(Microsoft Word _ApoE_ELISA_Kir \211p,\223\372_080808_.doc)
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- まいえ ひらみね
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1 Printed: October 28, 2001 Revised: February 18, 2002 Revised: September 12, 2002 Revised: August 8, 2008 For Research Use Only. Not for use in diagnostic procedures. Quantitative test kit for ApoE4 or Pan-ApoE ApoE4/Pan-ApoE ELISA Kit CODE No wells MEDICAL & BIOLOGICAL LABORATORIES CO., LTD Marunouchi 3 chome, Naka-ku Nagoya Japan TEL; (052) , FAX; (052) , URL
2 CONTENTS 1. Intended Use Summary and Explanation Principle Materials Provided Storage and Stability Specimen Species Cross Reactivity Materials Required but not provided Analytical Precautions Health and Safety Information Method Performance Characteristics Related products References
3 Before use, thoroughly read these Instructions. Intended Use MBL ApoE4/Pan-ApoE ELISA Kit is based on sandwich ELISA and capable of measuring ApoE4 or Pan-ApoE. For Research Use Only. Not for use in diagnostic procedures. Summary and Explanation Apolipoprotein E (ApoE), a 35-kDa plasma protein containing sialic acid, constitutes a part of major apoproteins such as chylomicrons, VLDL and HDL. ApoE plays a role in the transport and metabolism of triglyceride-cholesterol, and is known to be synthesized in the liver, brain and other organs. ApoE is a polymorphic apolipoprotein exhibiting three major isoforms Apo E2, E3 and E4 coded by three alleles of ε2, ε3 and ε4 at a single gene locus respectively. A combination of these isoforms makes six different phenotypes (3 homozygotes-e2/e2, E3/E3, E4/E4 and 3 heterozygotes-e2/e3, E3/E4, E2/E4). ApoE2 is known as an important marker to diagnose type III hyperlipoproteinemia and is reported to be related to cardiac disease. ApoE4 is now in the spotlight as one of the risk factors for Alzheimer's disease. MBL ApoE4/Pan-ApoE ELISA Kit measures the amount of human ApoE4 or Pan-ApoE specifically with high sensitivity using affinity purified polyclonal antibody against ApoE and monoclonal antibody against ApoE4. It can also measure the difference among the homozygotes (E4/E4) and the heterozygotes (E2/E4, E3/E4) of ApoE4 phenotypes, and non-apoe4 zygotes (E2/E2, E3/E3, E2/E3) by taking a concentration ratio between ApoE4 and Pan-ApoE. Principle MBL ApoE4/Pan-ApoE ELISA Kit measures Human ApoE4 or Pan-ApoE by sandwich ELISA. The assay uses affinity purified polyclonal antibody against ApoE and monoclonal antibody against ApoE4. In the wells coated with anti-apoe polyclonal antibody, samples to be measured or standards are incubated. After washing, a peroxidase conjugated anti-apoe4 monoclonal antibody or a peroxidase conjugated anti-apoe polyclonal antibody is added in the microwell and incubated. After another washing, the peroxidase substrate is added and allowed to incubate for an additional period of time. An acid solution is then added to each well to terminate the enzyme reaction and to stabilize the developed color. The optical density (O.D.) of each well is then measured at 450 nm using a microplate reader. The concentration of ApoE4 or Pan-ApoE is calibrated from a standard curve based on reference standards
4 Materials Provided Microwells Calibrator Name Materials Quantity Conjugate reagent (for ApoE4) Conjugate reagent (for Pan-ApoE) Breakaway microwell strips coated with anti-human Pan-ApoE antibody Human ApoE4/Pan-ApoE Calibrator (Lyophilized) Peroxidase conjugated anti-human ApoE4 monoclonal antibody (x 101) Peroxidase conjugated goat anti-human ApoE (x 101) 8-wells 12 strips 0.1 ml 2 vials 0.15 ml 1 vial 0.15 ml 1 vial Conjugate diluent 1 Buffer for diluting Conjugate diluent 2 20 ml 1 vial Conjugate diluent 2 Assay diluent Wash concentrate Buffer for diluting samples (5 x concentrated solution) Buffer for washing microwells (10 x concentrated solution) 1 ml 1 vial 50 ml 1 vial 100 ml 1 vial Substrate TMB/H 2 O 2 (ready for use) 20 ml 1 vial Stop solution 0.5 M H 2 SO 4 (ready for use) 20 ml 1 vial Instruction manual 1 Storage and Stability All kit components must be stored at 2-8 C. All reagents are stable for 12 months after manufacturing when stored at 2-8 C. Do not use the kit beyond the expiration date. When Microwell strips are not immediately required, it should be returned to the ziplock pouch. The pouch must be carefully resealed to avoid moisture absorption and stored at 2-8 C. Do not freeze Conjugated reagent. Specimen Use fresh serum, EDTA plasma and cerebrospinal fluid. Do not use heparin plasma. No preservative should be added. Avoid using hemolyzed, lipemic, or bacterially contaminated sera. If storage is needed, they should be aliquoted and frozen below -70 C. The specimens should be used within one week. Do not repeat freezing and thawing
5 Species Cross Reactivity Species Human Mouse Rat Samples plasma, serum, cerebrospinal fluid Not Tested Not Tested Reactivity + Materials Required but not Provided Microplate reader (wavelength: 450 nm, 620 nm/reference) Multichannel micropipette (e.g., 100 µl-300 µl) Single channel pipette (10 µl & 100 µl ) Reagent vessel Autowasher or wash bottle Deionized or distilled water One liter graduated cylinder for preparation of wash solution Test tubes for patient sample dilution (e.g., 1,000 µl) Disposable pipette tips Paper towels Basin and disinfectant Microplate cover Analytical Precautions 1. This kit is intended for research use only. Not for use in diagnostic procedure. 2. Identical lot number of microwell strips, Conjugated reagents must be used. 3. Do not expose the kit to direct sun during assay and storage. 4. Contamination of the TMB substrate solution with conjugate or other oxidants will cause the solution to change color prematurely. 5. Allow all the components to come to room temperature (20-25 C) before use. 6. All microwell strips which are not immediately required should be returned to the ziplock pouch, which must be carefully resealed to avoid moisture absorption. 7. Fresh samples should be used. 8. When Wash concentrate is stored at 2-8 C, some precipitation or turbidity may appear. However it does not affect the reagent efficiency. Dissolve the wash concentrate completely when prepare the wash solution. 9. Stop solution is 0.5 M sulfuric acid. As it is corrosive product, protect eyes and skin and handle with care
6 Health and Safety Information 1. In accordance with the principles of Good Laboratory Practice it is strongly recommended that all clinical specimens and materials should be handled as potentially infectious and used with all necessary precautions. 2. Liquid waste containing acid must be neutralized with base, e.g. sodium bicarbonate, before decontamination with sodium hypochlorite. Acid-containing liquid waste that has been neutralized and other liquid waste should be decontaminated by adding a sufficient volume of sodium hypochlorite to obtain a final concentration of at least 1.0%. A 30-minute exposure to 1.0% sodium hypochlorite is necessary to ensure effective decontamination. 3. Do not pipette by mouth. Wear disposable gloves and eye protection while handling specimens and performing the assay. Wash hands thoroughly when finished. 4. Calibrator is derived from human serum, in which HBs antigen, HCV antibody and HIV antibody cannot be detected. No test method, however, can guarantee the absence of these or any other infectious agents. These reagents and all patient samples should be handle at the Biosafety Level 2 as recommended for any potentially infectious human serum or blood specimen in the Center for Disease Control/National Institute of Health manual Biosafety in Microbiology and Biomedical Laboratories, 1993 and FDA LABELING GUIDELINES FOR IN VITRO DIAGNOSTIC REAGENT MANUFACTURERS, DEC Avoid contact of reagents with eyes, skin and clothing. If contact occurs, immediately flush with water. 6. Assay diluent contains sodium azide (0.09%) as a preservative. Azide may react with copper or lead in plumbing system to form explosive metal azides. Therefore, always flush with plenty of water when disposing materials containing azide into a drain. 7. Implement used for the test should be disposed after treated in the ways shown below. Soak in 2% final conc. glutaraldehyde solution for more than 1 hour. or Soak in 0.5% Sodium hypochlorite solution (available chlorine: approximately 5,000 ppm) for more than 1 hour. or Autoclave at 121 C for more than 20 minutes
7 Method Preparation of Reagents Bring all assay materials to room temperature (20-25 C) for 1 hour before prior to use. 1. Wash solution Prepare Wash solution by diluting Wash Concentrate, 1:10 with distilled water prior to use. (e.g., add 100 ml of Wash Concentrate to 900 ml of distilled water) The Wash solution is stable for 2 weeks at 2-8 C. * Wash concentrate may get turbid at 2-8 C, which does not cause inconsistent result. Prior to preparation of Wash Solution, dissolve thoroughly. 2. Assay diluent solution Prepare Assay diluent solution by diluting the Assay diluent, 1:5 with distilled water prior to use. (e.g., add 50 ml of Assay diluent to 200 ml of distilled water) 3. Conjugate diluent solution Prepare Conjugate diluent solution by diluting the Conjugate diluent 2, 1:20 with the Conjugate diluent 1 prior to use. (e.g., add 0.5 ml of Conjugate diluent 2 to 9.5 ml of Conjugate diluent 1) * Prepare only a sufficient amount of Conjugate diluent solution for the assay because it is not stable. 4. ApoE4 Conjugate Solution Prepare ApoE4 Conjugate solution by diluting Conjugate reagent (for ApoE4), 1:101 with the Conjugate diluent solution prior to use. (e.g., add 10 µl of the Conjugate reagent (for ApoE4) to 1,000 µl of the Conjugate diluent solution) The ApoE4 Conjugate Solution is stable for 2 weeks at 2-8 C. 5. Pan-ApoE Conjugate Solution Prepare Pan-ApoE Conjugate solution by diluting Conjugate reagent (for Pan-ApoE), 1:101 with the Conjugate diluent solution prior to use. (e.g., add 10 µl of the Conjugate reagent (for Pan-ApoE) to 1,000 µl of the Conjugate diluent solution) The Pan-ApoE Conjugate Solution is stable for 2 weeks at 2-8 C. 6. Standards Reconstitute Standards by dissolving lyophilized Calibrator with distilled water. The volume of distilled water and precise procedures are indicated in the attached document Preparation of Standards * If storage is needed after reconstitution, prepare appropriate aliquots and freeze them below -20 C. Avoid repeated freezing and thawing
8 7. Other reagents (Microwells, Substrate and Stop Solution) are ready for use. Preparation of samples 1. Sample type a) Serum and EDTA Plasma Prepare 1:500~1:600 dilution of serum or plasma sample. Each investigator should establish appropriate methods of sample dilution. Example: Blood was collected in Venoject -II Tube AUTOSEP (TERUMO, Japan; cat# VP-AS109K) or Venoject -II Tube (TERUMO; cat# VP-DK050K). Thereafter, according to the manufacture s instructions, collect the serum or plasma. (e.g. 1:600 dilution First dilution: Add 10 µl of each sample to 190 µl of the Assay diluent solution. Second dilution: Add the 20 µl first diluted sample to 580 µl of the Assay diluent solution.) b) Cerebrospinal fluid Prepare 1:50~1:100 dilution of cerebrospinal fluid sample. Each investigator should establish appropriate methods of sample dilution. (e.g. 1:100 dilution Add 5 µl of each sample to 495 µl of the Assay diluent solution.) 2. Storage Diluted samples must be used within 6 hours. Set-up of the Assay Microwell Strips: After the strips have warmed to room temperature, cut open the pouch and remove required microwell strips and place them in the frame. Strips that are not needed for the assay should be place into the pouch, sealed and returned to storage at 2-8 C
9 Assay procedure STEP 1. (Sample incubation) Duplicate assay is recommended. 1) Pipette 150 µl of standards and each diluted sample to a disposable 96-well polyvinyl plate, then transfer 100 µl each with multichannel pipette into the appropriate Microwells coated with antibody. * Because the reaction starts on the transfer to the Microwells, pipetting should be completed as quickly as possible. 2) Cover the plate and incubate for 60 minutes at C. STEP 2. (Washing) Manual wash: Aspirate or discard the well contents. Fill the well with Wash solution and then aspirate or discard the contents. Repeat this step another 3 times. Tap the plate on a paper towel to remove any remaining Wash solution. Autowasher: Wash 4 times with Wash solution. Tap the plate on a paper towel to remove any remaining Wash solution. * Each laboratory is recommended to confirm its own appropriate washing times and set-up. * Wash solution should be used at C. STEP 3. (Conjugate incubation) 1) Pipette 100 µl of the Conjugate solution to each well with multichannel pipette. 2) Cover the plate and incubate for 60 minutes at C. STEP 4. (Washing) Wash the wells following the STEP 2. procedure. STEP 5. (Substrate incubation) 1) Pipette 100 µl of the Substrate to each well with multichannel pipette. * Because the enzyme reaction starts on the transfer to the Microwells, pipetting should be completed as quickly as possible. * Use disposable new pipette and vessel, as Substrate is easily oxidized by metal ions and can be contaminated by microbes. * The Substrate once poured in vessel should not be returned to the bottle. 2) Cover the plate and incubate for 30 minutes at C. STEP 6. (Stopping reaction) Pipette 100 µl of the Stop solution to each well with multichannel pipette
10 Reading Read the absorbance of each well at 450 nm using a plate reader. If a dual wavelength plate reader is available, set the test wavelength at 450 nm and the reference at 620 nm. * Reading should be done as quickly as possible after adding the Stop solution. * Ensure that the back of the plate is clean and dry, and that no air bubbles are present on the surface of the liquid in the wells before reading. Calculation of results 1) Calculate the mean absorbance value of each standard. 2) Plot the semi-log graph paper and construct a standard curve. [Absorbance on the vertical axis, concentration (ng/ml) on the horizontal axis.] 3) Read the ng/ml corresponding to the absorbance of sample from the calibration curve. Report the concentration of samples by multiplying the dilution factor. * If absorbance of sample exceeds the one of the Top standard, dilute the sample and measure again. Concentration of Pan-ApoE and ApoE4 in normal plasma Plasma samples from 135 healthy donors were assayed with the ApoE4/Pan-ApoE ELISA Kit. The results were as follows. (Calculated as described in Calculation of results) n Total 135 genotype ε2/ε4, ε3/ε4 19 genotype ε4/ε4 3 genotype ε2/ε2, ε2/ε3, ε3/ε3 113 ApoE4 (µg/ml) Pan-ApoE (µg/ml) Average SD ApoE4/Pan-ApoE ratio Average SD Average SD Average SD
11 Performance Characteristics Pan-ApoE 1. Sensitivity The sensitivity of the assay is 4 ng/ml. The minimum detection limit estimated by serial dilution was 4 ng/ml since the mean +2 SD of the 0 ng/ml was lower than the mean -2 SD of the 4 ng/ml. 2. Reproducibility Intra-assay reproducibility was determined by assaying the plasma 5 times. Pan-ApoE concentrations of the plasma samples were calculated as described in Calculation of results in assay procedure. Sample plasma 1 plasma 2 plasma 3 Number of determinations Mean (ng/ml) C.V. (%) Recovery test ApoE4 (genotype ε4/ε4) was added to sample (genotype ε3/ε3) at different concentration. (A) additional ApoE4 (ng/ml) Pan-ApoE concentration observed (ng/ml) (B) Recovery (ng/ml) (B/A) Recovery (%) ApoE4 1. Sensitivity The sensitivity of the assay is 8 ng/ml. The minimum detection limit estimated by serial dilution was 8 ng/ml since the mean +2 SD of the 4 ng/ml was lower than the mean -2 SD of the 8 ng/ml. 2. Reproducibility Intra-assay reproducibility was determined by assaying the plasma 5 times. ApoE4 concentrations of the plasma samples were calculated as described in Calculation of results in assay procedure
12 Sample plasma 1 plasma 2 plasma 3 Number of determinations Mean (ng/ml) C.V. (%) Recovery test ApoE4 (genotype ε4/ε4) was added to sample (genotype ε3/ε3) at different concentration. (A) additional ApoE4 (ng/ml) ApoE4 concentration observed (ng/ml) (B) Recovery (ng/ml) (B/A) Recovery (%) Identification of ApoE4 phenotype The ApoE4/Pan-ApoE ELISA Kit can measure a difference between homozygote (E4/E4), heterozygotes (E2/E4, E3/E4) that are members of ApoE4 phenotype and non-apoe4 zygotes (E2/E2, E3/E3, E2/E3), by taking a concentration ratio between ApoE and Pan-ApoE. ApoE/Pan-ApoE ratio
13 Related products M067-3 anti-apolipoprotein E4 (1F9) M068-3 anti-apolipoprotein E (3D12) M046-3 anti-amyloid β-protein, N-terminal specific (2C8) M066-3 anti-amyloid β/amyloid Precursor Protein (4E12) M009-3 anti-amyloid Precursor Protein (3E9) References 1) Mihovilovic, M., et al., J. Lipid Res. 48, (2007) 2) Mann, K. M., et al., Hum. Mol. Gen. 13, (2004) 3) Yamauchi, K., et al., Clin. Chem. 45, (1999) 4) Corder, E. H., et al., Science 261, (1993) 5) Saunders, A. M., et al., Neurology 43, (1993) 6) Davignon, J., et al., Arterioscler. Thromb. Vasc. Biol. 8,1-21 (1988) 7) Breslow, J. L., et al., J. Lipid Res. 23, (1982) Manufacture MEDICAL & BIOLOGICAL LABORATORIES CO., LTD Marunouchi 3 chome, Naka-ku Nagoya Japan Tel; (052) , Fax; (052) , URL
14 はじめに シアル酸を含有する 35 kda 血漿蛋白質である Apolipoprotein E(ApoE) は カイロシクロン VLDL HDL の主要なアポ蛋白成分の一部であり LDL レセプターや ApoE レセプターのリガンドとして トリグリセリド コレステロールの輸送ならびに代謝に重要な役割をはたしており 肝臓 脳 マクロファージをはじめ全身の臓器で生合成されます ApoE は 主に 3 つの遺伝子型 genotype ε2,ε3 および ε4 を主な対立遺伝子アレルとして持ち 発現されるアイソフォーム蛋白質 ApoE2 E3 および E4 の組み合わせにより 6 種類の表現 (phenotype) ( ホモ接合体 3 種 E2/E2, E3/E3, E4/E4 とヘテロ接合体 3 種 E2/E3, E3/E4, E2/E4) があります ApoE2 のホモ接合体は III 型高脂血症診断の重要な指標として知られ ApoE phenotype と冠動脈疾患との関連も報告されています さらに ApoE4 は アルツハイマー病の危険因子として注目されています ApoE4/Pan-ApoE ELISA Kit は 2 種類の標識抗体 ( ペルオキシダーゼ標識抗ヒト ApoE4 モノクローナル抗体及びペルオキシダーゼ標識抗ヒト ApoE ポリクローナル抗体 ) を用いることにより Apolipoprotein E(Pan-ApoE) あるいは Apolipoprotein E4(ApoE4) を特異的かつ高感度に測定することができます さらに ApoE4 と Pan-ApoE の比を求めることにより ApoE4 phenotype のホモ接合体 (E4/E4) ヘテロ接合体 (E2/E4, E3/E4) 及び非 ApoE4 接合体 (E2/E2, E2/E3, E3/E3) を区別できます 測定原理 ApoE4/Pan-ApoE ELISA Kit は サンドイッチ ELISA 法によりヒト ApoE4 及び全 ApoE 量を測定する試薬です 本試薬はアフィニティー精製した ApoE に対するポリクローナル抗体及び ApoE4 に対するモノクローナル抗体を用いています 抗ヒト ApoE ポリクローナル抗体を感作したマイクロカップにサンプルを添加し 抗原 抗体反応させます 洗浄後 ペルオキシダーゼ標識抗ヒト ApoE4 モノクローナル抗体あるいはペルオキシダーゼ標識抗ヒト ApoE ポリクローナル抗体を添加して反応させ 抗体 - 抗原 - 標識抗体の複合物を形成させます 洗浄後 TMB( テトラメチルベンチジン ) と過酸化水素の溶解液を基質溶液として添加し 酵素 ( ペルオキシダーゼ ) により発色させ 反応停止後 吸光度 (A450) を測定して ApoE4 あるいは全 ApoE を定量します
15 キット構成 Name Materials Quantity Breakaway microwell strips coated with Microwells anti-human Pan-ApoE antibody Human ApoE4/Pan-ApoE Calibrator Calibrator (Lyophilized) Peroxidase conjugated anti-human ApoE4 Conjugate reagent (for ApoE4) monoclonal antibody (x 101) Peroxidase conjugated goat anti-human Conjugate reagent (for Pan-ApoE) ApoE (x 101) 8-wells 12 strips 0.1 ml 2 vials 0.15 ml 1 vial 0.15 ml 1 vial Conjugate diluent 1 Buffer for diluting Conjugate diluent 2 20 ml 1 vial Conjugate diluent 2 Assay diluent Wash concentrate Buffer for diluting samples (5 x concentrated solution) Buffer for washing microwells (10 x concentrated solution) 1 ml 1 vial 50 ml 1 vial 100 ml 1 vial Substrate TMB/H 2 O 2 (ready for use) 20 ml 1 vial Stop solution 0.5 M H 2 SO 4 (ready for use) 20 ml 1 vial Instruction manual 1 保存と有効 2-8 で保存してください 有効期間は製造後 12 ヶ月です Conjugate reagent は冷凍しないでください 準備するもの マイクロプレートリーダー ( 波長 : 450 nm, 620 nm/ 副波長 ) マルチチャンネルマイクロピペット ( 例 100 µl-300 µl) シングルマイクロピペット (10 µl & 100 µl ) リザーバー プレートウォッシャーまたは洗浄ビン 精製水 Wash solution 準備用の 1 L シリンダー 検体希釈用チューブ ( 例 1,000 µl) ディスポーザブルのチップ ペーパータオル 1 次反応準備用マイクロプレート マイクロプレートカバー - 13-
16 交差性 種 ヒト マウス ラット 検体 血漿 (EDTA), 血清, 脳脊髄液 未検討 未検討 交差性 + 使用上またはまたは取り扱い上の注意 1. 有効期限切れの試薬は使用しないでください 2. 本キットの構成品は室温 (20-25 ) に戻してから使用してください 3. 保存及び使用中は 試薬に直射日光が当たらないようにご注意ください 4. 検体や試薬 試薬間のコンタミネーションにご注意ください 5. 反応温度 反応時間は厳守してください 6. マイクロカップ間のコンタミネーションを防ぐため 液の泡立ちや 周囲への付着が起こらないようにしてください 7. Substrate は金属イオンにより酸化されやすいのでディスポーザブルの新しい器具を使用し 試薬ボトルからリザーバーへ移す際にもディスポーザブルのピペットを使用してください また 一度リザーバーへ移した Substrate は試薬ボトルへ戻さないでください 8. 試薬類は皮膚などに付けないよう注意して取り扱ってください もし 皮膚などにかかった時は大量の水で洗い流してください 9. Calibrator は 体外診断用医薬品を用いて HBs 抗原 HCV 抗体および HIV 抗体が陰性であることを確認していますが これらのウイルス及び他のウイルスを完全に否定できる試験方法ではないことから 検体同様 感染の可能性があるものとして十分注意して取り扱ってください 10. Assay diluent には 0.09% アジ化ナトリウムを添加しています 誤って目や口に入ったり 皮膚に付着した場合には水で十分に洗い流す等の応急措置を行い 必要があれば医師の手当てを受けてください また アジ化ナトリウムは 配管中で爆発性のアジ化銅やアジ化鉛を形成する事が報告されています これらの物質の形成を防ぐため アジ化ナトリウムを含んだ廃液は十分量の水で洗い流してください 11. 抗体感作マイクロカップは湿気を嫌いますので 開封後はアルミ袋のチャックを確実に閉めて保存してください 12. Wash concentrate は 2-8 で保存した時 白濁を認める場合がありますが試薬性能に影響はありません Wash solution 調製時 十分に溶解してから使用してください 13. 検体は感染の可能性があるものとして注意して取り扱ってください 測定に使用した器具は 次のいずれかの方法で処理した後 廃棄してください 最終濃度 2% のグルタルアルデヒド溶液に 1 時間以上浸漬する 0.5% 次亜塩素酸ナトリウム溶液 ( 有効塩素約 5,000 ppm) に 1 時間以上浸漬する 121 で 20 分間以上高圧蒸気滅菌する 14. 本キットの構成品のうち Stop solution には 0.5 M 硫酸を用いています 使用の際には十分注意して取り扱ってください 15. 本製品は研究用試薬です ヒトの体内に用いたり 診断の目的に使用しないでください
17 操作法 試薬の調製 * すべての構成試薬は使用前に必ず室温 (20-25 ) に戻してから使用して下さい 洗浄液 ] 使用前に Wash concentrate を精製水で 10 倍希釈して下さい ( 例 ;900 ml の精製水に 100 ml の Wash concentrate を加えて希釈し Wash Solution とします )Wash Solution は 2-8 o C で保存すると およそ 2 週間安定です 1. Wash Solution [ 洗浄液 * Wash concentrate は 2-8 に保存したとき 濁りを生じる場合がありますが 測定には影響を 与えません 調製前に完全に溶解してください 検体希釈液 ] 2. Assay diluent solution [ 検体希釈液 Assay diluent を精製水で 5 倍希釈して下さい ( 例 ; 200 ml の精製水に 50 ml の Assay diluent を加えて希釈し Assay diluent solution とします ) 標識抗体希釈液 ] 3. Conjugate diluent solution [ 標識抗体希釈液 Conjugate diluent 2 を Conjugate diluent 1 で 20 倍希釈して下さい ( 例 ; 9.5 ml の Conjugate diluent 1 に 0.5 ml の Conjugate diluent 2 を加えて希釈し Conjugate diluent solution とします ) Conjugate diluent solution は不安定ですので 必要量のみを調製するようにして下さい 4. ApoE4 Conjugate solution Conjugate reagent (for ApoE4) を Conjugate diluent solution で 101 倍希釈して下さい ( 例 ; 1,000 µl の Conjugate diluent solution に 10 µl の Conjugate reagent (for ApoE4) を加えて希釈し ApoE4 Conjugate solution とします ) ApoE4 Conjugate solution は 2-8 で保存すると およそ 2 週間安定です 5. Pan-ApoE Conjugate solution Conjugate reagent (for Pan-ApoE) を Conjugate diluent solution で 101 倍希釈して下さい ( 例 ;1,000 µl の Conjugate diluent solution に 10 µl の Conjugate reagent (for Pan-ApoE) を加えて希釈し Pan-ApoE Conjugate solution とします Pan-ApoE Conjugate solution は 2-8 で保存すると およそ 2 週間安定です 6. Standards( 調製指示書参照 ) Calibrator を精製水で溶解して Standards を作製します Standards の調製方法はキット添付の文書 Standards の調製方法 に従ってください * 正確な測定を実施するため Standards は必要量のみを測定ごとに調製するようにしてくださ い 7. Substrate 及び Stop solution は調製済ですので そのままご使用ください
18 検体の調製 * 検体は新鮮な検体 ( 採取後 1 週間以内 ) を使用して下さい 希釈した検体は 6 時間以内に使用して下さい 1. 検体の種類 a) 血清および EDTA 血漿血清又は EDTA 血漿を Assay diluent solution で 500~600 倍に希釈します 最適希釈倍数は各施設にて設定してください 例 ; 血液を Venoject -II Tube AUTOSEP(TERUMO, Japan; cat# VP-AS109K) あるいは Venoject -II Tube(TERUMO; cat# VP-DK050K) に採取します その後 メーカーの指示に従って血清あるいは血漿を回収します ) 例 ;600 倍希釈の場合希釈 ( 第 1 段階 ): 190 µlの Assay diluent solution に 10 µlの検体を加えて 20 倍希釈します 希釈 ( 第 2 段階 ): 580 µl の Assay diluent solution に 20 倍希釈した検体を 20 µl 加えて 600 倍希釈にします b) 脳脊髄液脳脊髄液を Assay diluent solution で 50~100 倍希釈します 最適希釈倍数は各施設にて設定してください 例 ;100 倍希釈の場合 495 µl の Assay diluent solution に 5 µl の検体を加えて 100 倍希釈にします 検体の保存新鮮な検体をご使用になる事をお勧めしますが やむを得ず保存しなければならない場合は -70 以下で保存して 1 週間以内にお使いください また 繰り返し凍結融解することは避けてください なお 希釈した検体は 6 時間以内にお使いください 2. 検体 測定準備 マイクロプレートをキットから取り出して室温に戻した後 アルミ袋を開けて必要量のストリップを取り出し フレームに装填します 使用しないストリップはアルミ袋に入れて封をし 2~8 に保存します
19 測定手順 STEP 1. (1 次反応 ) 測定は二重測定でおこなう事をお勧めします 1) Standards 及び希釈した検体 150 µl を 1 次反応準備用マイクロプレートの各ウェルに添加し そこからマルチチャンネルピペットを用いて検体 100 µl を抗ヒト Pan-ApoE 抗体感作マイクロプレートに添加します * 反応は抗ヒト Pan-ApoE 抗体感作マイクロプレート ( 以下マイクロプレートと略します ) に 添加した時点から始まりますので 検体の添加はできるだけ短時間でおこなってください 2) で 60 分間反応させます STEP 2. ( 洗浄 ) 各ウェルの反応液を捨て Wash solution で各ウェルを満たしたあと Wash solution を除去します 4 回洗浄の後 プレートをペーパータオルなどにパンパンとたたきつけて残った Wash solution を完全に除去してください 自動洗浄機を用いる場合は 4 回の洗浄をおこないます * 最適洗浄回数及び洗浄条件については各施設で設定いただくようお勧めします * Wash solution は で使用してください STEP 3. (2 次反応 ) 1) 容器に Conjugate solution を注ぎ マルチチャンネルピペットを用いてマイクロプレートの各ウェルに Conjugate solution を 100 µl ずつ添加します 2) で 60 分間反応させます STEP 4. ( 洗浄 ) STEP 2. と同様に洗浄します STEP 5. ( 発色反応 ) 1) 容器に Substrate を注ぎ マルチチャンネルピペットを用いてマイクロプレートの各ウェルに Substrate を 100 µl ずつ添加します * Conjugate solution の添加に用いた容器とは必ず別の容器を用いてください * Substrate は金属イオンによって酸化されやすいので 新しい使い捨て容器を用いてくださ い * 微生物や金属イオンのコンタミネーションを避けるため Substrate は使い捨てのピペット を用いて容器に移してください * 1 度容器に注いだ Substrate は 決してボトルに戻さないでください 2) で 30 分間反応させます
20 STEP 6. ( 反応停止 ) Stop solution を容器に注ぎ マルチチャンネルピペットを用いて Stop solution を 100 µl ずつ各ウェルに添加し 反応を停止します 測定 波長 450 nm における 各ウェルの吸光度を測定します 2 波長分光光度計を用いる場合は 主波長を 450 nm 副波長を 620 nm に設定します * 測定は 反応停止後できるだけ速やかにおこなってください 測定前にマイクロプレートの底 に汚れのないこと 水分の付着がないこと ウェルの液表面に気泡がないことを確認してくだ さい 濃度の算出 1) 各 standard の平均吸光度を算出します 2) 片対数グラフにプロットして検量線を作成します [ 縦軸に吸光度 横軸に濃度 (ng/ml) をプロットします ] 3) 標準曲線から読んだ濃度に希釈倍数を乗じて得た値を検体の濃度として報告します * 検体の吸光度が standards の最高濃度を超えた場合は さらに検体を希釈して再測定してく ださい 正常人血漿常人血漿の濃度 正常人血漿 135 例を測定したところ 下記の結果になりました n Total 135 genotype ε2/ε4, ε3/ε4 19 genotype ε4/ε4 3 genotype ε2/ε2, ε2/ε3, ε3/ε3 113 ApoE4 Pan-ApoE (µg/ml) (µg/ml) ApoE4/Pan-ApoE ratio Average SD Average SD Average SD Average SD
21 性能 Pan-ApoE 1. 感度 感度検出限界は 4 ng/ml です 陽性血漿を希釈して測定した時 0 ng/ml 測定値の平均 +2 SDが 4 ng/ml 測定値の平均 2 SD より小さくなりました 2. 同時再現性血漿 3 例を用いて 同時に 5 回測定したところ 下表の結果が得られました 血漿サンプルの Pan-ApoE 濃度は 濃度の算出算出の項に示されている方法に従って求めました Sample plasma 1 plasma 2 plasma 3 Number of determinations Mean (ng/ml) C.V. (%) 添加回収試験血漿を用いて 添加回収試験をおこなったところ 下表の結果が得られました 異なる濃度の ApoE4(genotype ε4/ε4) を血漿サンプル (genotype ε3/ε3) に添加しました なお 血漿サンプルの Pan-ApoE 濃度は 濃度の算出算出の項に示されている方法に従って求めました (A) additional ApoE4 (ng/ml) Pan-ApoE concentration observed (ng/ml) (B) Recovery (ng/ml) (B/A) Recovery (%) ApoE4 1. 感度 感度検出限界は 8 ng/ml です 陽性血漿を希釈して測定した時 4 ng/ml 測定値の平均 +2 SDが 8 ng/ml 測定値の平均 2 SD より小さくなりました 2. 同時再現性血漿 3 例を用いて 同時に 5 回測定したところ 下表の結果が得られました 血漿サンプルの ApoE4 濃度は 濃度の算出算出の項に示されている方法に従って求めました - 13-
22 Sample plasma 1 plasma 2 plasma 3 Number of determinations Mean (ng/ml) C.V. (%) 添加回収試験血漿を用いて 添加回収試験をおこなったところ 下表の結果が得られました 異なる濃度の ApoE4(genotype ε4/ε4) を血漿サンプル (genotype ε3/ε3) に添加しました なお 血漿サンプルの ApoE4 濃度は 濃度の算出算出の項に示されている方法に従って求めました (A) additional ApoE4 (ng/ml) ApoE4 concentration observed (ng/ml) (B) Recovery (ng/ml) (B/A) Recovery (%) ApoE4 phenotype の同定 ApoE4/Pan-ApoE 濃度比を算出することによって ApoE4 ホモ接合体 (E4/E4) ヘテロ接合体 (E2/E4, E3/E4) 非 ApoE4 接合体 (E2/E2, E2/E3, E3/E3) を区別することができます ApoE/Pan-ApoE ratio - 20-
23 関連製品 M067-3 anti-apolipoprotein E4 (1F9) M068-3 anti-apolipoprotein E (3D12) M046-3 anti-amyloid β-protein, N-terminal specific (2C8) M066-3 anti-amyloid β/amyloid Precursor Protein (4E12) M009-3 anti-amyloid Precursor Protein (3E9) 参考文献 1) Mihovilovic, M., et al., J. Lipid Res. 48, (2007) 2) Mann, K. M., et al., Hum. Mol. Gen. 13, (2004) 3) Yamauchi, K., et al., Clin. Chem. 45, (1999) 4) Corder, E. H., et al., Science 261, (1993) 5) Saunders, A. M., et al., Neurology 43, (1993) 6) Davignon, J., et al., Arterioscler. Thromb. Vasc. Biol. 8,1-21 (1988) 7) Breslow, J. L., et al., J. Lipid Res. 23, (1982) 製造元 株式会社医学生物学研究所名古屋市中区丸の内 3 丁目 5 番地 10 号住友商事丸の内ビル 5 階 Tel; (052) , Fax; (052) , URL
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