本文選題：重金屬 + 鉛��； 參考：《南昌大學(xué)》2012年碩士論文

【摘要】：重金屬鉛是具有持續(xù)毒性的有害物質(zhì),在自然環(huán)境中很難通過生物、化學(xué)方式降解轉(zhuǎn)化為無毒物質(zhì)；通過飲食進(jìn)入人體后,則會(huì)在人體內(nèi)富集引起中毒,因此各國均對(duì)重金屬鉛在環(huán)境與食品中的殘留量有明確的、嚴(yán)格的限制。ELISA檢測(cè)方法相對(duì)原子吸收法、電感偶聯(lián)等傳統(tǒng)重金屬鉛檢測(cè)方法,具有經(jīng)濟(jì)、快速、便捷等優(yōu)勢(shì),高特異性重金屬鉛人工抗原和抗體的研制是建立重金屬鉛酶聯(lián)免疫快速檢測(cè)的關(guān)鍵。本研究通過對(duì)制備的重金屬鉛人工抗原的純化,提高人工抗原免疫效果,免疫Balb/c小鼠,制備抗鉛單克隆抗體,具體如下： 1)重金屬鉛人工抗原的制備 在重金屬鉛人工抗原制備過程中,本實(shí)驗(yàn)選取[(R)-2-氨基-3-(4-氨基苯基)丙基]-(S-S)環(huán)己烷-1,2二乙三胺五乙酸(P-NH2-Bn-Chx-A"-Dtpa)作為雙功能螯合劑,并對(duì)重氮化法、傳統(tǒng)戊二醛法和改良戊二醛法進(jìn)行了比較。重氮化法合成的抗原偶聯(lián)率相對(duì)較高,最高可以達(dá)到1：47,但是沉淀產(chǎn)生嚴(yán)重,同時(shí),通過實(shí)驗(yàn)發(fā)現(xiàn),當(dāng)偶聯(lián)比接近1：21左右時(shí),人工抗原溶液就極易產(chǎn)生絮狀沉淀,并且離心后,沉淀依然存在,無法應(yīng)用于后期的動(dòng)物免疫實(shí)驗(yàn),另外,重氮化法反應(yīng)條件劇烈,容易造成載體蛋白變性,從而減弱甚至其喪失免疫原性。傳統(tǒng)戊二醛法,雖然條件溫和,抗原反應(yīng)液澄清,但是其合成的抗原偶聯(lián)率相對(duì)較低,一般只有1：14左右,因此,研究對(duì)傳統(tǒng)戊二醛法加以改進(jìn),在使用相同螯合劑的情況下將偶聯(lián)率提高到1：20左右。 2)重金屬鉛人工抗原的純化 人工抗原的純度是影響免疫效果的重要因素之一,免疫抗原純度越高,誘發(fā)機(jī)體產(chǎn)生特異性抗體的可能也越高。在人工抗原制備過程中,容易出現(xiàn)蛋白鏈接蛋白的現(xiàn)象,若這種情況發(fā)生比較嚴(yán)重,則會(huì)對(duì)抗原鑒定結(jié)果帶來很大干擾,而且對(duì)產(chǎn)生抗體的特異性帶來不利影響。本實(shí)驗(yàn)選用濾膜孔徑較大的超速離心管,去除抗原溶液中分子量過大的交聯(lián)蛋白,通過SDS-PAGE電泳檢測(cè),表明抗原純度有明顯提高,且由于無效蛋白的去除,抗原偶聯(lián)比進(jìn)一步提高,由1：20提高至1：23。 3)重金屬鉛免疫檢測(cè)方法的建立 將重金屬鉛免疫檢測(cè)方法的實(shí)驗(yàn)條件進(jìn)行優(yōu)化,為建立高效、準(zhǔn)確的重金屬鉛抗體ELISA檢測(cè)方法打下基礎(chǔ)。本實(shí)驗(yàn)在原有ELISA檢測(cè)方法的基礎(chǔ)上對(duì)實(shí)驗(yàn)中使用的緩沖液類別、溶液pH值、檢測(cè)用包被抗原的包被時(shí)間及溫度等條件參數(shù)進(jìn)行優(yōu)化,最終結(jié)果表明,實(shí)驗(yàn)用緩沖液選用HBS緩沖液,并將pH值調(diào)整至7.4效果最好；包被抗原的包被時(shí)間在環(huán)境溫度37℃時(shí)為1小時(shí),當(dāng)環(huán)境溫度為4℃時(shí)包被時(shí)間為12小時(shí)為宜。 4)抗重金屬鉛單克隆抗體的制備與鑒定 本實(shí)驗(yàn)選用5-8周齡的Balb/c雌性小鼠進(jìn)行動(dòng)物免疫實(shí)驗(yàn)。將(1)中制備的重金屬鉛人工抗原用皮下多點(diǎn)注射的方式免疫小鼠,第四次免疫后,得到效價(jià)在1：102400至1：204800之間的小鼠血清,通過計(jì)算,不同種類包被抗原OD值的差異率達(dá)到131%,證明小鼠已對(duì)抗原產(chǎn)生特異性免疫應(yīng)答,并產(chǎn)生抗體。取陽性小鼠脾細(xì)胞與飼養(yǎng)的骨髓瘤細(xì)胞融合,細(xì)胞融合率達(dá)85.91%,單克隆數(shù)目比例為41.1%,繼續(xù)亞克隆五次之后,得到兩株敏感性、特異性都比較理想的細(xì)胞株15E8和20C11,再采用體內(nèi)誘生的方法獲得腹水型單抗。
[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.
【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】：2012
【分類號(hào)】：R155.5

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