一种高灵敏度的酶联免疫吸附测定法能够准确测量人类唾液中脑源性神经营养因子的水平。
A highly sensitive enzyme‑linked immunosorbent assay allows accurate measurements of brain‑derived neurotrophic factor levels in human saliva.
作者信息
Akutsu Fumie, Sugino Shiro, Watanabe Mitsuo, Barde Yves-Alain, Kojima Masaaki
机构信息
FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan.
School of Biosciences, Cardiff University, Cardiff, UK.
出版信息
F1000Res. 2025 May 22;14:161. doi: 10.12688/f1000research.160304.2. eCollection 2025.
BACKGROUND
Hitherto, BDNF levels in humans have been primarily measured in serum and/or plasma where these levels are readily measurable, but primarily reflect the content of BDNF in blood platelets. By contrast, previous attempts to measure BDNF levels in readily accessible human body fluids such as saliva have been complicated by a lack of sensitivity and/or specificity of BDNF ELISAs (see Discussion). Recently, the suitability of a highly sensitive BDNF ELISA assay was validated using mouse plasma and serum where conventional BDNF ELISA fail to detect BDNF. In this report, we demonstrate that BDNF levels in human saliva are extremely low, in the low pg/mL range, yet detectable in all saliva samples tested.
METHODS
Saliva samples were collected from healthy volunteers by a passive drool method. All samples were aliquoted and immediately frozen to keep at -80°C until use. At the time of use, the samples were thawed, centrifuged to remove any remaining particles and BDNF measurement conducted by using a previously validated BDNF ELISA assay (see below). Recombinant mature BDNF was used as a reference.
RESULTS
The intra-assay variability was in the range of CV = 1.8 to 4.9%. Saliva samples could be kept frozen at -80°C for 2 months until use for measurements, but more than 4 freeze and thaw cycles caused BDNF losses presumably due to structural change of the antigen. The measurements were not affected by the method of collection provided the samples were diluted at least 2-fold.
CONCLUSIONS
The results indicate that human saliva samples collected in a non-invasive fashion can be used as a source of material to try and correlate BDNF levels with human conditions of interest. These results also confirm those of an independent study published recently using the same BDNF ELISA kit to measure BDNF levels in human saliva samples.
背景
迄今为止,人类体内的脑源性神经营养因子(BDNF)水平主要在血清和/或血浆中进行测量,这些水平易于测量,但主要反映血小板中BDNF的含量。相比之下,此前尝试在唾液等易于获取的人体体液中测量BDNF水平时,由于BDNF酶联免疫吸附测定(ELISA)缺乏敏感性和/或特异性而变得复杂(见讨论)。最近,一种高灵敏度的BDNF ELISA检测方法的适用性在小鼠血浆和血清中得到了验证,而传统的BDNF ELISA无法检测到BDNF。在本报告中,我们证明人类唾液中的BDNF水平极低,处于低皮克/毫升范围内,但在所有测试的唾液样本中均可检测到。
方法
通过被动流涎法从健康志愿者中收集唾液样本。所有样本均进行分装,并立即冷冻保存在-80°C直至使用。使用时,将样本解冻,离心以去除任何残留颗粒,然后使用先前验证的BDNF ELISA检测方法进行BDNF测量(见下文)。重组成熟BDNF用作参考。
结果
批内变异系数(CV)在1.8%至4.9%范围内。唾液样本可在-80°C冷冻保存2个月直至用于测量,但超过4次冻融循环会导致BDNF损失,可能是由于抗原结构变化所致。只要样本至少稀释2倍,测量不受收集方法的影响。
结论
结果表明,以非侵入性方式收集的人类唾液样本可作为一种材料来源,用于尝试将BDNF水平与感兴趣的人类状况相关联。这些结果也证实了最近发表的一项独立研究结果,该研究使用相同的BDNF ELISA试剂盒测量人类唾液样本中的BDNF水平。
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