基于細(xì)胞的酶聯(lián)免疫檢測(cè)實(shí)驗(yàn)

基于細(xì)胞的酶聯(lián)免疫檢測(cè)實(shí)驗(yàn)（Cell Based Elisa）可以：

（1）檢測(cè)高通量的樣本，定量細(xì)胞膜上或細(xì)胞內(nèi)的蛋白質(zhì)的表達(dá)情況。
（2）用于磷酸化蛋白質(zhì)的檢測(cè)，易降解蛋白質(zhì)的檢測(cè)，少量細(xì)胞目的蛋白質(zhì)的檢測(cè)。
 
試驗(yàn)方法原理
 
先將細(xì)胞種于 96 孔板中，對(duì)細(xì)胞進(jìn)行不同條件的處理。待檢測(cè)目的蛋白時(shí)，對(duì)細(xì)胞進(jìn)行多聚甲醛的固定、滅活、打孔及封閉非特異性結(jié)合位點(diǎn)。根據(jù)抗原抗體反應(yīng)的原理，一抗孵育與目的蛋白質(zhì)結(jié)合，加入酶標(biāo)記二抗與一抗結(jié)合，加入底物顯色后酶標(biāo)儀讀取數(shù)值。
 
實(shí)驗(yàn)材料

細(xì)胞
試劑、試劑盒：  多聚甲醛 Cell Based Elisa 試劑盒
儀器耗材： 移液槍 酶標(biāo)儀 恒溫箱
 
實(shí)驗(yàn)步驟
 
1 將細(xì)胞種于 96 孔培養(yǎng)板中。
2 培養(yǎng)細(xì)胞，根據(jù)實(shí)驗(yàn)設(shè)計(jì)對(duì)細(xì)胞進(jìn)行不同條件的處理。
3 待檢測(cè)目的蛋白時(shí)，對(duì)細(xì)胞進(jìn)行多聚甲醛的固定、滅活、打孔及封閉非特異性結(jié)合位點(diǎn)。加入一抗孵育與目的蛋白質(zhì)結(jié)合，加入酶標(biāo)記二抗與一抗結(jié)合，加入底物顯色后酶標(biāo)儀讀取數(shù)值。
4 細(xì)胞清洗后加入結(jié)晶紫與細(xì)胞核結(jié)合讀取數(shù)值，作為內(nèi)參值進(jìn)行比對(duì)。
5 數(shù)據(jù)處理。
 
注意事項(xiàng)
 
1 根據(jù)細(xì)胞的大小和生長(zhǎng)情況進(jìn)行種板和培養(yǎng)，待檢測(cè)時(shí)細(xì)胞的密度控制在 75-90%。
2 對(duì)于懸浮細(xì)胞，加入 100ul 10ug/ml 的多聚-L-賴氨酸，37℃ 的條件下處理 30 min 將懸浮細(xì)胞貼壁到培養(yǎng)板上，PBS 清洗兩次，每次 5 min。
3 若是懸浮細(xì)胞，加入 100ul 的 8% 多聚甲醛室溫孵育 25-30 min 將細(xì)胞固定。
4 甲醛具有揮發(fā)性，終止液具有腐蝕性，請(qǐng)帶好防護(hù)設(shè)備。
 
優(yōu)勢(shì)
 
1 樣本的制備過(guò)程中，不涉及裂解細(xì)胞，只需將細(xì)胞及蛋白質(zhì)原位固定，降低蛋白質(zhì)發(fā)生降解的可能性。
2 在操作過(guò)程中，不涉及傳統(tǒng)免疫印跡法的制膠，跑膠，轉(zhuǎn)膜，曝光等步驟，為研究人員節(jié)省很多時(shí)間。
3 整個(gè)操作在 96 孔板中進(jìn)行，容易實(shí)現(xiàn)高通量樣本的檢測(cè)，或者少量細(xì)胞目的蛋白質(zhì)檢測(cè)。
4 操作簡(jiǎn)單，很容易實(shí)現(xiàn)多個(gè)平行，多次重復(fù)，所得數(shù)據(jù)具有統(tǒng)計(jì)分析意義。 
 
應(yīng)用 Cell Based Elisa 發(fā)表的文章:
 
1 Alexia Arpel, Paul Sawma, Caroline Spenle, Justine Fritz, Lionel Meyer, Norbert Garnier, et al. Transmembrane Domain Targeting Peptide Antagonizing ErbB2/Neu Inhibits Breast Tumor Growth and Metastasis. Cell Reports 2014; 8, 1714–1721.
 
2 Shixin Xu, Aiqin Zhong, Xiaokun Bu, HuiningMa,Wei Li, Xiaomin Xu, Junping Zhang. Salvianolic acid B inhibits plaets-mediated inflammatory response in vascular endothelial cells. Thrombosis Research 2015; 135, 137–145.
 
3 Henri H. VERSTEEG, Evert NIJHUIS., Gijs R. VAN DEN BRINK, Maaike EVERTZEN, Gwenda N. PYNAERT,Sander J. H. VAN DEVENTER, et al. A new phosphospecific cell-based ELISA for p42/p44 mitogen-activated protein kinase (MAPK), p38 MAPK, protein kinase B and cAMP-response-element-binding protein. Biochem. J 2000; 350, 717±722.
 
4  Rubina W. Saeed,Santosh Varma,Tina Peng-Nemeroff,Barbara Sherry,David Balakhaneh, Jared Huston, et al. Cholinergic stimulation blocks endothelial cell activation and leukocyte recruitment during inflammation. JEM 2005, Vol. 201, 1113–1123.
 
5 Sanjiv Dhingra, Ashim K. Bagchi, Ana L. Ludke, Anita K. Sharma, Pawan K. Singal. Akt Regulates IL-10 Mediated Suppression of TNFa- Induced Cardiomyocyte Apoptosis by Upregulating Stat3 Phosphorylation. PLoS ONE 2011; 6(9): e25009.
 
6 H Yokoyama, Y Ikehara1, Y Kodera, S Ikehara, Y Yatabe, Y Mochizuki et al. Molecular basis for sensitivity and acquired resistance to gefitinib in HER2-overexpressing human gastric cancer cell lines derived from liver metastasis. British Journal of Cancer 2006; 95, 1504 – 1513.
 
7 MICHAEL WEICHHAUS, JOHN BROOM, KLAUS WAHLE and GIOVANNA BERMANO. A novel role for insulin resistance in the connection between obesity and postmenopausal breast cancer. 
INTERNATIONAL JOURNAL OF ONCOLOGY 2012; 41: 745-752.
 
8 S. Fineschi, F. Cozzi, D. Burger1, J.-M. Dayer, P. L. Meroni and C. Chizzolini. Anti-fibroblast antibodies detected by cell-based ELISA in systemic sclerosis enhance the collagenolytic activity and matrix metalloproteinase-1 production in dermal fibroblasts. Rheumatology 2007; 46:1779–1785.
 
9 Abderrahim El Guerrab1, Rabah Zegrour, Carine-Christiane Nemlin, Flavie Vigier, Anne Cayre, Frederique Penault-Llorca3, et al. Differential Impact of EGFR-Targeted Therapies on Hypoxia Responses: Implications for Treatment Sensitivity in Triple-Negative Metastatic Breast Cancer. PLoS ONE 2011; 6(9): e25080.
 
10 Gopalan Soman, Xiaoyi Yang, Hengguang Jiang, Steve Giardina, and Gautam Mitra. Comparison of GD2 Binding Capture ELISA Assays for Anti-GD2-Antibodies Using GD2-Coated Plates and a GD2-Expressing Cell-Based ELISA. J Immunol Methods. 2011; 373(1-2): 181–191.