發(fā)布時(shí)間：2018-03-31 17:07

  本文選題：DAB2IP　切入點(diǎn)：熒光免疫層析　出處：《福建中醫(yī)藥大學(xué)》2014年碩士論文

【摘要】：目的：研發(fā)DAB2IP蛋白的熒光免疫層析檢測(cè)試劑盒,探討不同中醫(yī)證型肺癌與DAB2IP蛋白表達(dá)量的相關(guān)性。 方法：應(yīng)用B細(xì)胞雜交瘤技術(shù)建立分泌抗人DAB2IP單克隆抗體的雜交瘤細(xì)胞株；采用鼠源性單克隆抗體亞型檢測(cè)試劑盒鑒定單抗的亞類；體內(nèi)誘生腹水法制備單抗；免疫親和層析法純化抗體；Western blot分析抗體特異性；熒光免疫層析法研制DAB2IP熒光免疫試劑盒；在此基礎(chǔ)上,應(yīng)用DAB2IP熒光免疫試劑盒檢測(cè)不同臨床中醫(yī)證型肺癌中DAB2IP蛋白的表達(dá)。 結(jié)果：獲取兩株穩(wěn)定分泌抗人DAB2IP單抗的雜交瘤(2A4、2G8)；鼠源性單克隆抗體亞型檢測(cè)試劑盒鑒定2A4、2G8細(xì)胞株均屬小鼠IgGl亞類；經(jīng)體內(nèi)誘生腹水法制備單抗,小鼠腹水形成的陽(yáng)性率為95%以上,腹水的收獲量平均為5.Oml/只小鼠；經(jīng)免疫親和層析法純化后的抗體跑膠結(jié)果顯示其分子量約為150kD; Western blot顯示2A4單抗能與蛋白發(fā)生特異性反應(yīng),特異性良好；成功研制DAB2IP分子的熒光免疫層析檢測(cè)試劑盒,靈敏度為ing/m1,精密度為6.08%,線性范圍在1.00-500.00ng/mL,提示該檢測(cè)系統(tǒng)具有良好的靈敏度、精密性和準(zhǔn)確性。應(yīng)用該試劑盒檢測(cè)不同臨床中醫(yī)證型肺癌中的DAB21P分子的表達(dá)量,顯示氣陰兩虛組與其它組間差別有顯著性(P0.05),具有明顯統(tǒng)計(jì)學(xué)意義。 結(jié)論：本研究成功制備了具有自主知識(shí)產(chǎn)權(quán)的兩株抗人DAB2IP單抗和DAB2IP熒光免疫層析檢測(cè)試劑盒。該試劑盒通過(guò)對(duì)血清中DAB2IP含量進(jìn)行半定量分析,為其在腫瘤基礎(chǔ)研究和臨床中的應(yīng)用提供了實(shí)驗(yàn)依據(jù),為肺癌的靶向治療提供了新的思路和途徑。由于肺癌氣陰兩虛組與其它組在DAB2IP分子表達(dá)上存在顯著差異(P0.05),提示,采用望聞問(wèn)切與現(xiàn)代免疫學(xué)方法相結(jié)合,可能使中醫(yī)分型更加客觀、更加深入,該研究對(duì)于中醫(yī)證型現(xiàn)代化研究具有一定參考價(jià)值。
[Abstract]:Objective : To develop a fluorescent immunochromatography assay kit for the research and research on the correlation between different types of traditional Chinese medicine syndrome type lung cancer ( NSCLC ) and the expression level of DAB 2IP protein .

Methods : A hybridoma cell line secreting anti - human DAB 2IP monoclonal antibody was established by B - cell hybridoma technique .
a murine monoclonal antibody subtype detection kit is adopted to identify the subclass of the monoclonal antibody ;
preparing monoclonal antibody by in vivo induced ascites method ;
purifying the antibody by immunoaffinity chromatography ;
The specificity of antibody was analyzed by Western blot .
Development of fluorescence immunoassay kit by fluorescence immunochromatography
On the basis of this , the expression of DAB 2IP protein in lung cancer of different clinical syndrome types was detected by the fluorescence immunoassay kit .

Results : Two hybridoma clones ( 2A4 , 2G8 ) were obtained .
The mouse - derived monoclonal antibody subtype detection kit was used to identify the 2A4 and 2G8 cell strains , which belong to the mouse IgG1 subclass ;
The positive rate of ascites formation in mice was more than 95 % , and the average recovery of ascites was 5.Oml / mouse ;
The results showed that the molecular weight was about 150 kD after purified by immunoaffinity chromatography . Western blot showed that the monoclonal antibody of 2A4 could specifically react with the protein , and its specificity was good ;
The sensitivity is ing / m1 , the precision is 6.08 % and the linear range is 1.00 - 500.00ng / mL . It is suggested that the detection system has good sensitivity , precision and accuracy .

Conclusion : This study has successfully prepared two kinds of anti - human DAB 2IP monoclonal antibodies with independent intellectual property . The kit has provided an experimental basis for its application in tumor basic research and clinical application by semi - quantitative analysis of the content in serum . It suggests that the combination of the two virtual groups of lung cancer and modern immunology may make the classification of traditional Chinese medicine more objective and deeper , and the research has certain reference value for the modern research of TCM syndrome type .

【學(xué)位授予單位】：福建中醫(yī)藥大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2014
【分類號(hào)】：R734.2

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本文編號(hào)：1691654