植物絲裂原活化蛋白激酶 OsMPK6(mitogen-activated protein kinase,OsMPK6)ELISA 試劑盒
Plant mitogen-activated protein kinase (OsMPK6)ELISA Kit

    尊敬的客戶，感謝你選用本公司的產品。本產品適用于體外定量檢測植物細胞培養上清液、組織等中天然和重組的OsMPK6 濃度。檢測其他特殊樣本請咨詢本公司技術支持。試劑盒僅供科研使用。 使用前請仔細閱讀說明書并檢查試劑盒組分。 如有疑問，請聯系南京卡米洛生物工程有限公司。 您將得到我們的服務。
       Dear customers, thank you for choosing our products. This product is suitable for in vitro quantitative detection of Plant cell culture supernatant and organizations in the natural and recombinant OsMPK6 concentration. Detection of other special sample please contact our technical support. The kit is for research use only. Please read the instructions carefully before using and check the kit components. If you have any questions, please contact Nanjing Camillo biological Co., Ltd. 


本試劑盒采用雙抗體夾心法，雙抗體夾心原理，是基于要測試的抗原具有二價以上的特性，能識別包被抗體，同時能識別檢測抗體，具體過程如下：

1. 將特異性抗體與固相載體連接，形成固相抗體，洗滌除去未結合的抗體及雜質，并用無關蛋白封閉空余結合位點。
2. 加受檢標本使之與固相抗體接觸反應一段時間，讓標本中的抗原與固相載體上的抗體結合，形成固相抗原復合物。洗滌除去其他未結合的物質。
3. 加生物素標記抗體，使固相免疫復合物上的抗原與生物素標記抗體結合。*洗滌未結合的生物素標記抗體。此時固相載體上帶有的酶量與標本中受檢物質的量正相關。
4. 加辣根過氧化物酶標記親和素，使生物素標記抗體與辣根過氧化物酶標記的親和素結合。*洗滌未結合的酶標記物。此時固相載體上帶有的酶量與標本中受檢物質的量正相關。
5. 加入底物顯色，即可計算標本濃度。
6. 注：一個抗體分子上可以標記上若干個生物素分子，一個生物素分子可以結合一個辣根過氧化物酶標記的親和素，從而帶來了大量的辣根過氧化物酶結合到抗體上，比傳統的直接辣根過氧化物酶標記抗體具有更高的敏感性和放大效應。

This kit employs Double Antibody Sandwich Technique. The principle of Double Antibody Sandwich is based on characteristics of the tested antigen with more than two valances which can identify coated antibody and detection antibody at same time. The specific process is as follows:

1. Connect the specific antibodies and solid phase carriers to form immobilized antibodies. Wash out uncombined antibodies and impurities. Seal the rest binding sites with irrelevant proteins.
2. Join under test with immobilized antibodies for contact reaction. After a while, combine antigens in and antibodies on carriers into the antigens complex. Wash out uncombined antibodies and impurities.
3. Add biotin labeling antibodies to combine with the antigens on immune complexes. Wash out the uncombined biotin labeling antibodies thoroughly. The enzyme amount on the carrier is now positively related to the amount of the tested substance in specimens.
4. Add horseradish peroxidase to label the avidins and incorporate them with the biotin labeling antibodies. Wash out the incorporated enzyme markers thoroughly. The enzyme amount on the carrier is now positively related to the amount of the tested substance in specimens.
5. Add substrates for coloring, and compute the concentration of specimens.
Note: an antibody molecule can be marked on several biotin molecules and a biotin molecule can be connected with a HRP-Avidin to form numbers of horseradish peroxidases combining with antibodies which shows higher sensitivity and amplification effect comparing with traditional direct HRP-Antibodies.


【植物OsMPK6酶聯免疫試劑盒檢測原理】
本實驗采用雙抗體夾心ELISA法。所提供的ELISA Kit是典型的夾心法酶聯免疫吸附測定試劑盒（ELISA）。預先包被的抗體為Anti-OsMPK1 Antibody（北京華大蛋白質研發中心有限公司；產品編號：AbP80140-A-SE）。檢測相抗體為多克隆抗體，經生物素（biotin）標記。樣品和生物素標記抗體先后加入酶標板孔反應后，PBS或TBS洗滌。隨后加入過氧化物酶標記的親和素反應；經過PBS或TBS的*洗滌后用底物TMB顯色。TMB在過氧化物酶的催化下轉化成藍色，并在酸的作用下轉化成最終的黃色。顏色的深淺和樣品中的待測因子呈正相關。
【Detection principle of Plant mitogen-activated protein kinase (OsMPK6) ELISA kit】
This experiment use double-sandwich elisa technique and the ELISA Kit provided is typical. The pre-coated antibody is Plant OsMPK6 monoclonal antibody (Beijing Protein Innovation Co.,Ltd. (BPI)；Article number：AbP80140-A-SE) and the detecting antibody is polyclonal antibody with biotin labeled.  Samples and biotin labeling antibody are added into ELISA plate wells and washed out with PBS or TBS. Then Avidin-peroxidase conjugates are added to ELISA wells in order; Use TMB substrate for coloring after reactant thoroughly washed out by PBS or TBS. TMB turns into blue in peroxidase catalytic and finally turns into yellow under the action of acid. The color depth and the testing factors in samples are positively correlated.


【植物OsMPK6酶聯免疫試劑盒檢測原理示意圖】
【Schematic diagram of the Plant OsMPK6 ELISA kits】

【操作步驟】

1. 從已平衡至室溫的密封袋中取出試驗所需板條，未用的板條和干燥劑請放回鋁箔袋內封存于2-8℃。
2. 預留空白孔（若使用雙波長讀板，空白孔可以不設）。
3. 分別將標本或不同濃度植物OsMPK6標準品（0U/ml孔加標準品稀釋液）加入相應孔中(100μl/孔)， 37℃孵箱孵育90分鐘。
4. 提前30分鐘制備生物素化植物OsMPK6抗體工作液。
5. 洗板2次。
6. 加入生物素化植物OsMPK6抗體工作液(100μl/孔)。37℃孵箱孵育60分鐘。
7. 提前30分鐘制備酶結合物工作液。室溫避光放置。  
8. 洗板3次。
9. 除空白孔外，加入酶結合物工作液(100μl/孔)。37℃孵箱，避光孵育30分鐘。
10. 洗板5次。
11. 加入TMB顯色工作液（包括空白孔）100μl/孔，37℃孵箱，避光孵育，當標準曲線高濃度的顏色較深，有明顯的顏色梯度的時候，即可終止。試驗顯色反應請勿超過30分鐘。

加入終止液（包括空白孔）100μl/孔，混勻后即刻測量OD（450nm）值(10分鐘內)。
【Steps】
1.Take out needed strips from zip lock bag which balances to room temperature. The unused strips and desiccant should be put back into the sealed aluminum foil bag at 2-8℃ for storage.
2. Set aside blank wells (if dual-wavelength reading plate is used, the blank wells could be ignored)
3. Add samples or different concentration of Plant OsMPK6 standard samples to corresponding wells (100μl for each well), 0U/ml well should be filled with standard diluent. Seal the reaction wells with adhesive tapes, hatching in incubator at 37℃ for 90 min.
4. Prepare biotinylated Plant OsMPK6 antibody liquid 30min in advance.
5. Wash the Elisa plate 2 times
6. Add the biotinylated Plant OsMPK6 antibody liquid to each well (100μl for each). Seal reaction wells with adhesive tapes,  hatching  in incubator at 37℃ for 60 min.
7. Prepare enzyme-conjugate liquid 30min in advance.
8. Wash the Elisa plate 3 times
9. Add enzyme-conjugate liquid to each well except blank wells (100μl for each). Seal the reaction wells with  adhesive tapes,  hatching  in incubator at 37℃ for 30 min.
10. Wash the Elisa plate 5 times.
11. Add 100μl Colour Reagent liquid to individual well (also into blank well), hatching in dark incubator at 37℃. When color for high concentration of standard curve become darker and color gradient appears, the hatching can be stopped. The chromogenic reaction should be controlled within 30 min.
12. Add 100μl Stop solution to individual well (also into blank well). Mix well. Read OD（450nm）within 10 min.

試劑盒參數
【檢測范圍】200U/ml-3.12U/ml
【靈敏度】最小可測植物OsMPK6 < 0.6U/ml.
【特異性】系統和其它因子無交叉反應。
【批間差】≤12%.
【批內差】≤8%.
【回收率】70-110%.
【保存】-20℃ [短期內（如兩周）可4℃]
【用途】用于體外定量分析液體標本（通用型）。
【規格】96T.
【生產日期】見酶標板鋁箔袋封口鋼印。
【有效期】12個月（-20℃）.

【Detection range】200U/ml-3.12U/ml
【Sensitivity】the minimum detectable Plant OsMPK6 up to 0.6U/ml.
【Specific】No cross-reaction with other factors.
【Intra assay Precision】≤ 8%
【Inter assay Precision】≤ 12%
【Recovery】70 - 110 percent.
【Storage】-20℃ [Short-term should be placed 4℃(such as two weeks)]
【Uses】used in vitro quantitative analysis of liquid samples (Universal).
【Specifications】96T.
【Production Date】See outer packaging label.
【Validity】12 months (-20℃).