Musculoskeletal Research, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN 46285, USA.
Osteoarthritis Cartilage. 2010 Sep;18(9):1150-8. doi: 10.1016/j.joca.2010.06.011. Epub 2010 Jul 13.
Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA.
The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA.
The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity.
We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.
关节软骨中聚集蛋白聚糖的蛋白水解降解是骨关节炎（OA）的一个显著特征。本研究旨在开发一种灵敏的酶联免疫吸附试验（ELISA），用于检测人血清和尿液中聚集蛋白聚糖酶切片段，以促进聚集蛋白聚糖酶抑制剂在 OA 中的临床开发。
通过互补决定区（CDR）饱和诱变工程和优化 BC3 单克隆抗体，该抗体检测聚集蛋白聚糖酶切聚集蛋白聚糖中的 ARGS 新表位序列，以提高其与新表位的结合亲和力。使用优化的α-ARGS 抗体（BC3-C2）作为捕获抗体和一种针对聚集蛋白聚糖透明质酸结合区（HABR）的商业抗体作为检测抗体，开发了夹心 ELISA（BC3-C2 ELISA）。使用该 ELISA 检测和定量体外消化物、人软骨外植体培养上清液以及人滑液、血清和尿液中存在的聚集蛋白聚糖酶切片段。
与亲本 BC3 抗体相比，优化后的抗体对含 ARGS 的肽的亲和力提高了 4 个对数级，同时保持了不与跨膜肽交叉反应的能力。BC3-C2 ELISA 能够在无需糖基化的情况下检测天然状态下的聚集蛋白聚糖酶切片段。该 ELISA 能够测量基础状态（无细胞因子刺激）下人类关节软骨（HAC）外植体培养物中产生的聚集蛋白聚糖酶的 ARGS 含聚集蛋白聚糖片段。用聚集蛋白聚糖酶抑制剂处理会导致培养上清液中释放的 ARGS 新表位呈剂量依赖性抑制。该 ELISA 检测法还能够检测人滑液、血清和尿液中的 ARGS 含片段，表明其作为聚集蛋白聚糖酶活性生物标志物的潜在用途。
我们使用优化的 ARGS 抗体开发了一种新型 ELISA，并首次证明了基于 ELISA 的人血清和尿液中聚集蛋白聚糖降解产物的测量。该测定法有可能作为临床中的机械药物活性生物标志物，并有望显著影响/加速聚集蛋白聚糖酶抑制剂和其他用于 OA 的疾病修饰药物的临床开发。
