Leeds Institute of Molecular Medicine, University of Leeds, Leeds, UK.
Clin Exp Rheumatol. 2012 Jul-Aug;30(4):534-42. Epub 2012 Aug 29.
Accurately measuring cytokines in clinical material remains an important challenge in the development of biomarkers. Enzyme-linked immunoabsorbent assays (ELISAs) are considered 'gold standard'; however, their use is limited by the relatively large sample volume required for multiple analyte testing. Several alternatives (including membrane or bead-ELISA) have been developed particularly to enable multiplexing. Concerns were raised regarding their use in rheumatology due to interference by heterophilic antibodies, notably rheumatoid factor (RF). In this report, we compared several multiplex assays using serum from rheumatoid arthritis (RA) patients with respect to the presence of residual RF following attempted removal employing commonly used procedures.
Healthy control and RF-positive/negative RA sera were used to compare 4 multiplex assays with ELISA: bead-based 'Luminex' immunoassay, cytometric bead assays (CBAs), membrane-based and Mosaic™ ELISAs. Sera were tested following Ig blockade (mixed species serum) or removal (using PEG6000 or sepharose-L).
Ig removal was only partially efficient and residual RF was detected in most sera. RF had no impact on cytokine measurement by ELISA. In single and multiplex Luminex, cytokine levels associated with false positive results correlated directly with RF titres. Following Ig-blockade/removal, these relationship remained suggesting false positivity was still associated with the presence of residual RF. Conversely, detection of cytokines in multiplex membrane-based or Mosaic- ELISA were not affected by the presence of RF; however, levels of cytokines readily detected by ELISA were often below the detection threshold of these assays. CBA assays were also low on sensitivity but unaffected by RF.
False positivity, due to the presence of heterophilic antibodies, mainly affected Luminex assays. Other assays however remained limited in their sensitivity. Multiplexing of cytokine measurement remains a challenge, particularly in rheumatological pathologies, until assays of adequate sensitivity are developed. ELISA remains the gold standard.
在生物标志物的开发中，准确测量临床标本中的细胞因子仍然是一个重要的挑战。酶联免疫吸附测定（ELISA）被认为是“金标准”；然而，由于需要大量样本进行多分析物检测，其应用受到限制。为了实现多重检测，已经开发出了几种替代方法（包括膜或珠 ELISA）。由于异嗜性抗体（尤其是类风湿因子，RF）的干扰，人们对它们在风湿病学中的应用提出了担忧。在本报告中，我们比较了几种使用类风湿关节炎（RA）患者血清的多重检测方法，这些方法涉及到使用常用方法去除 RF 后残留 RF 的情况。
使用健康对照和 RF 阳性/阴性 RA 血清，比较了 4 种与 ELISA 结合的多重检测方法：基于珠的“Luminex”免疫测定、细胞计数珠测定（CBA）、膜和 Mosaic ELISA。在 Ig 阻断（混合种属血清）或去除（使用 PEG6000 或琼脂糖-L）后，检测血清。
Ig 去除仅部分有效，并且在大多数血清中检测到残留 RF。RF 对 ELISA 检测细胞因子没有影响。在单重和多重 Luminex 中，与假阳性结果相关的细胞因子水平与 RF 滴度直接相关。在 Ig 阻断/去除后，这些关系仍然存在，表明假阳性仍然与残留 RF 的存在有关。相反，在多重膜或 Mosaic-ELISA 中检测细胞因子不受 RF 的影响；然而，ELISA 容易检测到的细胞因子水平通常低于这些检测方法的检测阈值。CBA 检测方法也灵敏度较低，但不受 RF 的影响。
由于异嗜性抗体的存在，假阳性主要影响 Luminex 检测方法。然而，其他检测方法在灵敏度方面仍然受到限制。在开发出足够敏感的检测方法之前，细胞因子的多重检测仍然是一个挑战，特别是在风湿病理方面。ELISA 仍然是金标准。
