenta-derived type 4 collagen.
However, type 4 collagen (including 7S and NC1) that appears in urine due to nephritis is derived from the kidney. The current type 4 collagen measurement kit treated with placenta and treated with pepsin was not sufficient for detecting nephritis.
In some cases, kidney-derived type 4 collagen (including 7S and NC1) also appears in the bloodstream, but it can be distinguished from type 4 collagen (including 7S and NC1) such as liver fibrosis. It was necessary.

それゆえ、腎炎の検出に用いるタイプ４コラーゲン（７ＳやＮＣ１を含めて）測定キットなら、腎臓由来の抗原とその抗原に基ずく抗体を利用するほうが理に適うだろうと思い至った。Therefore, it was thought that it would be more reasonable to use a kidney-derived antigen and an antibody based on the antigen for a type 4 collagen (including 7S and NC1) measurement kit used for nephritis detection.

そこで、出発材料に腎臓組織を用いる事とした。Therefore, it was decided to use kidney tissue as a starting material.

予備試験としての胎盤由来と腎臓由来の比較（１）
ペプシン処理にて取り出した、胎盤由来タイプ４コラーゲンとウシ腎糸球体基底膜（ＧＢＭ）由来タイプ４コラ−ゲンを抗原として腎炎患者尿中の抗タイプ４コラーゲン抗体をＥＬＩＳＡ法で測定したところ、明らかに腎ＧＢＭ由来タイプ４コラーゲンに良く反応した。従って、抗原に腎由来を用いることの妥当性を裏付けた。
［測定手順；マイクロプレート固相化抗原（抗原５ｕｇ／ｍｌＰＢＳ，ｐＨ７．６を１２０ｕｌ／ｗｅｌｌ添加し４Ｃ×一夜放置）−洗浄後、４倍希釈検体１００ｕｌ添加−室温×２時間放置−洗浄後、酵素標識抗ヒト抗体１００ｕｌ添加−室温×１時間放置−洗浄後、基質溶液（ＴＭＢ）１００ｕｌ−青色を目安に反応（室温×５〜３０分）−反応停止液（１Ｎ−Ｈ２ＳＯ４）５０ｕｌ添加し、直ちに４５０ｎｍで吸光度測定］Comparison of placenta-derived and kidney-derived as a preliminary test (1)
When an anti-type 4 collagen antibody in nephritis urine was measured by ELISA using placenta-derived type 4 collagen and bovine kidney glomerular basement membrane (GBM) -derived type 4 collagen as antigens, it was revealed by pepsin treatment. It reacted well with kidney GBM type 4 collagen. Therefore, the validity of using kidney origin as the antigen was confirmed.
[Measurement procedure: Microplate solid-phased antigen (antigen 5 ug / ml PBS, pH 7.6 added 120 ul / well, left 4 C x overnight)-Washed, 4-fold diluted specimen 100 ul added-room temperature x 2 hours-washed Add 100 ul of enzyme-labeled anti-human antibody-Leave at room temperature for 1 hour-Wash, then react with 100 ul of substrate solution (TMB)-Blue (standard temperature-5-30 minutes)-Add 50 ul of reaction stop solution (1N-H2SO4) Immediately measure absorbance at 450 nm]

予備試験としての胎盤由来と腎臓由来の比較（２）
更に、ペプシン処理にて得られたウシ腎ＧＢＭ由来タイプ４コラーゲンでラットとウサギで抗体を作製した（プロテインＧ精製）。この２種の抗体で、サンドイッチＥＬＩＳＡ法でウシ腎ＧＢＭ由来タイプ４コラ−ゲンを測定した。その結果、０．１３ｎｇ／ｍｌまで測定できたが、ヒト胎盤由来タイプ４コラーゲン（Ｋ３４コラ−ゲン技術研修会製）を検体とした時は、吸光度は半分以下でバックも極めて高かった。
この結果は、尿中のタイプ４コラーゲンを測定するには、腎由来のタイプ４コラーゲンを標準とし、腎由来の抗タイプ４コラーゲン抗体を用いる事が良いと判断できた。つまり、腎由来の物質を測定するには腎由来の抗原、標準、腎由来抗原に対応する抗体を用いるのが適切である。
［測定手順；マイクロプレート固相化抗体（ウサギＩｇＧ２０００倍希釈ＰＢＳ．ｐＨ７．６を１２０ｕｌ／ｗｅｌｌ添加し４Ｃ×一夜放置）−洗浄後、段階希釈抗原（タイプ４コラ−ゲン）１００ｕｌ添加−室温×２時間放置−洗浄後、抗体（ラットＩｇＧ１０００倍希釈）１００ｕｌ添加−室温×２時間放置−洗浄後、酵素標識抗ラット抗体１００ｕｌ添加−室温×１時間放置−洗浄後、基質溶液（ＴＭＢ）１００ｕｌ−青色を目安に反応（室温×５〜３０分）−反応停止液（１Ｎ−Ｈ２ＳＯ４）５０ｕｌ添加し、直ちに４５０ｎｍで吸光度測定］Comparison of placenta-derived and kidney-derived as a preliminary test (2)
Furthermore, antibodies were produced in rats and rabbits using bovine kidney GBM-derived type 4 collagen obtained by pepsin treatment (protein G purification). With these two types of antibodies, bovine kidney GBM-derived type 4 collagen was measured by sandwich ELISA. As a result, it was possible to measure up to 0.13 ng / ml, but when human placenta-derived type 4 collagen (manufactured by K34 Collagen Technical Workshop) was used as a sample, the absorbance was less than half and the back was extremely high.
From this result, it was determined that in order to measure type 4 collagen in urine, it is preferable to use type 4 collagen derived from kidney as a standard and anti-type 4 collagen antibody derived from kidney to be used. That is, to measure a kidney-derived substance, it is appropriate to use an antigen corresponding to a kidney-derived antigen, standard, or kidney-derived antigen.
[Measurement procedure: Microplate immobilized antibody (rabbit IgG 2000-fold diluted PBS, pH 7.6, 120 ul / well added, 4 C x left overnight)-After washing, step diluted antigen (type 4 collagen) 100 ul added-room temperature x 2 hours standing-washing, antibody (rat IgG 1000-fold dilution) 100 ul addition-room temperature x 2 hours standing-washing, enzyme-labeled anti-rat antibody 100 ul addition-room temperature x 1 hour standing-washing, substrate solution (TMB) 100 ul- Reaction (room temperature x 5 to 30 minutes) with blue as a guide-Add 50 ul of reaction stop solution (1N-H2SO4) and immediately measure absorbance at 450 nm]

さらに、酵素処理は細菌コラゲナ−ゼを用いる事とした。
ペプシン処理ではタイプ４コラーゲンが細断片となり、一定の分子量のものを得る事は困難である。又、７Ｓを取り出す時にも、最初にペプシン処理を行うと更にコラゲナ−ゼ処理を行う必要があり二度手間を要する。
最初から細菌コラゲナ−ゼで処理すれば１回の手間で済み、カラムクロマトグラフィーで７ＳとＮＣ１が簡単に分取でき、その電気泳動像は一定である。Further, bacterial collagenase was used for the enzyme treatment.
With pepsin treatment, type 4 collagen becomes a fine fragment and it is difficult to obtain a constant molecular weight. Further, when 7S is taken out, if pepsin treatment is performed first, it is necessary to further perform collagenase treatment, which is troublesome twice.
If treated with bacterial collagenase from the beginning, only one time is required, 7S and NC1 can be easily separated by column chromatography, and the electrophoresis image is constant.

そこで、電気泳動像が一定の７ＳやＮＣ１を用いる事とした。
７ＳやＮＣ１は、いわゆるタイプ４コラーゲンを使用するのに比べ、均質の分子量が得られるので、動物に投与して抗体を作製する時も、ロット間でのばらつきが少ないし、抗原でアフィニテ−精製しても力価に差が生じにくい。この事は、７ＳやＮＣ１の検出キット作製に有用である。同時に、７ＳやＮＣ１を抗原として生体試料から自己抗体を検出するにも作成時の抗原による差が生じないと言う利点がある。
ＮＣ１については、抗糸球体基底膜抗体腎炎（狭義のグッドパスチャ−症）などで研究が進んでいるので、ここでは、研究の進んでいない７Ｓについて述べ、基礎研究にも役立つようにする。ＮＣ１についても７Ｓについて述べることと同様である。Therefore, 7S or NC1 whose electrophoresis image is constant is used.
7S and NC1 have a homogeneous molecular weight compared to the use of so-called type 4 collagen, so there is less variation between lots when administering antibodies to animals, and affinity purification with antigen Even so, there is little difference in titer. This is useful for preparing detection kits for 7S and NC1. At the same time, detecting autoantibodies from biological samples using 7S or NC1 as an antigen has the advantage that there is no difference due to the antigen at the time of preparation.
Regarding NC1, since research is progressing in anti-glomerular basement membrane antibody nephritis (narrow sense of Goodpascher's disease) and the like, 7S, which has not been researched, will be described here to be useful for basic research. NC1 is the same as described for 7S.

以上の様にして、腎炎時に出現する腎特異的な７Ｓの検出にふさわしい「ウシ腎糸球体基底膜由来」を出発原料に、簡便な１回の酵素処理「細菌コラゲナ−ゼ」で得た均質な分子量の「７Ｓ」を抗原として、抗体を作製した。
７Ｓ抗体作製に用いる抗原は腎糸球体由来ならいずれの動物種でも良い。抗体作製動物もいずれの動物でも良い。
抗体作製には、ウサギとモルモットの２種を用いた例を示す。一般的にモルモットでの抗体作製は他の動物に比べ例示が少ない。
ウサギに作製する時には、背部皮内に同容量のＦＣＡと共に初回０．１−０．１５ｍｇ、追加免疫は２週後、４週後に０．２−４５ｍｇを投与し、ＥＬＩＳＡで充分抗体価が上っている事を確認して５週後に採血した。続いて、７Ｓでアフィニテ−精製を行い、１ｍｇ／ＰＢＳ１ｍｌ液で冷凍保存した。
モルモットを用いる時は、背部皮内に同容量のＦＣＡと共に初回０．０５−０．１ｍｇ、追加免疫は２週後、４週後に０．１−０．３ｍｇを投与し、以降同様にして精製した抗体をＢｉｏｔｉｎ標識し、検出にはＡｖｉｄｉｎ−ＨＲＰを用いる。腎由来７Ｓ測定ＥＬＩＳＡキットは、次の仕様とした。
固相化抗原（ウサギ由来５ｕｇ／ｍｌ）マイクロプレ−トを１夜放置−洗浄後、ブロックエ−ス２５％−ＰＢＳを加え保存。使用時、ＰＢＳ−０．０５％Ｔｗｅｅｎ２０で洗浄後、検体（標準、４倍希釈尿、１０倍希釈血清）を加え−２時間室温放置後洗浄し、Ｂｉｏｔｉｎ−抗体（モルモット由来）を加え−２時間室温放置後洗浄し、Ａｖｉｄｉｎ−ＨＲＰ（４千倍希釈）を加え−３０分放置後洗浄し、ＴＭＢ発色基質液を加え−室温２０分放置後、反応停止液（１Ｎ硫酸）を加えて、４５０ｎｍで吸光度を測定する。As described above, starting from “bovine kidney glomerular basement membrane” suitable for detection of kidney-specific 7S appearing at the time of nephritis, the homogeneity obtained by simple single enzyme treatment “bacterial collagenase” An antibody was prepared using “7S” having an appropriate molecular weight as an antigen.
Any animal species may be used as the antigen used for preparing the 7S antibody as long as it is derived from the glomeruli. The antibody-producing animal may be any animal.
Examples of using two types of rabbits and guinea pigs for antibody production are shown. In general, antibody production in guinea pigs is less exemplified than other animals.
When preparing rabbits, the initial dose of 0.1-0.15 mg with the same amount of FCA in the dorsal skin is administered, and 0.2-45 mg is given for booster immunization after 2 weeks and 4 weeks, and the antibody titer is sufficiently increased by ELISA. The blood was collected 5 weeks later. Subsequently, it was purified with 7S and stored frozen in 1 mg / PBS 1 ml solution.
When using guinea pigs, the initial dose of 0.05-0.1 mg is given in the back skin together with the same volume of FCA, and booster is administered 0.1-0.3 mg after 2 weeks and 4 weeks. The antibody was labeled with Biotin, and Avidin-HRP was used for detection. The kidney-derived 7S measurement ELISA kit had the following specifications.
The solid-phased antigen (rabbit-derived 5 ug / ml) microplate was allowed to stand overnight-washed, and then stored with Block Ace 25% -PBS. At the time of use, after washing with PBS-0.05% Tween 20, add a specimen (standard, 4-fold diluted urine, 10-fold diluted serum), leave for 2 hours at room temperature, wash, and add Biotin-antibody (derived from guinea pigs) -2. Wash after standing at room temperature, add Avidin-HRP (diluted 4,000 times), wash after standing for -30 minutes, add TMB chromogenic substrate solution-stand for 20 minutes at room temperature, add reaction stop solution (1N sulfuric acid), Measure absorbance at 450 nm.

ヒト７Ｓ由来ペプチドの作製方法
α１鎖からα６鎖まで７Ｓのアミノ酸配列は公表されているので、これを用いる。α３鎖７Ｓ（約１９０個のアミノ酸配列）は、Ｎ端から２０個単位のアミノ酸配列を、１０個を重ねながらＣ端まで１８個に合成する（ｙ３０１−ｙ３１８）。全部の合成ペプチド（１８個）を抗原として、９６穴マイクロプレートに固相化する（５−１０ｕｇ／ｍｌＰＢＳ，ｐＨ７．６）。対象抗原はウシ腎糸球体基底膜由来７Ｓを用いた。検体には、あらかじめウシ腎糸球体基底膜由来７Ｓを抗原として抗７Ｓ抗体がある腎炎患者尿を用いる。ペプチドは、対象抗原より反応したものを選ぶ（以下７Ｓ様ペプチド）。このようにして用意した７Ｓ様ペプチドは、７Ｓの代用抗原として自己抗体の検出に、更に抗体作製の抗原として使用できる。７Ｓ様ペプチド及び又は抗７Ｓ様ペプチド抗体は血清や試料中の抗７Ｓ様ペプチド抗体及び又は７Ｓ様ペプチドの除去装置にも用いられる。７Ｓ様ペプチドの作製は、α４鎖からα６鎖の７Ｓについても同様にして行う。α５鎖ＮＣ１では、Ｎ端から１個から１２個が無い（公表文献の記載が空白）ので、最初の２０個のペプチドは無しにして２個目のペプチドを１８アミノ酸残基で作製する。他のα鎖ＮＣ１の欠損ついても同様に処理する。途中域のアミノ酸欠損残基は少ないので、そのままで合成する。
又、α１鎖、α２鎖は、α３鎖〜α６鎖までと相同性が約５０％程度あるので、α１鎖、α２鎖についてもペプチドを同様に実施しても良いが腎炎検出試薬の作製には十分で無い。Method for producing human 7S-derived peptide Since the amino acid sequence of 7S from α1 chain to α6 chain has been published, this is used. The α3 chain 7S (a sequence of about 190 amino acids) synthesizes 20 amino acid sequences from the N-terminal to 18 up to the C-terminal by overlapping 10 (y301-y318). All synthetic peptides (18) are immobilized on a 96-well microplate as an antigen (5-10 ug / ml PBS, pH 7.6) . As the target antigen, 7S derived from bovine kidney glomerular basement membrane was used. As a specimen, nephritis patient urine having an anti-7S antibody using 7S derived from bovine kidney glomerular basement membrane as an antigen is used in advance. The peptide is selected from the target antigen (hereinafter referred to as 7S-like peptide). The 7S-like peptide thus prepared can be used as a 7S surrogate antigen for detection of autoantibodies and further as an antigen for antibody production. The 7S-like peptide and / or anti-7S-like peptide antibody is also used in an apparatus for removing anti-7S-like peptide antibody and / or 7S-like peptide in serum and samples. The 7S-like peptide is prepared in the same manner for 7S from α4 chain to α6 chain. In the α5 chain NC1, there are no 1 to 12 from the N end (the published literature is blank), so the second peptide is prepared with 18 amino acid residues without the first 20 peptides. The same applies to other α-chain NC1 deletions. Since there are few amino acid-deficient residues in the middle, it is synthesized as it is.
In addition, since α1 chain and α2 chain have about 50% homology with α3 chain to α6 chain, peptides may be similarly applied to α1 chain and α2 chain. Not enough.

α３鎖からα６鎖までのそれぞれの抗７Ｓ様ペプチド抗体を用いたアフィニテ−カラムを用意して、腎糸球体基底膜由来７Ｓ（例えばウシ）を通すことで、各α鎖の７Ｓ（α３鎖〜α６鎖の７Ｓ）が得られる。これら各α鎖の７Ｓを用いる事で、又その抗体を用いる事で、抗７Ｓ抗体や７Ｓが測定できる。By preparing an affinity column using each anti-7S-like peptide antibody from α3 chain to α6 chain and passing 7S derived from glomerular basement membrane (for example, bovine), 7S (α3 chain˜ α6 chain 7S) is obtained. By using 7S of each α chain and using the antibody, anti-7S antibody and 7S can be measured.

７Ｓ投与又は抗７Ｓ抗体投与による腎炎の寛容化
動物（ウサギやラットなど）にあらかじめ一定期間微量の７Ｓ（以下、含む７Ｓ様ペプチド）又は抗７Ｓ抗体を経口投与もしくは注射投与（皮下、皮内、筋肉、静注他）することで、後日の免疫惹起が緩和もしくは抑制される。7S administration or tolerization of nephritis by administration of anti-7S antibody (rabbit, rat, etc.) A small amount of 7S (hereinafter, including 7S-like peptide) or anti-7S antibody is orally administered or injected (subcutaneous, intradermal, (Intramuscular, intravenous injection, etc.) alleviates or suppresses subsequent immune induction.

The invention's effect

本発明は、腎炎の早期発見、治療薬の開発、医薬品の腎臓への副作用評価指標、ジン疾患治療薬のスクリ−ニング方法、腎炎の透析装置、腎炎モデルの作製として、更には各種研究に於ても利用できるがこれに限定されない。The present invention relates to the early detection of nephritis, the development of therapeutic drugs, the evaluation of side effects of drugs on the kidney, the screening method for therapeutic drugs for gin diseases, the preparation of nephritis dialysis devices, the nephritis model, and in various studies. However, it is not limited to this.

〔添付１〕ヒト７Ｓのアミノ酸配列例[Attachment 1] Example of amino acid sequence of human 7S 〔添付２〕ウシ腎糸球体基底膜を細菌コラゲナ−ゼ処理で得た７ＳとＮＣ１[Attachment 2] 7S and NC1 obtained by treatment of bovine kidney glomerular basement membrane with bacterial collagenase

Claims

Anti-renal-derived type IV collagen 7S region (7S) antibody and / or 7S (hereinafter also including 7S-derived peptide) measurement reagent using anti-renal-derived 7S-derived peptide antibody

Renal-derived 7S antibody measurement nephritis detection reagent using kidney-derived 7S as an antigen

Renal glomerular basement membrane type IV collagen α7 chain and / or α4 chain and / or α5 chain and / or α6 chain 7S as antigen and / or its peptide (hereinafter referred to as 7S) antigen as a method for measuring urinary anti-7S antibody

Renal glomerular basement membrane type IV collagen αS chain and / or α4 chain and / or α5 chain and / or α6 chain 7S as an antigen and / or 7S measurement in urine using an antibody that reacts with its peptide (hereinafter 7S) as an antigen Law

Kidney-derived 7S drug

Anti-renal 7S antibody drug

Anti-7S antibody and / or 7S removal apparatus using renal-derived 7S and / or anti-renal-derived 7S antibody

Nephritis model elicited by renal-derived 7S and / or anti-renal-derived 7S antibody