【摘要】：Exendin-4是由39個氨基酸組成的多肽,與胰高血糖素樣肽-1(GLP-1)有53%的同源性,是一種有效的GLP-1受體激動劑,已作為II型糖尿病治療藥物在臨床應(yīng)用。目前其檢測方法主要包括高效液相色譜法,液相-質(zhì)譜法和免疫分析法。色譜法無法同時檢測大批量樣品,而且需要專業(yè)儀器設(shè)備;雖然免疫分析法已有商品化的檢測試劑盒,但其價格昂貴,檢測準(zhǔn)確度欠佳。因此本文設(shè)計利用抗exendin-4的單克隆抗體構(gòu)建一種夾心的酶聯(lián)免疫吸附法(ELISA)用于檢測exendin-4。以exendin-4免疫雌性的BALB/c小鼠,經(jīng)過3次免疫檢測血清效價后,取脾細(xì)胞與SP2/0骨髓瘤細(xì)胞融合。用有限稀釋法將陽性細(xì)胞單克隆化篩選,經(jīng)過4輪篩選共得到15株單克隆雜交瘤細(xì)胞。收集其中1株細(xì)胞培養(yǎng)基上清,經(jīng)純化得到單克隆抗體2-B7。將單抗2-B7與生物素標(biāo)記的多抗聯(lián)合構(gòu)建了夾心的ELISA檢測方法,并對其進(jìn)行了方法學(xué)考察。該方法的可檢測最低濃度為0.25ng/ml,特異性良好,批內(nèi)變異系數(shù)和批間變異系數(shù)均小于5%,具有很好的重復(fù)性,可以用于本實(shí)驗(yàn)室相關(guān)研究樣品的檢測。在上述研究基礎(chǔ)上,提出進(jìn)一步優(yōu)化ELISA檢測方法的設(shè)想,并完成了一部分工作,包括半抗原偶聯(lián)載體的免疫和特異性識別exendin-4 C末端和N末端的陽性細(xì)胞篩選。
[Abstract]:Exendin-4 is a 39 amino acid peptide with 53% homology with glucagon like peptide-1 (GLP-1). It is an effective GLP-1 receptor agonist and has been used as a therapeutic drug for type II diabetes. At present, the main detection methods include high performance liquid chromatography, liquid-mass spectrometry and immunoassay. Chromatography can not detect large quantities of samples at the same time, and requires professional instruments and equipment. Although commercial detection kits have been developed for immunoassay, their prices are expensive and the detection accuracy is poor. So we designed a sandwich enzyme linked immunosorbent assay (ELISA) using monoclonal antibody against exendin-4 to detect exendin-4.. Female BALB/c mice were immunized with exendin-4. After three times of immunoassay, spleen cells were fused with SP2/0 myeloma cells. A total of 15 monoclonal hybridoma cells were obtained after four rounds of screening by monoclonal screening with limited dilution method. One cell culture medium supernatant was collected and purified to obtain monoclonal antibody 2-B 7. A sandwich ELISA detection method was constructed by combining monoclonal antibody 2-B7 with biotinylated polyclonal antibody, and its methodology was investigated. The lowest detectable concentration of this method is 0.25 ng / ml, the specificity is good, the coefficient of variation within and between batches is less than 5, and the method has good repeatability. It can be used for the detection of related samples in our laboratory. Based on the above studies, the idea of further optimization of ELISA detection method is proposed, and part of the work is completed, including the immunological screening of hapten coupling vectors and the specific identification of exendin-4 C-terminal and N-terminal positive cells.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.11

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 戴楠;曹曉靜;楊宇馨;李夢俠;易維今;王東;胡川閩;;人脫嘌呤脫嘧啶核酸內(nèi)切酶單克隆抗體的制備及其生物學(xué)特性鑒定[J];免疫學(xué)雜志;2010年03期

2 何忠效;淺談單克隆抗體[J];生物學(xué)通報;2004年09期

相關(guān)碩士學(xué)位論文 前1條

1 田洪斌;注射用艾塞那肽凍干粉針劑的研究[D];吉林大學(xué);2008年

，

本文編號：2358795