Engmark Mikael, Jespersen Martin C, Lomonte Bruno, Lund Ole, Laustsen Andreas H
Department of Bio and Health Informatics, Technical University of Denmark, Kgs. Lyngby, Denmark; Department of Biotechnology and Biomedicine, Technical University of Denmark, Kgs. Lyngby, Denmark.
Department of Bio and Health Informatics, Technical University of Denmark, Kgs. Lyngby, Denmark.
Toxicon. 2017 Nov;138:151-158. doi: 10.1016/j.toxicon.2017.08.028. Epub 2017 Sep 1.
Polyvalent snakebite antivenoms derive their therapeutic success from the ability of their antibodies to neutralize venom toxins across multiple snake species. This ability results from a production process involving immunization of large mammals with a broad suite of toxins present in venoms. As a result of immunization with this wide range of toxins, many polyvalent antivenoms have a high degree of cross-reactivity to similar toxins in other snake venoms - a cross-reactivity which cannot easily be deconvoluted. As a proof of concept, we aimed at exploring the opposite scenario by performing a high-throughput evaluation of the extent of cross-reactivity of a polyclonal mixture of antibodies that was raised against only a single snake venom fraction. For this purpose, a venom fraction containing short neurotoxin 1 (SN-1; Uniprot accession number P01416, three-finger toxin (3FTx) family), which is the medically most important toxin from the notorious black mamba (Dendroaspis polylepis), was employed. Following immunization of a rabbit, a specific polyclonal antibody response was confirmed by ELISA and immunodiffusion. Subsequently, these antibodies were investigated by high-density peptide microarray to reveal linear elements of recognized epitopes across 742 3FTxs and 10 dendrotoxins. This exploratory study demonstrates in a single immunized animal that cross-reactivity between toxins of high similarity may be difficult to obtain when immunizing with a single 3FTx containing venom fraction. Additionally, this study explored the influence of employing different lengths of peptides in high-density peptide microarray experiments for identification of toxin epitopes. Using 8-mer, 12-mer, and 15-mer peptides, a single linear epitope element was identified in SN-1 with high precision.
多价蛇咬伤抗蛇毒血清的治疗成功源于其抗体中和多种蛇类毒液毒素的能力。这种能力源于一个生产过程，该过程涉及用毒液中存在的多种毒素对大型哺乳动物进行免疫。由于用如此广泛的毒素进行免疫，许多多价抗蛇毒血清对其他蛇毒中的相似毒素具有高度交叉反应性——这种交叉反应性很难解析。作为概念验证，我们旨在通过对仅针对单一蛇毒组分产生的多克隆抗体混合物的交叉反应程度进行高通量评估，来探索相反的情况。为此，使用了一种毒液组分，其含有短神经毒素1（SN-1；Uniprot登录号P01416，三指毒素（3FTx）家族），这是臭名昭著的黑曼巴蛇（Dendroaspis polylepis）医学上最重要的毒素。对一只兔子进行免疫后，通过ELISA和免疫扩散确认了特异性多克隆抗体反应。随后，通过高密度肽微阵列研究这些抗体，以揭示742种3FTx和10种树突毒素中识别表位的线性元件。这项探索性研究表明，在用含有单一3FTx的毒液组分进行免疫时，高相似性毒素之间的交叉反应性可能很难获得。此外，本研究还探讨了在高密度肽微阵列实验中使用不同长度的肽对毒素表位鉴定结果的影响。使用8聚体、12聚体和15聚体肽，在SN-1中高精度地鉴定出了单个线性表位元件。
