【摘要】： 隨著人們生活水平的不斷提高和環(huán)保意識的增強,對重金屬污染尤為關(guān)注,殘留檢測急需加強。但常用的重金屬檢測方法需依靠大型儀器,大都存在樣品處理繁瑣、費用高、檢測時間長等缺陷。本研究利用鰲合劑螯合Cd2+、偶聯(lián)載體蛋白體外合成Cd2+免疫抗原和檢測抗原;利用紫外分光光度法、三硝基苯磺酸法、以及抗血清效價檢測對其進(jìn)行評估;利用單克隆抗體制備技術(shù)初步建立鎘離子的ELISA檢測方法;旨在為重金屬Cd~(2+)的殘留檢測提供一種方便、準(zhǔn)確、特異、低廉、快速的新技術(shù)。 結(jié)果:(1)合成抗原紫外吸收峰與載體蛋白紫外吸收峰有明顯差異,游離氨基含量在33%～40%之間。(2)100μg/只劑量免疫小鼠后,抗血清效價明顯高于50、200μg/只劑量免疫的小鼠,抗血清效價在1:81 920以上。(3)50%的PEG,SP2/0與小鼠脾細(xì)胞細(xì)胞比例為1:7.1時,融合率高達(dá)98.4%。(4)用BSA-IEDTA-Cd和BSA-IEDTA檢測細(xì)胞上清,篩選出了2孔陽性雜交瘤細(xì)胞1A1和4E7,陽性率為0.35%;經(jīng)3次陽性率100%的克隆后只獲得了抗Cd2+的雜交瘤細(xì)胞株1A1。(5)利用已建立的間接競爭ELISA法檢測發(fā)現(xiàn),1A1細(xì)胞上清效價在1:1 024以上,腹水效價高于1:256 000。(6)腹水蛋白濃度為15.04 mg/mL,單克隆抗體為IgG1。 結(jié)論:(1)IEDTA可以用于重金屬Cd2+免疫抗原或檢測抗原合成的螯合劑。(2)KLH作為免疫抗原的載體蛋白,免疫效果優(yōu)于以BSA為載體蛋白;BSA用于檢測抗原載體蛋白,可以降低成本,檢測結(jié)果也相對穩(wěn)定。(3)合理的免疫方案有助于產(chǎn)生高效價的抗血清,免疫效果與免疫劑量間無正相關(guān)性。(4)實驗雖已制備出抗Cd2+的單克隆抗體,但細(xì)胞融合陽性較低,克隆不夠穩(wěn)定,抗原合成工藝尚需進(jìn)一步優(yōu)化。
[Abstract]:With the improvement of people's living standard and environmental protection consciousness, heavy metal pollution is paid more attention to, and the residue detection needs to be strengthened. However, the commonly used heavy metal detection methods rely on large scale instruments, most of them have the defects of complicated sample processing, high cost, long detection time and so on. In this study, Cd2 immune antigen and detection antigen were synthesized by chelating Cd2 and coupling carrier protein in vitro, and evaluated by UV spectrophotometry, trinitrobenzene sulfonic acid method and antiserum titer test. In order to provide a convenient, accurate, specific, inexpensive and rapid method for the detection of heavy metal Cd~ (2), a ELISA method for the detection of cadmium ion was established by using monoclonal antibody preparation technique. Results: (1) the ultraviolet absorption peak of synthetic antigen was significantly different from that of carrier protein, and the content of free amino group was between 33% and 40%. The titer of antiserum was significantly higher than that of mice immunized with a dose of 50200 渭 g / g. The titer of antiserum was above 1:81 920. (3) when the ratio of 50% PEG,SP2/0 to spleen cells was 1: 7.1, The fusion rate was as high as 98.4%. (4) the supernatant was detected by BSA-IEDTA-Cd and BSA-IEDTA, and 1A1 and 4E7 were screened out, the positive rate was 0.35. After three clones with 100% positive rate, only hybridoma cell line 1A1. (5) the titer of 1A1 cell supernatant was found to be above 1:1 by using the established indirect competitive ELISA assay. The titer of ascites was higher than 1: 256000. (6) the concentration of ascitic protein was 15.04 mg/mL, and monoclonal antibody was IgG1.. Conclusion: (1) IEDTA can be used as a chelating agent for heavy metal Cd2 immune antigen or antigen synthesis. (2) KLH as carrier protein of immune antigen is better than BSA as carrier protein. BSA can be used to detect antigen-carrier proteins, which can reduce the cost, and the results are relatively stable. (3) A reasonable immunization program helps to produce high titer antiserum. There was no positive correlation between immune effect and immune dose. (4) although monoclonal antibodies against Cd2 had been prepared, the fusion positive of cells was low, the clone was not stable, and the technology of antigen synthesis needed to be further optimized.
【學(xué)位授予單位】：甘肅農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2008
【分類號】：R392

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