【摘要】： 目的:制備和初步鑒定小鼠抗雞蛋清提取物單克隆抗體。 方法:以純化后的蛋清提取物為免疫原,經(jīng)背部皮下多點(diǎn)注射免疫BALB/c小鼠。采用棋盤滴定法確定抗原的最佳包被濃度,并通過間接ELISA法檢測免疫小鼠血清效價(jià),選取血清效價(jià)高的小鼠用于單克隆抗體的制備。無菌分離免疫小鼠脾臟,制成單個(gè)細(xì)胞懸液,與小鼠骨髓瘤NS-1細(xì)胞融合。通過間接ELISA法篩選10株陽性融合孔中的融合細(xì)胞,采用有限稀釋法進(jìn)行克隆與亞克隆培養(yǎng);間接ELISA法篩選可特異性分泌抗蛋清提取物單克隆抗體(monoclonal antibody, McAb)的雜交瘤細(xì)胞;增量培養(yǎng)法制備單克隆抗體; Ig類與亞類鑒定試劑盒鑒定McAb的Ig類與亞類;間接ELISA法檢測雜交瘤細(xì)胞凍融前后產(chǎn)生McAbs的效價(jià);Western blot鑒定McAbs的特異性。 結(jié)果:采用棋盤滴定法確定抗原的最佳包被濃度為100 ng?mL-1。經(jīng)克隆及3～4輪亞克隆,8株克隆失去產(chǎn)生抗體的能力,最終獲得2株可穩(wěn)定分泌抗蛋清提取物McAb的雜交瘤細(xì)胞,分別命名為5E12和4F1;其分泌的McAb均為IgG1。Western blot分析表明,McAb 4F1可與轉(zhuǎn)印至膜上的蛋白形成單一條帶,所識(shí)別的靶分子的相對分子質(zhì)量(Mr)約為44KD。間接ELISA法檢測兩株細(xì)胞凍融前后所產(chǎn)生的McAb5E12、4F1的效價(jià)分別為1:5000、1:300。 結(jié)論:成功地制備了兩株抗蛋清提取物McAb 5E12和4F1,為下一步制備、純化特異性抗蛋清提取物單克隆抗體提供了雜交瘤細(xì)胞,并為進(jìn)一步建立快速檢測、提取、純化及鑒定食物中蛋清變應(yīng)原的方法,奠定了有效的實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Objective: to prepare and identify mouse monoclonal antibody against egg white extract. Methods: BALB/c mice were immunized by subcutaneous injection of purified egg white extract. The best coating concentration of antigen was determined by chessboard titration and the titer of immunized mice was detected by indirect ELISA method. The mice with high serum titer were selected for the preparation of monoclonal antibody. The spleen of mice was isolated and immunized with aseptic method. A single cell suspension was made and fused with mouse myeloma NS-1 cells. The fusion cells from 10 positive fusion cells were screened by indirect ELISA method, cloned and subcloned by limited dilution method, and hybridoma cells secreting monoclonal antibody (monoclonal antibody, McAb) against egg white extract were screened by indirect ELISA method. Monoclonal antibodies were prepared by incremental culture, Ig classes and subclasses of McAb were identified by Ig and subclass identification kit, and the specificity of McAbs was identified by indirect ELISA assay for detection of McAbs production before and after freezing and thawing of hybridoma cells. Results: the optimum coating concentration of antigens was determined by chessboard titration for 100 ng?mL-1.. After cloning and three rounds of subcloning, 8 clones lost the ability to produce antibodies. Finally, two hybridoma cells secreting McAb stably from egg white extract were obtained. They were named 5E12 and 4F1, respectively, and the McAb secreted by them were both IgG1.Western blot analysis. The results showed that McAb 4F1 could form a single band with the protein transferred to the film, and the relative molecular weight (Mr) of the target molecule recognized was about 44kD. The titer of McAb5E12,4F1 produced by indirect ELISA assay before and after freezing and thawing was 1: 5 000 and 1: 300, respectively. Conclusion: two strains of anti-egg white extract (McAb 5E12 and 4F1) were successfully prepared, which provided hybridoma cells for further preparation and purification of specific monoclonal antibodies against egg white extract. The method of purification and identification of egg white allergens in food has laid an effective experimental foundation.
【學(xué)位授予單位】：南昌大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

【引證文獻(xiàn)】

相關(guān)碩士學(xué)位論文 前1條

1 朱曉琳;豬傳染性胃腸炎病毒重組N蛋白的表達(dá)純化及其單克隆抗體的研究[D];福建農(nóng)林大學(xué);2012年

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本文編號(hào)：2289834