摘要
目的研制标准化的血吸虫循环抗原(SCAg)酶免疫法检测试剂,建立规模化生产能力。方法通过体外培养及分子筛液相色谱获取虫源性及虫卵源性SCAg,用免疫增强剂和淋巴结内免疫法制备高效、特异抗SCAg的捕捉抗体,应用半抗原抗体桥放大技术制备高灵敏度SCAg识别探针,通过抗氧化剂及螯合剂比较研究制备稳定的底物系统,按医学决定水平确定免疫学质量控制标准及参考,优选原材料质量,规范生产工艺,研究制定反应体系的标准化,建立SCAg快速研发的技术平台。结果TCA提取的SCAg测出最低含量为1ng/ml(10-9g),对实验高感染(1000±10条尾蚴)家兔混合血清检测的终点滴度可达1∶1280以上,有效诊断率为96.29%,研制的SCAg试剂的批内变异4.69%～7.66%,批间变异4.12%～7.19%,一年内同批试剂分次对定值血清测定OD值变化小于10%,显示一年内为有效期。通过SCAg技术平台,进一步研究开发了囊尾蚴CAg、弓形体CAg的检测方法,初步实验观察对囊尾蚴囊液及感染弓形体5d的小鼠腹水抗原检测滴度分别达到1∶640及1∶1280。结论SCAg标准化平台技术可大量地、快速地提供标准化的血吸虫病诊断试剂,对其他寄生虫CAg检测显示有较好的应用前景。
Objective Standardization study of enzyme immune assay kit for detection of schistosome circulating antigen (SCAg) and es tablishment of production ability in large scale. Method Adult a nd egg sources antigen of schistosome was obtained by using culture in v itro and molecular sieve liquid chromatography. High effective and speci fic capture-antibody against SCAg was prepared by using immune enhancement agen t and by inducing immune through lymph-node injection. Probe to SCAg of high se nsitivity for recognition was acquired by using hapten-antibody bridge techniqu e for magnifying. Stable substract system was chosen by comparison method either among antioxidants or among chelators. Quality control standard and reference w as made according to medical criterion. Raw and processed materials were optimiz ed in quality, production technique was normalized and standardization of reacti on complex and system was worked out. With all the above studies, the technique platform for SCAg detection has been developed. Result SCAg abst racted with TCA could detect 1 ng/ml (10^(-9) g) minimum and final titer coul d attain as high as 1∶1 280 or above for serum from rabbits infected with 1 000±10 cercariae. The rate of effective diagnosis was 96.29%. Coeffici ent of variation within one batch was 4.69%～7.66% and between batches was 4. 12%～7.19%. OD value of given serum determined in different period of one year was less than 10% indicating that the effective time of the assay kit was one ye ar. On the basis of this technique platform for SCAg established, further develo pments have been worked out for cysticercus CAg, toxoplasmatic CAg, and titer of intracapsular (liquid) of Cysticercus cellulosae and ascites from mice infected with Toxoplasma gondii five days later attai ned to 1∶640 to 1∶1 280. (Conclusion) T he standard technique platform possesses large scale production ability in provi ding standard enzyme immune assay kit for detection of SCAg. Besides, it reveal s good prospects for detection of circulating a
出处
《寄生虫病与感染性疾病》
CAS
2005年第2期49-53,共5页
Parasitoses and Infectious Diseases
基金
国家高技术研究发展专项经费资助(编号2004AA2Z3580)
关键词
血吸虫
抗原
免疫酶技术
标准化
schistosome
antigen
immune enzyme technique
stand ardization
