Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies.
Evaluation of immunoglobulin purification methods and their impact on quality and yield of antigen-specific antibodies.
复制标题
DOI：
10.1186/1475-2875-7-129
复制
发表时间：
2008-07-14
期刊：
影响因子：
3
通讯作者：
Angov E
中科院分区：
文献类型：
--
作者：
Bergmann-Leitner ES;Mease RM;Duncan EH;Khan F;Waitumbi J;Angov E
抗体是对抗疟疾血液期寄生虫的主要效应物。对来自2a/B期疫苗试验的免疫血清中的功能活性进行评估可能会为寻找保护的免疫相关性提供宝贵的信息。然而，抗疟疾药物的存在、不适当的采集/储存条件或伴随的针对其他病原体的免疫应答可能导致血清/血浆在体外检测时的非特异性抗寄生虫活性。免疫球蛋白的纯化是用于降低这种非特异性背景活性的标准方法，但是纯化方法本身可以改变回收的Ag特异性抗体的质量和产率。为了解决这一问题，评价了各种免疫球蛋白（IG）纯化方法（蛋白G琼脂糖凝胶、蛋白A/G琼脂糖凝胶、聚乙二醇和辛酸-硫酸铵沉淀）对纯化兔和人Ig质量、数量和功能活性的影响。通过SDS-PAGE分析回收的Ig的产率和纯度，通过Ag特异性ELISA（测定滴度、亲合力和同种型分布的变化）分析其质量，并通过体外寄生虫生长抑制试验（GIA）分析其功能活性。该比较表明，人血清/血浆样品的总体聚乙二醇纯化和兔血清的蛋白G琼脂糖凝胶纯化对于回收功能性Ag特异性抗体是最佳的。因此，需要对纯化方法进行严格考虑，以避免选择回收IG的非代表性群体，这可能影响疫苗效力的解释，或影响对保护的免疫相关性的搜索。
Antibodies are the main effectors against malaria blood-stage parasites. Evaluation of functional activities in immune sera from Phase 2a/b vaccine trials may provide invaluable information in the search for immune correlates of protection. However, the presence of anti-malarial-drugs, improper collection/storage conditions or concomitant immune responses against other pathogens can contribute to non-specific anti-parasite activities when the sera/plasma are tested in vitro. Purification of immunoglobulin is a standard approach for reducing such non-specific background activities, but the purification method itself can alter the quality and yield of recovered Ag-specific antibodies. To address this concern, various immunoglobulin (Ig) purification methods (protein G Sepharose, protein A/G Sepharose, polyethylene glycol and caprylic acid-ammonium sulphate precipitation) were evaluated for their impact on the quality, quantity and functional activity of purified rabbit and human Igs. The recovered Igs were analysed for yield and purity by SDS-PAGE, for quality by Ag-specific ELISAs (determining changes in titer, avidity and isotype distribution) and for functional activity by in vitro parasite growth inhibition assay (GIA). This comparison demonstrated that overall polyethylene glycol purification of human serum/plasma samples and protein G Sepharose purification of rabbit sera are optimal for recovering functional Ag-specific antibodies. Consequently, critical consideration of the purification method is required to avoid selecting non-representative populations of recovered Ig, which could influence interpretations of vaccine efficacy, or affect the search for immune correlates of protection.
登录
查看更多内容
影响因子：
2.2
作者：
PULLEN, GR;FITZGERALD, MG;HOSKING, CS
通讯作者：
HOSKING, CS
影响因子：
--
作者：
TEMPONI, M;KAGESHITA, T;FERRONE, S
通讯作者：
FERRONE, S
影响因子：
9.4
作者：
Turpin, EA;Lauer, DC;Swayne, DE
通讯作者：
Swayne, DE
影响因子：
2.2
作者：
PEROSA, F;CARBONE, R;DAMMACCO, F
通讯作者：
DAMMACCO, F
影响因子：
4.1
作者：
Azevedo, Ana M.;Rosa, Paula A. J.;Aires-Barros, M. Raquel
通讯作者：
Aires-Barros, M. Raquel