CAJ | 학술논문

目的研制广州管圆线虫单克隆抗体诊断循环抗原提高诊断的特异性.方法将广州管圆线虫分泌性抗原免疫小鼠,免疫鼠脾细胞与骨髓瘤细胞融合为杂交瘤细胞,用广州管圆线虫阳性患者血清筛选阳性杂交瘤细胞,培养阳性的杂交瘤细胞分离制备单克隆抗体命名为12D5和21B7,用免疫组织化学的方法分析12D5和21B7单抗结合抗原在广州管圆线虫体内的分布,并用筛选的双12D5和21B7单抗进行抗体夹心ELISA检测实验感染广州管圆线虫的大鼠、广州管圆线虫感染病人血清循环抗原(CAg),用其他寄生虫抗原鉴别单抗的特异性,并与抗体检测比较其敏感性和特异性.结果经鉴定单抗12D5为IgG1,21 B7为IgM,两株单抗同时识别广州管圆线虫成虫相对分子质量为55 × 103的蛋白,两个单抗针对的抗原分布在虫体肠表面,12D5和21 B7双抗体夹心ELISA法对实验感染的广州管圆线虫的大鼠血清中CAg检出率为100%(48/48),广州管圆线虫感染病人血清CAg检出率为100%(32/32),与日本血吸虫、肝吸虫、肺吸虫、旋毛虫、蛔虫、包虫病人血清无交叉反应,与健康人血清无反应;而用抗原检测32个广州管圆线虫感染病人的抗体检出率为75%(24/32),同时抗体检测与其他寄生虫出现一定的交叉反应.结论 12D5和21 B7单抗结合的抗原为肠相关抗原,双抗体夹心ELISA法对感染广州管圆线虫人和动物血清中CAg检测的特异性强,敏感性高,优于抗体检测试剂,并能够确定现症感染.
목적 연제엄주관원선충단극륭항체진단순배항원제고진단적특이성.방법 장엄주관원선충분비성항원면역소서,면역서비세포여골수류세포융합위잡교류세포,용엄주관원선충양성환자혈청사선양성잡교류세포,배양양성적잡교류세포분리제비단극륭항체명명위12D5화21B7,용면역조직화학적방법분석12D5화21B7단항결합항원재엄주관원선충체내적분포,병용사선적쌍12D5화21B7단항진행항체협심ELISA검측실험감염엄주관원선충적대서、엄주관원선충감염병인혈청순배항원(CAg),용기타기생충항원감별단항적특이성,병여항체검측비교기민감성화특이성.결과 경감정단항12D5위IgG1,21 B7위IgM,량주단항동시식별엄주관원선충성충상대분자질량위55 × 103적단백,량개단항침대적항원분포재충체장표면,12D5화21 B7쌍항체협심ELISA법대실험감염적엄주관원선충적대서혈청중CAg검출솔위100%(48/48),엄주관원선충감염병인혈청CAg검출솔위100%(32/32),여일본혈흡충、간흡충、폐흡충、선모충、회충、포충병인혈청무교차반응,여건강인혈청무반응;이용항원검측32개엄주관원선충감염병인적항체검출솔위75%(24/32),동시항체검측여기타기생충출현일정적교차반응.결론 12D5화21 B7단항결합적항원위장상관항원,쌍항체협심ELISA법대감염엄주관원선충인화동물혈청중CAg검측적특이성강,민감성고,우우항체검측시제,병능구학정현증감염.
Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.