Department of Medical Microbiology and Immunology, Aarhus University, Wilhelm Meyers Allé 4, DK-8000 Aarhus C, Denmark.
J Immunol Methods. 2011 Sep 30;372(1-2):204-8. doi: 10.1016/j.jim.2011.06.030. Epub 2011 Jul 12.
The enzyme-linked immunosorbent assay (ELISA) and its derivatives are powerful tools used in research, in the clinic, and in many other analytical and quality control settings. In general, ELISAs are robust, reproducible and reliable. However, a number of pitfalls of ELISAs have been described over the years. The issue of rheumatoid factor (RF), autoantibodies against the Fc portion of IgG, is well recognized (yet often forgotten), as are problems arising from heterophilic antibodies induced by external antigens that cross-react with self-antigens. A few years ago focus was on human anti-mouse antibodies (HAMA) concomitant with the increased use of mouse monoclonal antibody therapy, a problem that is now diminishing due to development of humanized antibodies. Issues pertaining to food antigens or environmentally encountered antigens are less recognized. We report a recently encountered example of the latter resulting in interference in a solid-phase sandwich assay. Due to the set-up employing a monoclonal rat IgG for capture and a monoclonal rat IgM for development the interference had to be human antibodies reacting with rat light-chain. Out of 102 Danish Caucasian blood donors we found a prevalence of anti-rat kappa light chain antibodies of close to 40% (39/102, defined as at least 2-fold elevated measurements), with around 6% (6/102) having very high levels (defined as at least 4-fold elevated measurements), yielding significantly higher measurements in the assay designed to measure the complement component MAp19 in serum samples. The interference could be blocked by the addition of rat immunoglobulin to the sample buffer. An individual, who had been followed over time, demonstrated a periodic increase of interfering antibodies, highlighting that it is an independently varying parameter and thereby a variable interference in assays. Our results highlight a major pitfall of potential relevance to many sandwich-type assays, as well as an approach to rectify such problems.
酶联免疫吸附测定（ELISA）及其衍生方法是用于研究、临床以及许多其他分析和质量控制环境的强大工具。通常情况下，ELISA 具有稳健、可重现和可靠的特点。然而，多年来已经描述了 ELISA 的一些陷阱。类风湿因子（RF），即针对 IgG Fc 部分的自身抗体，是一个众所周知的问题（但经常被遗忘），还有由与自身抗原发生交叉反应的外部抗原引起的异嗜性抗体引起的问题。几年前，人们关注的是与使用鼠单克隆抗体治疗同时出现的人抗鼠抗体（HAMA），由于开发了人源化抗体，这个问题现在正在减少。与食物抗原或环境中遇到的抗原相关的问题则较少被认识到。我们报告了一个最近遇到的例子，该例子导致固相夹心测定中出现干扰。由于采用单克隆大鼠 IgG 进行捕获和单克隆大鼠 IgM 进行开发的设置，因此必须是与人抗体反应的大鼠轻链。在 102 名丹麦白种人献血者中，我们发现抗大鼠 κ 轻链抗体的患病率接近 40%（39/102，定义为至少 2 倍升高的测量值），其中约 6%（6/102）具有非常高的水平（定义为至少 4 倍升高的测量值），这导致在设计用于测量血清样本中补体成分 MAp19 的测定中产生了更高的测量值。通过在样品缓冲液中添加大鼠免疫球蛋白可以阻断干扰。一位个体经过一段时间的随访，显示出干扰抗体的周期性增加，这突出表明它是一个独立变化的参数，因此在测定中会出现可变干扰。我们的结果突出了一个重大陷阱，这对许多夹心型测定具有潜在相关性，同时也提出了一种纠正这些问题的方法。
