摘要
目的寻找和确定引起特异性不足的因素,实施科学的对策加以解决,真正实现鼠疫的快速诊断技术并以此为基础构建新一代的检测技术。方法(1)研制高质量的鼠疫F1抗原的单克隆抗体,考虑到位点问题要求尽可能多的、针对不同位点的高效价瘤株,构建稳定的“抗体源;”(2)利用获得的高效瘤株通过纯系小鼠生产抗体或用无蛋白细胞培基生产抗体;(3)对抗体进行纯化并使其达到“免疫纯水平”并建立提取、纯化工艺,最终建立稳定的低成本的无交叉“抗体源;”(4)纯化抗原至“免疫纯水平”或“电泳纯水平”,建立提取、纯化工艺,最终建立稳定低成本的无交叉“抗原源;”(5)对新一代快速检测技术进行实验室和现场评价,对特异性即实验的准确性做出评价。结果(1)确定了影响鼠疫免疫学诊断技术特别是间接红细胞凝集试验(IHA及R IHA)特异性不足的两大因素:a.技术落后,试验中采用的载体不适导致了相当数量的假性凝集;b.技术方法中使用的抗原、抗体严重不纯,尤其是制备抗体时免疫动物“自身抗体”的掺入是造成交叉反应更重要的因素;(2)研制并开发出分泌抗鼠疫F1单克隆抗体(F1-M cAb)的杂交瘤株164株,并从其中筛选出针对不同位点的高效瘤株6株,经活性鉴定及配对试验形成了4个组合,具高效价和高活性;(3)使用制备电泳技术将鼠疫F1抗原提纯至电泳纯水平;(4)将鼠疫F1-M cAb提纯至免疫纯水平;(5)引进、消化了具有先进水平的胶体金标免疫层析技术;(6)使用电泳纯的鼠疫F1抗原及免疫纯的鼠疫F1-M cAb构建了能自人和动物血液中直接检测鼠疫特异抗体的金标免疫层析技术(G ICA)和能自人和动物脏器或穿剌物中直接检测鼠疫F1抗原或鼠疫病原的反向金标免疫层析技术(RG ICA);(7)由于采用了纯化的抗原、抗体,提高了标记效果,其敏感性超过IHA和R IHA,阳性检出率在排除了假阳性和交叉反应后依然高出血凝约5%左右;(8)上述两项技术实验室内经44株非鼠疫菌抗血清和90株非鼠疫菌交叉测定、中和试验以及蛋白印迹鉴定(W estern b lotting)均证实反应特异,基本消除了传统技术存在的非特异反应;(9)经4省(区)不同宿主动物3 168份(其中血清材料2 265份,脏器材料903份)现场标本验证,与实验室取得了一致的结论。结论胶体金免疫层析测试条较好地解决了特异性之难题,有效地避免了其它病原微生物导致的以及酸、碱和嗜异性凝集素、低温条件等造成的假性凝集,同时也有效地消除了由于抗原、抗体不纯,特别是实验动物自然感染其它病原产生的“自身抗体”导致的交叉反应,该技术操作简便,特别适合现场和边远地区应用,对比传统技术该法不需专业性操作,不需仪器设备,因而乡村医生可自行进行操作并做出诊断。
Objective To look for and make sure the factor of interfering specificity, by employing scientific technique and developing the indeed rapid diagnosis for plague. Methods （ 1 ） Developing Constructing the source of antibodies. （ 2 ） Producing antibodies by ascites of mice or free serum culture with obtained hybridoma. （ 3 ） Purifying and getting the antibody at immune level, setting up the methods of distilling and purifying antibody, and forming stable, cheap and special antibody source. （ 4 ） Purifying and getting the antigen at immune level and electrophrosis level, setting up the methods of distilling and purifying antigen, and forming stable, cheap and special antigen source. （5） Constructing new diagnosis techniques based on the above antigen and antibody. （6） Evaluating new diagnosis techniques at laboratory and in the field, especially evaluating the specificity of new diagnosis techniques. Results （ 1 ） Two factors affecting the specificity of IHA and RIHA were found. They were the used carrier and crude antigen and antibody, especially including autoantibody in crude antibody. （2） We constructed 164 strains of hybridoma to produce F1 -McAb, including 6 strains of high productive F1 - McAb, which formed 4 pairs to detect F1 antigen with high titer. （ 3 ） We obtained pure F1 antigen at electrophoresis level with preparation electrophoresis technique. （4） We obtained pure F1 antibody at electrophoresis level. （5） We introduced the advanced GICA. （6） We set up GICA to detect serum Of human and animals, and RGICA to detect animal viscera and human puncture fluid. （ 7 ） The sensitive of GICA and RGICA were more than that of IHA and RIHA, respectively, because of the application of pure antigen and antibody. The positive rate of GICA and RGICA was higher than that of IHA and RIHA by about 5%, respectively, after removing false positive. （ 8 ） The specificity of GICA and RGICA were conformed by serum of 44 strains of non - plague and 90 strains of non - plague respectively, neutralization test and Western blotting test. GICA and RGICA excluded false positive reaction by and large. （9）The same conclusions were obtained from 3 168 samples（2 265 of serum samples, 903 of viscera samples） from different rodents in the field test in four provinces. Conclusions We resolve the difficulty specificity in GICA and RGICA to avoid false positive resulted from other bacteria, acid, alkali, inhomogenous agglutinin, low temperature and so on. At the same time, GICA and RGICA eliminate cross -reaction caused by crude antigen and antibody, especially autoantibody in crude antibody. GICA and RGICA are simple, rapid, high sensitive with high specificity. They are suitable for use in field foci, especially in rural area. Since the technique doesnt need special operation and equipments, rural doctor can use it skillfully.
出处
《地方病通报》
2006年第3期1-10,94,F0003,共12页
Endemic Diseases Bulletin
基金
国家自然科学基金(30160078)
云南省重大攻关项目(2002NG17)
