植物丝裂原活化蛋白激酶 OsMPK6(mitogen-activated protein kinase,OsMPK6)ELISA 试剂盒
Plant mitogen-activated protein kinase (OsMPK6)ELISA Kit

    尊敬的客户，感谢你选用本公司的产品。本产品适用于体外定量检测植物细胞培养上清液、组织等中天然和重组的OsMPK6 浓度。检测其他特殊样本请咨询本公司技术支持。试剂盒仅供科研使用。 使用前请仔细阅读说明书并检查试剂盒组分。 如有疑问，请联系南京卡米洛生物工程有限公司。 您将得到我们的服务。
       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. 


本试剂盒采用双抗体夹心法，双抗体夹心原理，是基于要测试的抗原具有二价以上的特性，能识别包被抗体，同时能识别检测抗体，具体过程如下：

1. 将特异性抗体与固相载体连接，形成固相抗体，洗涤除去未结合的抗体及杂质，并用无关蛋白封闭空余结合位点。
2. 加受检标本使之与固相抗体接触反应一段时间，让标本中的抗原与固相载体上的抗体结合，形成固相抗原复合物。洗涤除去其他未结合的物质。
3. 加生物素标记抗体，使固相免疫复合物上的抗原与生物素标记抗体结合。*洗涤未结合的生物素标记抗体。此时固相载体上带有的酶量与标本中受检物质的量正相关。
4. 加辣根过氧化物酶标记亲和素，使生物素标记抗体与辣根过氧化物酶标记的亲和素结合。*洗涤未结合的酶标记物。此时固相载体上带有的酶量与标本中受检物质的量正相关。
5. 加入底物显色，即可计算标本浓度。
6. 注：一个抗体分子上可以标记上若干个生物素分子，一个生物素分子可以结合一个辣根过氧化物酶标记的亲和素，从而带来了大量的辣根过氧化物酶结合到抗体上，比传统的直接辣根过氧化物酶标记抗体具有更高的敏感性和放大效应。

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酶联免疫试剂盒检测原理】
本实验采用双抗体夹心ELISA法。所提供的ELISA Kit是典型的夹心法酶联免疫吸附测定试剂盒（ELISA）。预先包被的抗体为Anti-OsMPK1 Antibody（北京华大蛋白质研发中心有限公司；产品编号：AbP80140-A-SE）。检测相抗体为多克隆抗体，经生物素（biotin）标记。样品和生物素标记抗体先后加入酶标板孔反应后，PBS或TBS洗涤。随后加入过氧化物酶标记的亲和素反应；经过PBS或TBS的*洗涤后用底物TMB显色。TMB在过氧化物酶的催化下转化成蓝色，并在酸的作用下转化成最终的黄色。颜色的深浅和样品中的待测因子呈正相关。
【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酶联免疫试剂盒检测原理示意图】
【Schematic diagram of the Plant OsMPK6 ELISA kits】

【操作步骤】

1. 从已平衡至室温的密封袋中取出试验所需板条，未用的板条和干燥剂请放回铝箔袋内封存于2-8℃。
2. 预留空白孔（若使用双波长读板，空白孔可以不设）。
3. 分别将标本或不同浓度植物OsMPK6标准品（0U/ml孔加标准品稀释液）加入相应孔中(100μl/孔)， 37℃孵箱孵育90分钟。
4. 提前30分钟制备生物素化植物OsMPK6抗体工作液。
5. 洗板2次。
6. 加入生物素化植物OsMPK6抗体工作液(100μl/孔)。37℃孵箱孵育60分钟。
7. 提前30分钟制备酶结合物工作液。室温避光放置。  
8. 洗板3次。
9. 除空白孔外，加入酶结合物工作液(100μl/孔)。37℃孵箱，避光孵育30分钟。
10. 洗板5次。
11. 加入TMB显色工作液（包括空白孔）100μl/孔，37℃孵箱，避光孵育，当标准曲线高浓度的颜色较深，有明显的颜色梯度的时候，即可终止。试验显色反应请勿超过30分钟。

加入终止液（包括空白孔）100μl/孔，混匀后即刻测量OD（450nm）值(10分钟内)。
【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.

试剂盒参数
【检测范围】200U/ml-3.12U/ml
【灵敏度】最小可测植物OsMPK6 < 0.6U/ml.
【特异性】系统和其它因子无交叉反应。
【批间差】≤12%.
【批内差】≤8%.
【回收率】70-110%.
【保存】-20℃ [短期内（如两周）可4℃]
【用途】用于体外定量分析液体标本（通用型）。
【规格】96T.
【生产日期】见酶标板铝箔袋封口钢印。
【有效期】12个月（-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℃).