Renner E D
Pathology and Laboratory Medicine, VAMC, Fargo, North Dakota 58102.
Cytometry. 1994 Jun 15;18(2):103-8. doi: 10.1002/cyto.990180209.
A rapid (2 h) amplified flow cytometric fluoroimmunoassay (AFCF) for Clostridium difficile toxin A was developed and compared with the cytotoxin assay (CTA) and culture of the organism from stool specimens from patients with suspected C. difficile-associated gastrointestinal disease (CAD). For this assay polyclonal antitoxin A was attached to 10-microns diameter and monoclonal antitoxin A was attached to fluorescent 0.1 micron-diameter polystyrene microspheres. The microspheres and sample were reacted together as in a conventional double-antibody sandwich assay. However, laser flow cytometric measurement allowed the omission of separation and washing steps by gating on light scattered by the larger microspheres and measuring only the fluorescence associated with these particles. The amount of fluorescence from the attached 0.1 micron microspheres was dependent on the concentration of toxin A in the sample. The AFCF detected purified toxin A at levels of 1 pg/ml and was linear from 1 to 40 pg/ml. The AFCF was compared with the CTA and culture of C. difficile for clinical use by comparing results from 198 stool specimens from patients with suspected CAD. The AFCF was 85.7% sensitive and 95.8% specific relative to the CTA, and 85.2% sensitive and 98.3% specific compared to the culture assay. If the isolation of toxigenic C. difficile or the patients clinical course was considered indicative of CAD, the sensitivities of the AFCF, CTA, and culture assay were 77.4%, 67.7% and 96.8%, respectively. The AFCF demonstrated a specificity of 98.8%, while both CTA and culture had a specificity of 100%.(ABSTRACT TRUNCATED AT 250 WORDS)
我们开发了一种用于艰难梭菌毒素A的快速（2小时）放大流式细胞荧光免疫测定法（AFCF），并将其与细胞毒素测定法（CTA）以及对疑似艰难梭菌相关性胃肠道疾病（CAD）患者粪便标本进行的该菌培养法进行比较。在该测定法中，多克隆抗毒素A附着于直径10微米的微球上，单克隆抗毒素A附着于荧光直径0.1微米的聚苯乙烯微球上。微球和样本如在传统双抗体夹心测定法中那样一起反应。然而，激光流式细胞术测量通过对较大微球散射的光进行选通并仅测量与这些颗粒相关的荧光，从而省略了分离和洗涤步骤。附着的0.1微米微球的荧光量取决于样本中毒素A的浓度。AFCF能检测到浓度为1 pg/ml的纯化毒素A，且在1至40 pg/ml范围内呈线性。通过比较198份疑似CAD患者粪便标本的结果，将AFCF与CTA及艰难梭菌培养法用于临床比较。相对于CTA，AFCF的敏感性为85.7%，特异性为95.8%；与培养测定法相比，敏感性为85.2%，特异性为98.3%。如果将产毒艰难梭菌的分离或患者的临床病程视为CAD的指征，则AFCF、CTA和培养测定法的敏感性分别为77.4%、67.7%和96.8%。AFCF的特异性为98.8%，而CTA和培养法的特异性均为100%。（摘要截短至250字）
