目的:建立人血浆抗英夫利西单抗抗体(antibodies to infliximab，ATI)免疫分析方法并进行方法学验证。方法:用甘氨酸酸解英夫利西单抗(infliximab，IFX)与ATI结合形成的复合物，然后用生物素标记IFX和辣根过氧化物酶(HRP)标记IFX结合ATI形成桥联复合物，再将溶液转移到链霉亲和素包被的孔板上进行检测。方法建立后采用51例未曾使用过IFX的克罗恩病患者血浆进行验证。结果:检测ATI的ELISA方法经验证筛选临界值为1。16，确证临界值为17。21％，滴度临界值为1。34，批内和批间精密度均小于20％，灵敏度达50ng·mL-1，当阳性对照抗体质量浓度为100ng·mL-1时，药物耐受水平为12。5μg·mL-1。结论:成功建立并验证了检测人血浆ATI的免疫分析方法，适合临床开展治疗药物检测。
Measurements of antibodies to infliximab in plasma by solution ELISA and its application in patients of Crohn's disease
OBJECTIVE To develop and validate a solution enzyme-linked immunosorbent assay(ELISA)method for detecting antibodies to infliximab(ATI).METHODS The infliximab(IFX)/ATI complexes were dissociated by glycine acid,then biotin-IFX and HRP-IFX conjugated with ATI and then resulting bridging complexes were transferred to a streptavidin matrix coated 96-well plate.After an establishment of ATI assay,plasma samples from 51 IFX-naïve Crohn's disease(CD)patients were utilized for method validation.RESULTS Based upon a panel of plasma samples from 51 IFX-naïve CD patients,the screening cut-point was 1.16(S/N).The confirmatory cut-point was determined as a decrease in signal of＞17.21％.The titer cut-point was 1.34.Both intra-and inter-assay precisions were＜20％of CV.The relative sensitivity of this assay was 50 ng·mL-1.Drug-tolerance at the concentration of 100 ng·mL-1 positive control was ≤12.50 μg·mL-1 IFX.The relative sensitiv-ity of this assay was 50 ng·mL-1.CONCLUSION The immunoassay for measuring ATI has been successfully developed and validated,this method can be used for therapeutic drug monitoring.
infliximabantibodies to infliximabCrohn's diseaseenzyme-linked immunosorbent assay
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