Ibsen P H
Bacterial Vaccines Department, Statens Seruminstitut, Copenhagen, Denmark.
Vaccine. 1996 Apr;14(5):359-68. doi: 10.1016/0264-410x(95)00230-x.
The effect of detoxification of pertussis toxin (PT) for vaccine usage by either genetic manipulation, hydrogen peroxide or formaldehyde treatment on epitope recognition by a large collection of murine monoclonal pertussis toxin antibodies (PT MAbs) was assessed in a solid-phase and a soluble phase enzyme-linked immunosorbent assay (ELISA). The MAb binding patterns were found to be different in the two assays as the immobilization step appeared to cause conformational alterations in the native as well as the toxoided forms of PT. According to the solid-phase ELISA, genetic, hydrogen peroxide and 0.35% formaldehyde detoxification of PT resulted in reduced epitope binding in 2.9, 31.4 and 78.1% of the MAbs, respectively. In the soluble-phase ELISA, in which the MAbs were allowed to react with the toxoids or native toxin in solution, the percentages of MAbs showing decreased binding activity were 9.1, 50.0 and 71.4%, respectively. Stabilization of native PT and the genetically inactivated PT by 0.035% formaldehyde reduced the epitope binding activity in 50.0 and 8.7% of the MAbs, respectively. Increased antibody binding in the soluble-phase ELISA was observed in some of the toxoids: this ranged from 0% in the 0.35% formaldehyde-treated PT to 13.6% in the hydrogen peroxide-treated and 27.3% in the genetically detoxified PT. Regarding the effects of detoxification on epitopes recognized by PT-neutralizing MAbs in the soluble-phase ELISA, we found that treatment of PT with either 0.035%, 0.35% formaldehyde or hydrogen peroxide induced impairment of epitope binding in 72.7, 81.8 and 45.5% of the MAbs, respectively. In the genetically inactivated PT, the epitopes recognized by the neutralizing MAbs either appeared to remain intact or to show increased MAb binding activity. The epitope-binding patterns of several PT MAbs with mouse-protective properties varied considerably and were shown to be dependent on the detoxification procedure employed. The relevance of epitope alterations on PT as a vaccine component is discussed. The results of the present study may have important implications for future quality assessment of PT for use in acellular pertussis vaccines.
在固相和可溶性酶联免疫吸附测定（ELISA）中，评估了通过基因操作、过氧化氢或甲醛处理对百日咳毒素（PT）进行解毒以用于疫苗时，大量鼠源单克隆百日咳毒素抗体（PT MAb）对表位的识别情况。在这两种测定中发现单克隆抗体的结合模式有所不同，因为固定步骤似乎会导致天然形式以及类毒素形式的PT发生构象改变。根据固相ELISA，PT的基因解毒、过氧化氢解毒和0.35%甲醛解毒分别导致2.9%、31.4%和78.1%的单克隆抗体的表位结合减少。在可溶性ELISA中，单克隆抗体与溶液中的类毒素或天然毒素反应，显示结合活性降低的单克隆抗体的百分比分别为9.1%、50.0%和71.4%。用0.035%甲醛对天然PT和基因灭活的PT进行稳定化处理，分别使50.0%和8.7%的单克隆抗体的表位结合活性降低。在一些类毒素中观察到可溶性ELISA中抗体结合增加：这一范围从0.35%甲醛处理的PT中的0%到过氧化氢处理的PT中的13.6%以及基因解毒的PT中的27.3%。关于在可溶性ELISA中解毒对PT中和单克隆抗体识别的表位的影响，我们发现用0.035%、0.35%甲醛或过氧化氢处理PT分别导致72.7%、81.8%和45.5%的单克隆抗体的表位结合受损。在基因灭活的PT中，中和单克隆抗体识别的表位要么似乎保持完整，要么显示出单克隆抗体结合活性增加。几种具有小鼠保护特性的PT单克隆抗体的表位结合模式差异很大，并且显示取决于所采用的解毒程序。讨论了PT表位改变作为疫苗成分的相关性。本研究结果可能对未来用于无细胞百日咳疫苗的PT的质量评估具有重要意义。
