本文關(guān)鍵詞： 尿微量白蛋白 單克隆抗體 多克隆抗體 熒光免疫檢測 酶聯(lián)免疫檢測　出處：《長春理工大學(xué)》2010年碩士論文　論文類型：學(xué)位論文

【摘要】： 尿微量白蛋白(MA)作為糖尿病、腎病、心血管疾病等慢性病的早期診斷指標(biāo),對其定量檢測有廣泛的臨床意義。因此,建立特異、靈敏的檢測尿微量白蛋白的方法具有重要意義。 本研究利用人血清白蛋白(HSA)制備抗HSA的兔多抗和小鼠單抗,并初步建立了基于尿微量白蛋白的雙抗體夾心免疫熒光及ELISA檢測方法。 采用HSA免疫大耳白兔制備免疫兔血清,并采用蛋白A親和層析柱純化,獲得高純度的兔多抗,效價測定達(dá)到1×10~6;同時用HSA免疫BALB/c小鼠,取其脾細(xì)胞與SP2/0骨髓瘤細(xì)胞進(jìn)行融合,經(jīng)克隆和間接ELISA篩選,篩選到六株能分泌抗HSA單克隆抗體的雜交瘤細(xì)胞株,其中三株效價高的雜交瘤株3F4、6F2和8B5制備腹水,經(jīng)過鑒定:三株單抗都識別同一抗原表位,且3F4的單抗亞類是IgG2b(k型);采用蛋白G親和層析柱純化腹水,測定效價均達(dá)到1×10~7。 以兔多抗和鼠單抗為基礎(chǔ),初步建立了尿微量白蛋白的雙抗體夾心免疫熒光檢測方法和ELISA檢測方法。免疫熒光與ELISA法的線性范圍范圍分別為:50～1000ng/ml、10～400ng/ml。經(jīng)敏感性實(shí)驗(yàn)、特異性實(shí)驗(yàn)、重復(fù)性實(shí)驗(yàn),結(jié)果表明,該兩種方法具有特異性好、靈敏度高和重復(fù)性好的特點(diǎn),為建立科學(xué)完善的尿微量白蛋白檢測方法提供基礎(chǔ)。
[Abstract]:As an early diagnostic index of diabetes, nephropathy, cardiovascular diseases, urinary microalbuminuria (MAA) has a wide range of clinical significance in quantitative detection. Therefore, the establishment of a specific. A sensitive method for the detection of urinary microalbumin is of great significance. In this study, rabbit polyclonal antibody and mouse monoclonal antibody against HSA were prepared by using human serum albumin (HSA), and a double antibody sandwich immunofluorescence and ELISA detection method based on urinary microalbumin was established. Rabbit serum was prepared by immunizing big ear white rabbits with HSA and purified by protein A affinity chromatography. The high purity rabbit polyclonal antibody was obtained and the titer was 1 脳 10 ~ (6). At the same time, BALB/c mice were immunized with HSA, and the spleen cells were fused with SP2/0 myeloma cells, and were screened by clone and indirect ELISA. Six hybridoma cell lines secreting monoclonal antibodies against HSA were screened, and three hybridoma strains with high titer 3F4, 6F2 and 8B5 were selected to produce ascites. All the three McAbs recognize the same epitope, and the McAb subclass of 3F4 is IgG2b(k type. Ascites were purified by affinity chromatography with protein G, and the titers reached 1 脳 10 ~ (7). It was based on rabbit polyclonal antibody and mouse monoclonal antibody. A double antibody sandwich immunofluorescence assay and a ELISA method for detection of urinary microalbumin were established. The linear ranges of immunofluorescence and ELISA were as follows:. 50ng / ml. The sensitivity, specificity and repeatability of the two methods were tested. The results showed that the two methods had good specificity, high sensitivity and good reproducibility. It provides a basis for the establishment of a scientific and perfect method for the detection of urinary microalbumin.
【學(xué)位授予單位】：長春理工大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2010
【分類號】：R392

【共引文獻(xiàn)】

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