【摘要】：目的：制備和鑒定卵清蛋白特異性單克隆抗體，建立雙抗體夾心ELISA法檢測(cè)流感疫苗中的卵清蛋白殘留量。 方法：以提純的卵清蛋白免疫Balb/c小鼠，第四次加強(qiáng)免疫后取脾淋巴細(xì)胞與NS－1骨髓瘤細(xì)胞按常規(guī)方法融合、篩選、克隆化及腹水注射制備McAb，采用間接ELISA方法進(jìn)行McAb的鑒定，Western blot進(jìn)行特異性鑒定；使用ProteinA親和層析柱純化腹水中的卵清蛋白單克隆抗體，用改良過(guò)碘酸鈉法對(duì)單抗進(jìn)行辣根過(guò)氧化物酶標(biāo)記。經(jīng)抗體配對(duì)實(shí)驗(yàn)確定包被抗體和酶標(biāo)抗體，再用方陣滴定法確定抗體工作濃度，制備針對(duì)卵清蛋白的雙抗體夾心ELISA檢測(cè)試劑，分析其穩(wěn)定性、靈敏度和特異性。用卵清蛋白標(biāo)準(zhǔn)品制備定量標(biāo)準(zhǔn)曲線，對(duì)待檢標(biāo)本進(jìn)行檢測(cè)并與國(guó)外試劑進(jìn)行比較。 結(jié)果：經(jīng)過(guò)細(xì)胞融合、多次克隆化獲得3株特異性分泌抗卵清蛋白的單克隆抗體雜交瘤細(xì)胞株，經(jīng)3次復(fù)蘇傳代仍能穩(wěn)定分泌抗體，分別命名為1C2、2F7和3B2。3株McAbs腹水ELISA效價(jià)在1：105~1：106；3株McAbs的亞型均為IgG1；相對(duì)親合力為2F73B21C2；初步建立了雙抗體夾心ELISA檢測(cè)方法，其中以1C2與3B2混合后作為包被抗體，，2F7為酶標(biāo)抗體時(shí)為ELISA最優(yōu)體系，最低檢測(cè)極限為31.25ng/ml，且檢測(cè)結(jié)果與卵清蛋白含量呈現(xiàn)良好的線性相關(guān)，R2=0.910，試劑盒有很好的特異性、穩(wěn)定性和精密度。對(duì)樣品測(cè)定結(jié)果與Serazym Ovalbumin試劑測(cè)定結(jié)果相比較（P0.05），無(wú)顯著差異。 結(jié)論：已成功制備出了卵清蛋白McAb，并建立了一種靈敏度較好、特異性強(qiáng)的測(cè)定流感疫苗中卵清蛋白殘留量的ELISA捕獲法，可應(yīng)用于流感疫苗生產(chǎn)過(guò)程卵清蛋白的監(jiān)測(cè)及疫苗的檢定。
[Abstract]:Aim: to prepare and identify specific monoclonal antibodies against ovalbumin and to establish a double antibody sandwich ELISA method for the detection of ovalbumin residues in influenza vaccine. Methods: Balb/c mice were immunized with purified ovalbumin. The spleen lymphocytes were fused with NS-1 myeloma cells by routine method after the fourth enhanced immunization. The McAb, was prepared by cloning and ascitic injection. Indirect ELISA method was used to identify McAb., Western blot was used for specific identification. Monoclonal antibodies against ovalbumin in ascites were purified by ProteinA affinity chromatography and labeled with horseradish peroxidase by modified sodium periodate method. The coated antibody and enzyme labeled antibody were determined by antibody pairing test, and the working concentration of the antibody was determined by square titration. The double antibody sandwich ELISA assay for ovalbumin was prepared and its stability, sensitivity and specificity were analyzed. The quantitative standard curve of ovalbumin standard sample was prepared and compared with foreign reagent. Results: three hybridoma cell lines secreting monoclonal antibodies specifically secreting ovalbumin were obtained after cell fusion. The hybridoma cells could still secrete antibodies stably after 3 times of resuscitation. They were named 1C2, 2F7 and 3B2.3, respectively, and the ELISA titers of ascitic fluid of McAbs strain were 1? 10 5 and 1? 10 6, respectively. The subtypes of McAbs were 2F73B21C22and 2F73B21C2respectively. A double antibody sandwich ELISA method was established, in which the mixture of 1C2 and 3B2 was used as the coated antibody, and 2F7 as the enzyme labeled antibody was the optimal system for ELISA, and the lowest detection limit was 31.25 ng / ml, and the detection limit was 31.25 ng / ml. There was a good linear correlation between the detection results and the content of ovalbumin. R2, 0.910, the kit had good specificity, stability and precision. There was no significant difference between the results of sample determination and that of Serazym Ovalbumin reagent (P0.05). Conclusion: ovalbumin McAb, has been successfully prepared and a sensitive and specific ELISA capture method for the determination of ovalbumin residues in influenza vaccine has been established. It can be applied to the monitoring of ovalbumin in the production process of influenza vaccine and the verification of vaccine.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類(lèi)號(hào)】：R392

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相關(guān)期刊論文 前10條

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本文編號(hào)：2463910