摘要
目的制备艰难梭菌A毒素的单克隆抗体酶标诊断试剂。方法采用夹心ELISA方法,用纯化的抗艰难梭菌A毒素的兔单特异血清包板,10%BSA-PBST封闭,加入检测样品,洗涤后加入辣根过氧化物酶标记的单克隆抗体,用底物TMP显色,2MH2SO4终止反应,测定OD450。结果对2株艰难梭菌产毒株、2株艰难梭菌非产毒株、26株大肠杆菌、2株痢疾杆菌、1株双歧杆菌、5株霍乱弧菌、2株伤寒沙门氏菌、7株肉毒梭菌、1株索氏梭菌、1株酪酸梭菌的培养滤液进行检测,该ELISA试剂具有很高的特异度和灵敏度,最低可检出艰难梭菌A毒素0.1ng。结论这种艰难梭菌A毒素的单克隆抗体酶标诊断试剂盒具有较高的特异度和灵敏度,适合临床推广应用。
Objective To establish the ELISA kit of monoclonal antibodies to Clostridium difficile toxin A. Methods A sandwich ELISA was used. Flat-bottomed 96 well polystyrene microtitre plates were coated with 100 μl of purified rabbit monospecific antitoxin（8μg/ml, capturing antibody） in carbonate buffer（pH9.6） and incubated overnight at 4℃, the plates were washed once in PBS con raining 0. 05% Tween-20, pH7.4 （PBST）. After 200μl of 10% BSA in PBS-T was added to the wells and incubated at 37℃ for 2h, washed 5 times in PBS-T with 3 min incubation at room temperature between each wash, 100μl of C. difficile toxin A or test samples in PBS-T were added to each well and incubated for 1h at 37℃ , washed 5 times. Then 100μl of 1 ： 1000 diluted monoclonal antibodies IgG- Horseradish peroxidase conjugate（detecting antibody） was added for 1h at 37℃, wells were washed five times with PBS-T, and 0. 1ml of tetramethylbenzidines substrate was added to each well. After 15 min at 37℃ in dark, the reactions were stopped by the addition of 1 drop of 2M sulfuric acid and the A450 was measured. Results The tested specimens included culture filtrates of 2 strains of toxigenic C. difficile, 2 non-toxigenic strains of C. difficile, 26 strains of E. coli, 2 strains of S. dysenteriae, 1 strains of Bifidobacterium, 5 strains of V. cholera, 2 strain of S. typhi, 7 strains of C. botulinum, 1 strain of toxigenic C. sordllii, and 1 strain of C. butyricum. The ELISA demonstrated high specificity and good sensitivity, it detected amounts of toxin A as low as 0. 1ng/ml. Conclusion An ELISA kits with high specificity and good sensitivity for the rapid detection of C. difficile toxin A was presented. It will be a beneficial tool to clinical detection of Clostridium difficile toxin A.
出处
《国际检验医学杂志》
CAS
2006年第5期391-393,共3页
International Journal of Laboratory Medicine
关键词
梭菌
艰难
细菌毒素类
抗体
单克隆
酶联免疫吸附测定
试剂盒
诊断
Clostridium, difficile
Bacterial toxins
Antibodies, monoclonal
Enzymelinked immunosorbent assay
Reagent kit,diagnostic
