Jelsma Tinka, van der Wal Fimme J, Fijten Helmi, Dailly Nicolas, van Dijk Evert, Loeffen Willie L
Wageningen Bioveterinary Research (WBVR previously CVI), P.O. Box 65, 8200 AB Lelystad, The Netherlands.
Pepscan, Zuidersluisweg 2, 8243 RC Lelystad, The Netherlands.
J Virol Methods. 2017 Sep;247:114-118. doi: 10.1016/j.jviromet.2017.05.008. Epub 2017 May 22.
Most serological assays detect antibody responses in biological samples through affinity of serum antibodies for antigens provided in the assay. Certain antigens, however, may be difficult to produce and/or may contain unwanted epitopes. In these cases, a practical alternative may be the use of peptides as representatives for specific epitopes. Peptides can be obtained after purification in large quantities for a modest price, but screening of a large set of peptides during development may be relatively expensive. To cut costs of screening peptides for a new serological assay, the concept was investigated of using cheap non-purified (crude) peptides instead of purified peptides. Peptides were selected that represent three well-described linear epitopes of viral proteins: VP2 of canine parvovirus (CPV), gp41 of human immunodeficiency virus (HIV) and E2 of classical swine fever virus (CSFV). Crude and purified biotinylated peptides with either a short or long spacer between the biotin and the epitope were used to test their capability to bind antibodies in a bead-based suspension array. The results show that, in a bead-based suspension array, crude peptides can function as antigen for specific monoclonal antibodies, and that the acquired signals are less than with purified peptides. CSFV-derived crude peptides were also able to detect specific antibodies in swine serum, indicating the applicability of crude peptides for pre-screening large numbers of different peptides during the development of serological peptide-based assays.
大多数血清学检测通过检测生物样品中血清抗体与检测中所提供抗原的亲和力来检测抗体反应。然而，某些抗原可能难以制备，和/或可能含有不需要的表位。在这些情况下，一种实际的替代方法可能是使用肽作为特定表位的代表。肽可以大量纯化后以适中的价格获得，但在开发过程中筛选大量肽可能相对昂贵。为了降低新血清学检测中筛选肽的成本，研究了使用廉价的未纯化（粗制）肽而非纯化肽的概念。选择了代表病毒蛋白三个已充分描述的线性表位的肽：犬细小病毒（CPV）的VP2、人类免疫缺陷病毒（HIV）的gp41和经典猪瘟病毒（CSFV）的E2。使用生物素与表位之间具有短间隔或长间隔的粗制和纯化的生物素化肽，在基于珠子的悬浮阵列中测试它们结合抗体的能力。结果表明，在基于珠子的悬浮阵列中，粗制肽可以作为针对特定单克隆抗体的抗原，并且获得的信号比纯化肽少。源自CSFV的粗制肽也能够检测猪血清中的特异性抗体，表明粗制肽在基于肽的血清学检测开发过程中用于预筛选大量不同肽的适用性。
