Pan Jing, Small Thomas, Qin Dujie, Li Shawn, Wang Li, Chen Dave, Pauley Cindy, Verch Thorsten, Kaplanski Catherine, Bakhtiar Ray, Vallejo Yli Remo, Yin Ray
ANP Technologies, Inc. 824 Interchange Blvd., Newark, DE 19711, USA.
J Pharmacol Toxicol Methods. 2011 Mar-Apr;63(2):150-9. doi: 10.1016/j.vascn.2010.09.003. Epub 2010 Sep 22.
Rapid lateral flow immunogenicity assays for the detection of anti-drug antibodies (ADAs) to two biotherapeutic antibodies, an anti-HER2 antibody and an anti-TNF-α antibody, were developed using ANP Technologies, Inc.'s proprietary Nano-Intelligent Detection System (NIDS®) and compared to their ELISA counterparts.
Biotin and hapten-labeled drugs are incubated with the patient serum sample to allow ADA to form a bridge complex with each drug conjugate. The reaction mixture is then added to a test strip with an anti-hapten capture zone which captures the mixed bridge complex. The bridge-complexed biotinylated drug then reacts with streptavidin-labeled gold particles in situ. The signal developed at the capture zone, which is directly proportional to ADA in the sample, is then quantitatively measured with a handheld reader. The counterpart ELISAs were run using the same reagents. Dose-response, specificity/free drug depletion, and screening cut-point assays were performed using both methods.
The rapid assays' performance compare very closely to their ELISA counterparts'. Both types of assays identified the same positive samples in screening a limited population of 50 normal serum samples for the anti-HER2 antibody. In the case of anti-TNF-α, both assays identified the same positive samples out of 50 normal and 20 rheumatoid arthritis patient serum samples but differed in the assessment of two others. The rapid assay correctly identified as negative an ELISA false positive sample, and correctly tested as positive an ELISA false negative sample. Positive results were verified with a specificity/free drug depletion assay.
The NIDS® rapid immunogenicity assay offers distinct advantages over current methods in simplicity, low cost, and short time to result. More importantly, the method requires no sample dilution and no washing steps which can perturb fragile complexes formed by low-affinity ADAs. Thus, the assay can potentially detect ADAs with various affinities.
利用ANP技术公司专有的纳米智能检测系统（NIDS®）开发了用于检测两种生物治疗性抗体（一种抗HER2抗体和一种抗TNF-α抗体）的抗药物抗体（ADA）的快速侧向流动免疫原性检测方法，并将其与酶联免疫吸附测定（ELISA）方法进行比较。
将生物素和半抗原标记的药物与患者血清样本孵育，使ADA与每种药物偶联物形成桥连复合物。然后将反应混合物添加到带有抗半抗原捕获区的测试条上，该捕获区捕获混合的桥连复合物。桥连复合物化的生物素化药物随后与链霉亲和素标记的金颗粒原位反应。然后用手持式阅读器定量测量在捕获区产生的信号，该信号与样品中的ADA成正比。使用相同的试剂进行对应的ELISA检测。两种方法均进行了剂量反应、特异性/游离药物消耗和筛选切点检测。
快速检测方法的性能与对应的ELISA方法非常接近。在对50份正常血清样本的有限群体进行抗HER2抗体筛查时，两种检测方法都鉴定出相同的阳性样本。对于抗TNF-α，两种检测方法在50份正常血清样本和20份类风湿性关节炎患者血清样本中都鉴定出相同的阳性样本，但对另外两份样本的评估有所不同。快速检测方法正确地将一份ELISA假阳性样本鉴定为阴性，并正确地将一份ELISA假阴性样本检测为阳性。通过特异性/游离药物消耗检测验证了阳性结果。
NIDS®快速免疫原性检测方法在简便性、低成本和短出结果时间方面比现有方法具有明显优势。更重要的是，该方法不需要样品稀释和洗涤步骤，而这些步骤可能会干扰由低亲和力ADA形成的脆弱复合物。因此，该检测方法有可能检测出具有不同亲和力的ADA。
