Development of a Direct Alhydrogel Formulation Immunoassay (DAFIA).
Development of a Direct Alhydrogel Formulation Immunoassay (DAFIA).
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DOI：
10.1016/j.jim.2009.03.005
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发表时间：
2009-05-15
影响因子：
2.2
通讯作者：
Sau, Allan
中科院分区：
文献类型：
--
作者：
Zhu, Daming;Huang, Shuhui;Gebregeorgis, Elizabeth;McClellan, Holly;Dai, Weili;Miller, Louis;Sau, Allan
Alhydrogel®（氢氧化铝）是美国广泛使用的佐剂。监管部门要求对疫苗进行检测，以确定最终疫苗产品中的抗原含量。目前在我们的实验室中通过邻苯二甲醛（OPA）荧光蛋白测定法测定配制抗原的水平，并通过蛋白质印迹和SDS-PAGE测定抗原同一性和完整性。然而，OPA测定是非特异性的并且仅限于检测总蛋白含量，并且其通常不够灵敏以检测低剂量制剂中的抗原。此外，鉴别和完整性检测中使用的抗原必须使用提取程序从疫苗中提取，该提取程序耗时且可能无法完全回收抗原用于分析或可能在提取期间改变抗原的结构。本研究开发了一种直接明矾制剂免疫测定法（DAFIA），旨在直接（无需抗原提取）、准确和灵敏地测定明矾上的抗原含量、同一性和完整性。AMA 1-C1/Alhydrogel制剂用作试验开发和验证中的模型疫苗。结果表明，DAFIA具有高度的抗原特异性，准确度（87-100%），灵敏度（0.16 μg/ml），重现性好，检测范围为0.16-10 μg/ml。这些结果表明，DAFIA是测定与明矾结合的抗原的抗原含量、同一性和完整性的极佳测定方法，可用于常规疫苗质量控制，以检测基于铝水凝胶的疫苗中的抗原。
Alhydrogel® (aluminum hydroxide) is a widely used adjuvant in the US. Regulatory authorities require that vaccines be tested to determine the antigen content in the final vaccine product. The level of formulated antigen is currently determined in our laboratory by the o-Phthalaldehyde (OPA) fluorescent protein assay, and antigen identity and integrity are determined by Western blot and SDS-PAGE. However, OPA assay is non-specific and only limited to detection of total protein content, and it is often not sensitive enough to detect antigens in low dose formulations. Furthermore, antigens used in identity and integrity tests must be extracted from vaccines using an extraction procedure which is time-consuming and may not completely recover antigens for analysis or may alter the structures of antigens during extraction. The present study developed a Direct Alum Formulation Immunoassay (DAFIA) which was designed to directly (without antigen extraction), accurately, and sensitively determine the antigen content, identity and integrity on alum. The AMA1-C1/Alhydrogel formulation was used as a model vaccine in assay development and validation. The results showed that the DAFIA is highly antigen-specific, accurate (87–100%), sensitive (0.16 µg/ml), reproducible, and simple with a linear detection range of 0.16–10 µg/ml. These results demonstrate that DAFIA is an excellent assay to determine antigen content, identity and integrity of antigens bound to alum and may be used in routine vaccine quality control for testing antigens in Alhydrogel-based vaccines.
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