The pineal gland hormone melatonin continues to be of considerable interest to biomedical researchers. Of particular interest is the pattern of secretion of melatonin in relation to sleep timing as well as its potential role in certain diseases. Measuring melatonin in biological fluids such as blood and saliva presents particular methodological challenges since the production and secretion of the hormone are known to be extremely low during the light phase in almost all situations. Active secretion only occurs around the time of lights out in a wide range of species. The challenge then is to develop practical high‐throughput assays that are sufficiently sensitive and accurate enough to detect levels of melatonin less than 1 pg/mL in biological fluids. Mass spectrometry assays have been developed that achieve the required sensitivity, but are really not practical or even widely available to most researchers. Melatonin radioimmunoassays and ELISA have been developed and are commercially available. But the quality of the results that are being published is very variable, partly not only because of poor experimental designs, but also because of poor assays. In this review, I discuss issues around the design of studies involving melatonin measurement. I then provide a critical assessment of 21 immunoassay kits marketed by 11 different companies with respect to validation, specificity and sensitivity. Technical managers of the companies were contacted in an attempt to obtain information not available online or in kit inserts. A search of the literature was also conducted to uncover papers that have reported the use of these assays, and where possible, both daytime and night‐time plasma or saliva melatonin concentrations were extracted and tabulated. The results of the evaluations are disturbing, with many kits lacking any validation studies or using inadequate validation methods. Few assays have been properly assessed for specificity, while others report cross‐reaction profiles that can be expected to result in over estimation of the melatonin levels. Some assays are not fit for purpose because they are not sensitive enough to determine plasma or saliva DLMO of 10 and 3 pg/mL, respectively. Finally, some assays produce unrealistically high daytime melatonin levels in humans and laboratory animals in the order of hundreds of pg/mL. In summary, this review provides a comprehensive and unique assessment of the current commercial melatonin immunoassays and their use in publications. It provides researchers new to the field with the information they need to design valid melatonin studies from both the perspective of experimental/clinical trial design and the best assay methodologies. It will also hopefully help journal editors and reviewers who may not be fully aware of the pitfalls of melatonin measurement make better informed decisions on publication acceptability.

中文翻译：

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通过免疫测定测量褪黑激素。

松果体激素褪黑激素继续引起生物医学研究人员的极大兴趣。特别令人感兴趣的是褪黑激素的分泌模式与睡眠时间及其在某些疾病中的潜在作用有关。测量血液和唾液等生物体液中的褪黑激素提出了特殊的方法挑战，因为已知在几乎所有情况下，在轻度阶段，激素的产生和分泌都非常低。活跃的分泌只发生在大范围物种的熄灯时间。然后面临的挑战是开发实用的高通量检测方法，该方法足够灵敏和准确，足以检测生物体液中低于 1 pg/mL 的褪黑激素水平。已开发出达到所需灵敏度的质谱分析，但对于大多数研究人员来说，实际上并不实用，甚至无法广泛使用。褪黑激素放射免疫测定法和 ELISA 已被开发出来并可以在市场上买到。但是，正在发表的结果的质量参差不齐，部分原因不仅是因为实验设计不佳，还因为检测方法很差。在这篇综述中，我讨论了涉及褪黑激素测量的研究设计问题。然后，我对 11 家不同公司销售的 21 种免疫测定试剂盒在验证、特异性和灵敏度方面进行了重要评估。联系了这些公司的技术经理，试图获取无法在线或在套件插入中获得的信息。还进行了文献检索以发现报道使用这些测定的论文，并在可能的情况下，提取白天和夜间血浆或唾液褪黑激素浓度并制成表格。评估结果令人不安，许多试剂盒缺乏任何验证研究或使用的验证方法不充分。很少有分析对特异性进行了适当的评估，而其他分析报告了交叉反应谱，预计会导致对褪黑激素水平的高估。一些测定不适合目的，因为它们不够灵敏，无法分别测定 10 和 3 pg/mL 的血浆或唾液 DLMO。最后，一些检测在人类和实验室动物体内产生不切实际的高水平褪黑激素，大约为数百 pg/mL。总之，本综述对当前的商业褪黑激素免疫测定及其在出版物中的使用进行了全面而独特的评估。它为该领域的新研究人员提供了从实验/临床试验设计和最佳检测方法的角度设计有效褪黑激素研究所需的信息。它也有望帮助可能不完全了解褪黑激素测量陷阱的期刊编辑和审稿人对出版物的可接受性做出更明智的决定。