重金属铅人工抗原的制备与纯化及抗体免疫检测体系的建立

发布时间：2018-06-12 23:55

本文选题：重金属 + 铅　；参考：《南昌大学》2012年硕士论文

【摘要】：重金属铅是具有持续毒性的有害物质,在自然环境中很难通过生物、化学方式降解转化为无毒物质；通过饮食进入人体后,则会在人体内富集引起中毒,因此各国均对重金属铅在环境与食品中的残留量有明确的、严格的限制。ELISA检测方法相对原子吸收法、电感偶联等传统重金属铅检测方法,具有经济、快速、便捷等优势,高特异性重金属铅人工抗原和抗体的研制是建立重金属铅酶联免疫快速检测的关键。本研究通过对制备的重金属铅人工抗原的纯化,提高人工抗原免疫效果,免疫Balb/c小鼠,制备抗铅单克隆抗体,具体如下： 1)重金属铅人工抗原的制备在重金属铅人工抗原制备过程中,本实验选取[(R)-2-氨基-3-(4-氨基苯基)丙基]-(S-S)环己烷-1,2二乙三胺五乙酸(P-NH2-Bn-Chx-A"-Dtpa)作为双功能螯合剂,并对重氮化法、传统戊二醛法和改良戊二醛法进行了比较。重氮化法合成的抗原偶联率相对较高,最高可以达到1：47,但是沉淀产生严重,同时,通过实验发现,当偶联比接近1：21左右时,人工抗原溶液就极易产生絮状沉淀,并且离心后,沉淀依然存在,无法应用于后期的动物免疫实验,另外,重氮化法反应条件剧烈,容易造成载体蛋白变性,从而减弱甚至其丧失免疫原性。传统戊二醛法,虽然条件温和,抗原反应液澄清,但是其合成的抗原偶联率相对较低,一般只有1：14左右,因此,研究对传统戊二醛法加以改进,在使用相同螯合剂的情况下将偶联率提高到1：20左右。 2)重金属铅人工抗原的纯化人工抗原的纯度是影响免疫效果的重要因素之一,免疫抗原纯度越高,诱发机体产生特异性抗体的可能也越高。在人工抗原制备过程中,容易出现蛋白链接蛋白的现象,若这种情况发生比较严重,则会对抗原鉴定结果带来很大干扰,而且对产生抗体的特异性带来不利影响。本实验选用滤膜孔径较大的超速离心管,去除抗原溶液中分子量过大的交联蛋白,通过SDS-PAGE电泳检测,表明抗原纯度有明显提高,且由于无效蛋白的去除,抗原偶联比进一步提高,由1：20提高至1：23。 3)重金属铅免疫检测方法的建立将重金属铅免疫检测方法的实验条件进行优化,为建立高效、准确的重金属铅抗体ELISA检测方法打下基础。本实验在原有ELISA检测方法的基础上对实验中使用的缓冲液类别、溶液pH值、检测用包被抗原的包被时间及温度等条件参数进行优化,最终结果表明,实验用缓冲液选用HBS缓冲液,并将pH值调整至7.4效果最好；包被抗原的包被时间在环境温度37℃时为1小时,当环境温度为4℃时包被时间为12小时为宜。 4)抗重金属铅单克隆抗体的制备与鉴定本实验选用5-8周龄的Balb/c雌性小鼠进行动物免疫实验。将(1)中制备的重金属铅人工抗原用皮下多点注射的方式免疫小鼠,第四次免疫后,得到效价在1：102400至1：204800之间的小鼠血清,通过计算,不同种类包被抗原OD值的差异率达到131%,证明小鼠已对抗原产生特异性免疫应答,并产生抗体。取阳性小鼠脾细胞与饲养的骨髓瘤细胞融合,细胞融合率达85.91%,单克隆数目比例为41.1%,继续亚克隆五次之后,得到两株敏感性、特异性都比较理想的细胞株15E8和20C11,再采用体内诱生的方法获得腹水型单抗。
[Abstract]:Heavy metal lead is a harmful substance with persistent toxicity. It is difficult to convert into non-toxic substances by biological and chemical degradation in natural environment. After entering the human body by diet, the heavy metal lead will be enriched in human body and cause poisoning. Therefore, all countries have a clear and strict restriction on the residual amount of heavy metal lead in the environment and food and strictly limit the.ELISA detection side. The determination of heavy metal lead by method relative to atomic absorption, inductance coupling and other traditional methods has the advantages of economic, rapid, convenient and so on. The development of high specific heavy metal lead antigen and antibody is the key to rapid detection of heavy metal lead enzyme linked immunosorbent assay. Balb/c mice were immunized with anti - lead monoclonal antibodies.
1) preparation of artificial antigen of heavy metal lead
During the preparation of artificial antigen of heavy metal lead, this experiment selects [(R) -2- amino -3- (4- amino phenyl) propyl] - (S-S) cyclohexane -1,2 two ethyl three amine five acetic acid (P-NH2-Bn-Chx-A "-Dtpa) as a bifunctional chelating agent, and compares the diazotization method, traditional glutaraldehyde method and modified glutaraldehyde method. The antigen coupling rate synthesized by diazotization method At the higher level, the highest can reach 1:47, but the precipitation is serious. At the same time, it is found that when the coupling ratio is near 1:21, the artificial antigen solution will easily produce floc precipitate, and after centrifugation, the precipitation still exists and can not be applied to the later animal immunity test. In addition, the diazotization method has a severe reaction condition and easy to cause load. Body protein denaturation, thereby weakening even its loss of immunogenicity. Although the traditional glutaraldehyde method is mild, the antigen reaction liquid is clarified, the synthesis of antigen coupling rate is relatively low, generally only about 1:14. Therefore, the study of the traditional glutaraldehyde method to improve the coupling rate with the same chelating agent to 1:20. About.
2) purification of artificial antigen of heavy metal lead
The purity of the artificial antigen is one of the important factors affecting the immune effect. The higher the purity of the immune antigen, the higher the possibility of inducing the specific antibody in the body. In the process of artificial antigen preparation, the phenomenon of protein linked protein is easy to appear. If this situation is more serious, it will bring great interference to the result of antigen identification. In this experiment, a ultrafiltration centrifuge tube with a larger filter membrane was selected to remove the crosslinked protein with too much molecular weight in the antigen solution. It was detected by SDS-PAGE electrophoresis to show that the purity of the antigen was obviously improved, and the anti original coupling ratio was further improved from 1:20 to 1:23. due to the removal of the invalid protein.
3) establishment of immunoassay for heavy metal lead
The experimental conditions of heavy metal lead immunoassay are optimized to lay a foundation for establishing an efficient and accurate ELISA detection method for heavy metal lead antibody. Based on the original ELISA detection method, the class of buffer solution used in the experiment, the pH value of the solution, and the parameters of the envelope time and temperature of the coated antigen are optimized. The final result shows that the HBS buffer solution is used in the experimental buffer and the best effect is to adjust the pH value to 7.4. The envelope of the coated antigen is 1 hours at the ambient temperature 37 C, and the time for the package is 12 hours when the ambient temperature is 4.
4) preparation and identification of monoclonal antibodies against heavy metal lead
In this experiment, the Balb/c female mice of 5-8 weeks old were used to carry out the animal immunization experiment. The mice were immunized with subcutaneous multipoint injection of heavy metal lead antigen prepared in (1). After fourth times of immunization, the serum titer between 1:102400 and 1:204800 was obtained. By calculation, the difference rate of the antigen of different types of antigen was reached to 131%, It was proved that the mice had a specific immune response to the antigen, and produced antibodies. The positive mouse splenocytes were fused with the cultured myeloma cells. The rate of fusion was 85.91%, the proportion of the monoclonal number was 41.1%. After the subclone was continued for five times, two sensitivities and ideal specific cell lines, 15E8 and 20C11, were obtained. The birth method obtained the ascites type monoclonal antibody.
【学位授予单位】：南昌大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R155.5

【参考文献】