Use of Luminex xMAP-derived Bio-Plex bead-based suspension array for specific detection of PPV W and characterization of epitopes on the coat protein of the virus
Use of Luminex xMAP-derived Bio-Plex bead-based suspension array for specific detection of PPV W and characterization of epitopes on the coat protein of the virus
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DOI：
10.1016/j.jviromet.2008.07.016
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发表时间：
2008-11-01
影响因子：
3.1
通讯作者：
James, Delano
中科院分区：
文献类型：
--
作者：
Croft, Heather;Malinowski, Tadeusz;James, Delano
用重组肽免疫小鼠，制备了一组针对PPV W（分离物3174）外壳蛋白n端区的单克隆抗体（mab）。鉴定出表现最好的单克隆抗体为2C3和10G7。选择MAb 2C3用于比较标准的TAS-ELISA方案与Luminex xMAP技术衍生的珠状悬浮阵列系统，称为三重抗体三明治微球免疫测定（TAS-MIA）。TAS-MIA在草本和木质寄主中特异检测PPV W的灵敏度与TAS-ELISA相同。在4 h内完成，使用较少的试剂。表位识别分析使用一组重叠的合成针结合肽（Mimotopes）进行。肽粒(DEEDD6)-D-2和(MFNPV50)-M-46是表现最好的单克隆抗体最常识别的表位。b细胞识别位点的线性表位预测证实，这两种肽都位于高抗原性和可接近的区域。表位的第二个谷氨酸残基对单克隆抗体的识别至关重要，并且表位的背景与表位的序列同样重要。ELISA、Western blot和TAS-MIA结果与b细胞识别预测相关。这是一种有效的方法来确定合适的抗原表位，产生抗体，用于可靠的诊断程序。这是使用Luminex xMAP微珠悬浮阵列系统检测植物病毒的第一份报告。爱思唯尔B.V.版权所有
A panel of monoclonal antibodies (MAbs) directed against the N-terminus region of the coat protein (CP) of strain PPV W (isolate 3174) was generated by immunizing mice with recombinant peptides. The best performing MAbs were identified as 2C3 and 10G7. MAb 2C3 was selected for comparison of a standard TAS-ELISA protocol with a Luminex xMAP technology-derived bead-based suspension array system described as a triple antibody sandwich-microsphere immunoassay (TAS-MIA). TAS-MIA was as sensitive as TAS-ELISA for the specific detection of PPV W in herbaceous and woody hosts. It was completed in 4 h, and used less reagents. Epitope recognition analysis was carried out using a set of overlapping synthetic pin-bound peptides (Mimotopes). Pepticles (DEEDD6)-D-2 and (MFNPV50)-M-46 were the epitopes recognized most commonly by the best performing MAbs. Linear epitope prediction of B-cell recognition sites confirmed that both peptides fall within highly antigenic and accessible regions. The second glutaimic acid residue of the epitope is crucial for MAb recognition, and the context of the epitope is as important as the sequence of the epitope. The results obtained in ELISA, Western blot, and TAS-MIA correlated with B-cell recognition prediction. This is an effective approach to identify suitable antigenic epitopes that generate antibodies for use in reliable diagnostic procedures. This is the first report of the detection of a plant virus using the Luminex xMAP bead-based suspension array system. Crown Copyright (C) 2008 Published by Elsevier B.V. All rights reserved.